Search

Developing novel therapeutics often follows three steps: target identification, design of strategies to suppress target activity and drug development to implement the strategies. In this review, we recount the evidence identifying the basic leucine zipper transcription factors ATF5, CEBPB, and CEBPD as targets for brain and other malignancies. We describe strategies that exploit the structures of the three factors to create inhibitory dominant-negative (DN) mutant forms that selectively suppress growth and survival of cancer cells. We then discuss and compare four peptides (CP-DN-ATF5, Dpep, Bpep and ST101) in which DN sequences are joined with cell-penetrating domains to create drugs that pass through tissue barriers and into cells. The peptide drugs show both efficacy and safety in suppressing growth and in the survival of brain and other cancers in vivo, and ST101 is currently in clinical trials for solid tumors, including GBM. We further consider known mechanisms by which the peptides act and how these have been exploited in rationally designed combination therapies. We additionally discuss lacunae in our knowledge about the peptides that merit further research. Finally, we suggest both short- and long-term directions for creating new generations of drugs targeting ATF5, CEBPB, CEBPD, and other transcription factors for treating brain and other malignancies.

BLNK (BASH/SLP‐65) encodes an adaptor protein that plays an important role in B‐cell receptor (BCR) signaling. Loss‐of‐function mutations in this gene are observed in human pre‐B acute lymphoblastic leukemia (ALL), and a subset of Blnk knock‐out (KO) mice develop pre‐B‐ALL. To understand the molecular mechanism of the Blnk mutation‐associated pre‐B‐ALL development, retroviral tagging was applied to KO mice using the Moloney murine leukemia virus (MoMLV). The Blnk mutation that significantly accelerated the onset of MoMLV‐induced leukemia and increased the incidence of pre‐B‐ALL Cebpb was identified as a frequent site of retroviral integration, suggesting that its upregulation cooperates with Blnk mutations. Transgenic expression of the liver‐enriched activator protein (LAP) isoform of Cebpb reduced the number of mature B‐lymphocytes in the bone marrow and inhibited differentiation at the pre‐BI stage. Furthermore, LAP expression significantly accelerated leukemogenesis in Blnk KO mice and alone acted as a B‐cell oncogene. Furthermore, an inverse relationship between BLNK and C/EBPβ expression was also noted in human pre‐B‐ALL cases, and the high level of CEBPB expression was associated with short survival periods in patients with BLNK‐downregulated pre‐B‐ALL. These results indicate the association between the C/EBPβ transcriptional network and BCR signaling in pre‐B‐ALL development and leukemogenesis. This study gives insight into ALL progression and suggests that the BCR/C/EBPβ pathway can be a therapeutic target., C/EBPB was identified as a common retroviral integration site by retroviral tagging in Blnk knock‐out mice. B‐cell specific expression of the C/EBPB LAP isoform enhanced differentiation block of pre‐B‐cell and accelerated leukemogenesis of the Blnk mutation.

Amyloid-β precursor protein (APP) is sequentially cleaved by secretases and generates amyloid-β, the major components in senile plaques in Alzheimer’s disease. APP is upregulated in human Alzheimer’s disease brains. However, the molecular mechanism of how APP contributes to Alzheimer’s disease pathogenesis remains incompletely understood. Here we show that truncated APP C586-695 fragment generated by δ-secretase directly binds to CCAAT/enhancer-binding protein beta (CEBPB), an inflammatory transcription factor, and enhances its transcriptional activity, escalating Alzheimer’s disease-related gene expression and pathogenesis. The APP C586-695 fragment, but not full-length APP, strongly associates with CEBPB and elicits its nuclear translocation and augments the transcriptional activities on APP itself, MAPT (microtubule-associated protein tau), δ-secretase and inflammatory cytokine mRNA expression, finally triggering Alzheimer’s disease pathology and cognitive disorder in a viral overexpression mouse model. Blockade of δ-secretase cleavage of APP by mutating the cleavage sites reduces its stimulatory effect on CEBPB, alleviating amyloid pathology and cognitive dysfunctions. Clearance of APP C586-695 from 5xFAD mice by antibody administration mitigates Alzheimer’s disease pathologies and restores cognitive functions. Thus, in addition to the sequestration of amyloid-β, APP implicates in Alzheimer’s disease pathology by activating CEBPB upon δ-secretase cleavage.

Transcriptional dysregulation, which can be caused by genetic and epigenetic alterations, is a fundamental feature of many cancers. A key cytoprotective transcriptional activator, NRF2, is often aberrantly activated in non-small cell lung cancers (NSCLCs) and supports both aggressive tumorigenesis and therapeutic resistance. Herein, we find that persistently activated NRF2 in NSCLCs generates enhancers at gene loci that are not normally regulated by transiently activated NRF2 under physiological conditions. Elevated accumulation of CEBPB in NRF2-activated NSCLCs is found to be one of the prerequisites for establishment of the unique NRF2-dependent enhancers, among which the NOTCH3 enhancer is shown to be critical for promotion of tumor-initiating activity. Enhancer remodeling mediated by NRF2-CEBPB cooperativity promotes tumor-initiating activity and drives malignancy of NRF2-activated NSCLCs via establishment of the NRF2-NOTCH3 regulatory axis., Aberrant activation of NRF2 in cancer cells contributes to tumorigenicity and therapeutic resistance. Here, the authors show that NRF2 cooperates with CEBPB and remodels enhancers to confer tumor-initiating activity on NRF2- activated non-small cell lung cancers.

Sepsis is the major cause of mortality in the intensive care unit. The aim of this study was to identify the key prognostic biomarkers of abnormal expression and immune infiltration in sepsis. In this study, a total of 36 differentially expressed genes were identified to be mainly involved in a number of immune-related Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. The hub genes ( MMP9 and C3AR1) were significantly related to the prognosis of sepsis patients. The immune infiltration analysis indicated a significant difference in the relative cell content of naive B cells, follicular Th cells, activated NK cells, eosinophils, neutrophils and monocytes between sepsis and normal controls. Weighted gene co-expression network analysis and a de-convolution algorithm that quantifies the cellular composition of immune cells were used to analyse the sepsis expression data from the Gene Expression Omnibus database and to identify modules related to differential immune cells. CEBPB is the key immune-related gene that may be involved in sepsis. Gene set enrichment analysis revealed that CEBPB is involved in the processes of T cell selection, B cell–mediated immunity, NK cell activation and pathways of T cells, B cells and NK cells. Therefore, CEBPB may play a key role in the biological and immunological processes of sepsis.

Objective There is a pressing need to identify and validate, minimally invasive, molecular biomarkers that will complement current practices and increase the diagnostic accuracy in Parkinson’s disease (PD). Brain‐enriched miRNAs regulate all aspects of neuron development and function; importantly, they are secreted by neurons in amounts that can be readily detected in the plasma. Τhe aim of the present study was to validate a set of previously identified brain‐enriched miRNAs with diagnostic potential for idiopathic PD and recognize the molecular pathways affected by these deregulated miRNAs. Methods RT‐qPCR was performed in the plasma of 92 healthy controls and 108 idiopathic PD subjects. Statistical and in silico analyses were used to validate deregulated miRNAs and pathways in PD, respectively. Results miR‐22‐3p, miR‐124‐3p, miR‐136‐3p, miR‐154‐5p, and miR‐323a‐3p levels were found to be differentially expressed between healthy controls and PD patients. miR‐330‐5p, miR‐433‐3p, and miR‐495‐3p levels were overall higher in male subjects. Most of these miRNAs are clustered at Chr14q32 displaying CREB1, CEBPB, and MAZ transcription factor binding sites. Gene Ontology annotation analysis of deregulated miRNA targets revealed that “Protein modification,” “Transcription factor activity,” and “Cell death” terms were over‐represented. Kyoto Encyclopedia of Genes and Genome analysis revealed that “Long‐term depression,” “TGF‐beta signaling,” and “FoxO signaling” pathways were significantly affected. Interpretation We validated a panel of brain‐enriched miRNAs that can be used along with other measures for the detection of PD, revealed molecular pathways targeted by these deregulated miRNAs, and identified upstream transcription factors that may be directly implicated in PD pathogenesis.

Cell lines are powerful tools for research into liver function at the molecular level. However, they are generally unsuitable for rigorously assessing the effects of amino acid composition, because many lines require serum-containing medium for their maintenance. Here, we aimed to investigate the effects of ornithine and arginine, which are included in the characteristic metabolic process in hepatocyte, on a human hepatoma-derived cell line (FLC-4) that can be cultured in serum-free medium. FLC-4 cells were cultured under the following three conditions: + ornithine/ – arginine, – ornithine/ – arginine, and –ornithine/ + arginine. Albumin expression evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay and showed no obvious differences based on the presence of ornithine or arginine. However, the mRNA levels of two liver-enriched transcription factors (CEBPB and HNF1A), which are involved in regulating albumin expression, were significantly higher in cells grown in medium-containing arginine than that in cells grown in ornithine-containing medium. Western blotting showed that the levels both activating and inhibitory C/EBPβ isoforms were significantly increased in cells grown in arginine medium. Furthermore, we have found that depletion of both ornithine and arginine, the polyamine sources, in the medium did not cause polyamine deficiency. When ornithine and arginine were depleted, albumin production was significantly reduced at the mRNA level, CEBPB mRNA levels were increased, and the level of activating form of C/EBPβ was increased. The results of this study suggest that in hepatocyte, these two amino acids might have different functions, and because of which they elicit disparate cellular responses. Electronic supplementary material The online version of this article (10.1007/s13577-020-00383-1) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) are critical regulators in organ development. Among them, miR-191 is known to be regulated in early embryogenesis and dysregulated in cancer. This role in undifferentiated tissues suggests a possible part of miR-191 also in bone marrow derived mesenchymal stem cells (BMSCs) physiology. Here, we report that miR-191 decreased MMP expression and migration of BMSCs. Conditioned media of miR-191 overexpressing BMSCs block VEGF expression, and inhibit angiogenesis of HUVECs. Under osteogenic culture conditions, inhibition of miR-191 significantly induces bone formation. Moreover, our studies showed miR-191 might influence chondrogenesis of BMSCs by directly targeting CCAAT Enhancer Binding Protein Beta (CEBPB). Taken together, here we demonstrate the role of miR-191 in differentiation, migration and paracrine function of BMSCs.

Chemotherapy is the first-tier treatment regime for gastric cancer (GC) patients at advance stages. Mesenchymal stem cell (MSC) cam affect drug-resistance of GC cells in tumor microenvironment, but the detailed mechanism remains poorly understood. Present study aimed to investigate the regulation of MSC-induced long non-coding RNA (lncRNA) in GC. Dysregulated lncRNAs in GC were analyzed based on GEO data. Stemness and drug-resistance of GC cells were detected by sphere formation, colony formation, CCK-8, and flow cytometry analyses. MicroRNA (miRNA)-related pathways were analyzed by online KEGG analysis tool DAVID6.8. Molecular interactions were determined by luciferase reporter assay, pulldown, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and co-immunoprecipitation (CoIP). Results revealed that MSC co-culture improved stemness and drug-resistance of GC cells. LncRNA histocompatibility leukocyte antigen complex P5 (HCP5) was induced in GC cells by MSC co-culture, contributing to stemness and drug-resistance. Mechanistically, HCP5 sequestered miR-3619-5p and upregulated PPARG coactivator 1 alpha (PPARGC1A), increasing transcription complex Peroxisome proliferator activated receptor (PPAR) coactivator‐1α (PGC1α)/CEBPB and transcriptionally inducing carnitine palmitoyltransferase 1 (CPT1), which prompted the fatty acid oxidation (FAO) in GC cells. In conclusion, MSC-induced lncRNA HCP5 drove FAO through miR-3619-5p/AMPK/PGC1α/CEBPB axis to promote stemness and chemo-resistance of GC, indicating that targeting HCP5 was a novel approach to enhancing the efficacy of chemotherapy in GC.

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (

Objective Obesity represents a growing health problem that is reaching pandemic dimensions and lacks effective cures, thus highlighting an urgent need for better mechanistic understanding and new therapeutic strategies. Unlike transcription, the function of translation in obesity has hardly been investigated. Here, we fill this knowledge gap by pinpointing a crucial function for gene regulation at the step of translation in diet-induced obesity. Methods We performed studies with human adipose tissue, high-fat-diet-induced obese mice and rats, CPEB4-knockout mice, and adipocyte lines. Cells were transfected with small-interfering RNAs that knockdown CPEB4. Transcriptome-wide identification and validation of CPEB4 targets in adipocytes were obtained by RNA-protein coimmunoprecipitation and high-throughput sequencing. The effect of CPEB4 depletion on high-fat-diet-induced dysbiosis was determined by 16S ribosomal-RNA gene sequencing and microbiome bioinformatics. Results We show that cytoplasmic polyadenylation element-binding protein 4 (CPEB4), which controls the translation of specific mRNAs by modulating their poly(A) tails, is highly expressed in visceral fat of obese but not lean humans and rodents (mice and rats), where it orchestrates an essential post-transcriptional reprogramming for aggravation of high-fat-diet-induced obesity. Mechanistically, CPEB4 overexpression in obese adipocytes activates the translation of factors essential for adipose tissue expansion (Cebpb, Stat5a) and adipocyte-intrinsic immune-like potential (Ccl2, Tlr4), as demonstrated by RNA-immunoprecipitation and high-throughput sequencing and experimentally validated in vivo. Consistently blocking CPEB4 production in knockout mice protects against diet-induced body weight gain and reduces adipose tissue enlargement and inflammation. In addition, the depletion of CPEB4 specifically in obese adipocytes using short hairpin RNAs decreases cell differentiation, lipid accumulation, and the proinflammatory and migratory capacity of macrophages. The absence of CPEB4 also attenuates high-fat diet-induced dysbiosis, shaping the microbiome composition toward a more beneficial profile, as shown by microbiome bioinformatics analysis. Conclusion Our study identifies CPEB4 as a driver and therapeutic target to combat obesity., Highlights • CPEB4 expression is elevated in adipose tissue from obese patients and rodents. • CPEB4 overexpression is required for the abnormal obesity-associated phenotype. • Targeting CPEB4 reduces predisposition to diet-induced obesity. • Targeting CPEB4 alleviates obesity-associated microbiome dysbiosis. • CPEB4 is an attractive therapeutic target for treating obesity and its sequelae.

BackgroundMetabolic syndrome (MetS) is a group of signs and symptoms that are associated with higher risk of Type 2 Diabetes Mellitus (T2DM) and Cardiovascular Diseases (CVDs). The major risk factor for developing MetS is abdominal obesity that is caused by increase in adipocyte size or number. Adipocyte number multiplication is caused by differentiation of mesenchymal stem cells into adipose tissue. Numerous studies have evaluated the expression of key transcription factors including PPARG and CEBPB during adipocyte differentiation in murine cells such as 3T3-L1 cell line. In order to comprehend the expression changes during the process of fat accumulation in adipose tissue derived stem cells (ASCs), we compared the expression of DYRK1B, PPARG, and CEBPB in undifferentiated and differentiated ASCs into mature adipocytes between the patient (harboring DYRK1b R102C) and control (healthy individuals) groups.MethodsGene expression was evaluated on eighth days pre-induction and day 1, 5 and 15 post-induction. The pluripotent capacity of ASCs and the potential for differentiation into adipocyte were confirmed by flow cytometry analysis of surface markers (CD34, CD44, CD105 and CD90), and Oil red O staining, respectively. Expression of DYRK1B, PPARG, and CEBPB were assessed by RT-PCR in patients’ and normal individuals’ samples.ResultsThe expression of DYRK1B kinase and transcription factors (CEBPB and PPARG) are significantly higher in adipose derived stem cells harboring DYRK1b R102C compared to non-carriers on day 5 and 15 during adipocyte differentiation. These proteins may be suitable targets for therapeutic strategies in obesity and obesity related disorders like metabolic syndrome. Furthermore, AZ191 exhibited a potent and selectively inhibitory activity toward DYRK1B and CEBPB.ConclusionCEBPB, PPARA and DYRK1B contribute to adipogenesis and the development of metabolic syndrome; thus, they can be harnessed in developing therapeutic agents against metabolic syndrome.

Serine protease inhibitor Kazal-type 5 (SPINK5) has been indicated to act as a prognostic predictor for patients with head and neck squamous cell carcinoma. However, its specific role in nasopharyngeal carcinoma (NPC), a malignancy that has a high propensity for chemoresistance, remains largely obscure. We, thus, sought to investigate the importance of SPINK5 expression in regulating chemoresistance in NPC. Differentially expressed genes in NPC were screened using the cancer genome atlas-head and neck squamous cell carcinoma database and microarray analysis. SPINK5 was downregulated in NPC tissues and cells. After SPINK5 upregulation, the cells treated with cisplatin showed reduced cell survival and the ability to migrate, invade and metastasize. Mechanistically, the transcription factors regulating SPINK5 were queried through the JASPAR website, followed by dual-luciferase and Chromatin immunoprecipitation assay validation. CCAAT enhancer-binding protein (CEBP) beta (CEBPB) bound to the SPINK5 promoter region in NPC cells. The silencing of CEBPB enhanced the expression of SPINK5. CEBPB overexpression reversed the inhibitory effects of cisplatin on NPC cell malignant phenotype in the presence of SPINK5 overexpression. In conclusion, CEBPB silencing promoted chemoresistance of NPC cells via activating SPINK5, signifying that targeting CEBPB was a new approach to enhance the chemotherapy efficacy in NPC.

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7–14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.

Background: Septic shock (SS) and cardiogenic shock (CS) are two types of circulatory shock with a different etiology. Several studies have described the molecular alterations in SS patients, whereas the molecular factors involved in CS have been poorly investigated. We aimed to assess in the whole blood of CS and SS patients, using septic patients without shock (SC) as controls, transcriptomic modifications that occur over 1 week after ICU admission and are common to the two types of shock. Methods: We performed whole blood RNA sequencing in 21 SS, 11 CS, and 5 SC. In shock patients, blood samples were collected within 16 h from ICU admission (T1), 48 h after ICU admission (T2), and at day 7 or before discharge (T3). In controls, blood samples were available at T1 and T2. Gene expression changes over time have been studied in CS, SS, and SC separately with a paired analysis. Genes with p value < 0.01 (Benjamini-Hochberg multiple test correction) were defined differentially expressed (DEGs). We used gene set enrichment analysis (GSEA) to identify the biological processes and transcriptional regulators significantly enriched in both types of shock. Results: In both CS and SS patients, GO terms of inflammatory response and pattern recognition receptors (PRRs) were downregulated following ICU admission, whereas gene sets of DNA replication were upregulated. At the gene level, we observed that alarmins, interleukin receptors, PRRs, inflammasome, and DNA replication genes significantly changed their expression in CS and SS, but not in SC. Analysis of transcription factor targets showed in both CS and SS patients, an enrichment of CCAAT-enhancer-binding protein beta (CEBPB) targets in genes downregulated over time and an enrichment of E2F targets in genes with an increasing expression trend. Conclusions: This pilot study supports, within the limits of a small sample size, the role of alarmins, PRRs, DNA replication, and immunoglobulins in the pathophysiology of circulatory shock, either in the presence of infection or not. We hypothesize that these genes could be potential targets of therapeutic interventions in CS and SS., SCOPUS: ar.j, info:eu-repo/semantics/published

The PPAR coactivator-1α (PGC1α) is an important transcriptional co-activator in control of fatty acid metabolism. Mitochondrial fatty acid oxidation (FAO) is the primary pathway for the degradation of fatty acids and promotes NADPH and ATP production. Our previous study demonstrated that upregulation of carnitine palmitoyl transferase 1 A (CPT1A), the key regulator of FAO, promotes radiation resistance of nasopharyngeal carcinoma (NPC). In this study, we found that high expression of PGC1α is associated with poor overall survival in NPC patients after radiation treatment. Targeting PGC1α could sensitize NPC cells to radiotherapy. Mechanically, PGC1α binds to CCAAT/enhancer binding protein β (CEBPB), a member of the transcription factor family of CEBP, to promote CPT1A transcription, resulting in activation of FAO. Our results revealed that the PGC1α/CEBPB/CPT1A/FAO signaling axis promotes radiation resistance of NPC. These findings indicate that the expression of PGC1α could be a prognostic indicator of NPC, and targeting FAO in NPC with high expression of PGC1α might improve the therapeutic efficacy of radiotherapy.

Claudin-4 (CLDN4), a crucial member of tight junction proteins, is aberrantly expressed in breast cancer cells and contributes to cell migration and invasion. However, the mechanisms controlling CLDN4 expression in breast cancer are poorly understood. Here, we reported that CLDN4 expression correlated positively with p21-activated kinase 4 (PAK4) expression in human breast cancer tissues. Knockdown of PAK4 in MDA-MB-231 and ZR-75-30 cells suppressed CLDN4 expression and significantly inhibited cell migration and invasion. Conversely, restoration of CLDN4 expression in PAK4-knockdown cells reversed the inhibition of migration and invasion. We identified CCAAT/enhancer-binding protein β (CEBPB) as a novel transcriptional regulator of CLDN4 and confirmed that CEBPB bound to the −1093 to −991 bp region of the CLDN4 promoter. Importantly, we found that PAK4 enhanced CEBPB phosphorylation on Thr-235. In summary, we showed that PAK4-mediated CEBPB activation upregulated CLDN4 expression to promote breast cancer cell migration and invasion. Our results might contribute to understanding the mechanisms of CLDN4 regulation and suggest PAK4-CEBPB-CLDN4 axis as a potential therapeutic target for breast cancer.

Even in the era of ABL tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells is necessary for complete cure of the disease. Interferon-α (IFN-α) has long been used for the treatment of chronic-phase CML, but its mechanisms of action against CML stem cells remain unclear. We found that IFN-α upregulated CCAAT/enhancer binding protein β (C/EBPβ) in BCR-ABL–expressing mouse cells by activating STAT1 and STAT5, which were recruited to a newly identified 3′ distal enhancer of Cebpb that contains tandemly aligned IFN-γ–activated site elements. Suppression or deletion of the IFN-γ–activated site elements abrogated IFN-α–dependent upregulation of C/EBPβ. IFN-α induced differentiation and exhaustion of CML stem cells, both in vitro and in vivo, in a C/EBPβ-dependent manner. In addition, IFN-α upregulated C/EBPβ and induced exhaustion of lineage− CD34+ cells from CML patients. Collectively, these results clearly indicate that C/EBPβ is a critical mediator of IFN-α–induced differentiation and exhaustion of CML stem cells.

Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPβ occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBPα and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.

HL-60 promyelocytic cells are a widely used as a model for studying induced granulocytic differentiation. Investigation of proteins of the nuclear fraction, particularly transcription factors, is necessary for a better understanding of molecular mechanisms of cell maturation. Mass spectrometry is a powerful tool for analyzing a proteome due to its high sensitivity, specificity and performance. In this paper, using the selected reaction monitoring (SRM) method, we have assessed the levels of RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 and UBC9 nuclear proteins isolated using hypertonic buffer, detergents (sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and fissionable detergent ProteaseMAX™) and using centrifugation in a sucrose density gradient. The minimum and maximum protein content was 1.13±0.28 and 14.34±1.63 fmol/mkg of total protein for the transcription factor RBPJ and ubiquitin-protein ligase type I UBC9, respectively. According to the results of shotgun mass spectrometric analysis of nuclear fractions, 2356 proteins were identified, of which 106 proteins were annotated as transcription factors. 37 transcription factors were uniquely identified in the fraction obtained by centrifugation in a sucrose density gradient, while only 9 and 8 transcription factors were uniquely identified in the nuclear fractions obtained using hypertonic buffer and detergents, respectively. The transcription factors identified in the HL-60 cell line represent regulatory molecules; their directed profiling under the influence of differentiation inducers, will shed light on the mechanism of granulocyte maturation.Promielotsitarnye kletki linii HL-60 iavliaiutsia shiroko ispol'zuemoĭ model'iu dlia izucheniia indutsiruemoĭ granulotsitarnoĭ differentsirovki. Issledovanie belkov iadernoĭ fraktsii, v osobennosti transkriptsionnykh faktorov, neobkhodimo dlia luchshego ponimaniia molekuliarnykh mekhanizmov sozrevaniia kletok. Mass-spektrometriia iavliaetsia moshchnym metodom dlia analiza proteoma v silu vysokoĭ chuvstvitel'nosti, proizvoditel'nosti i spetsifichnosti analiza. V rabote, s ispol'zovaniem metoda monitoringa vybrannykh reaktsiĭ (SRM), provedena otsenka urovnia soderzhaniia iadernykh belkov RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 i UBC9, vydelennykh s primeneniem gipertonicheskogo bufera, detergentov (dodetsilsul'fata natriia (SDS), dezoksikholata natriia (DOC) i rasshchepliaemogo detergenta ProteaseMAX™) i s pomoshch'iu tsentrifugirovaniia v gradiente plotnosti sakharozy. Minimal'noe i maksimal'noe soderzhanie belkov sostavilo 1,13±0,28 i 14,34±1,63 fmol'/mkg obshchego belka dlia transkriptsionnogo faktora RBPJ i ubikvitin-proteinligazy pervogo tipa UBC9 sootvetstvenno. Po rezul'tatam panoramnogo mass-spektrometricheskogo analiza iadernykh fraktsiĭ udalos' identifitsirovat' 2356 belkov, iz nikh 106 belkov byli annotirovany kak transkriptsionnye faktory. Tret' transkriptsionnykh faktorov (37 belkov) byli identifitsirovany tol'ko vo fraktsii, poluchennoĭ s pomoshch'iu tsentrifugirovaniia v gradiente plotnosti sakharozy, v to vremia kak lish' 9 i 8 transkriptsionnykh faktorov bylo identifitsirovano tol'ko vo fraktsiiakh iader, poluchennykh s ispol'zovaniem gipertonicheskogo bufera i detergentov, sootvetstvenno. Transkriptsionnye faktory, identifitsirovannye v kletkakh linii HL-60, predstavliaiut soboĭ reguliatornye molekuly, napravlennoe profilirovanie kotorykh pod deĭstviem induktorov differentsirovki pozvolit prolit' svet na mekhanizm sozrevaniia granulotsitov.

Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments. Using data on differentially expressed transcripts and proteins we have applied upstream regulator search and obtained transcriptome- and proteome-based regulatory networks of induced granulocytic differentiation that cover both up-regulated (HIC1, NFKBIA, and CASP9) and down-regulated (PARP1, VDR, and RXRA) elements. To verify the designed network we measured HIC1 and PARP1 protein abundance during granulocytic differentiation by selected reaction monitoring (SRM) using stable isotopically labeled peptide standards. We also revealed that transcription factor CEBPB and LYN kinase were involved in differentiation onset, and evaluated their protein levels by SRM technique. Obtained results indicate that the omics data reflect involvement of the DNA repair system and the MAPK kinase cascade as well as show the balance between the processes of the cell survival and apoptosis in a p53-independent manner. The differentially expressed transcripts and proteins, predicted transcriptional factors, and key molecules such as HIC1, CEBPB, LYN, and PARP1 may be considered as potential targets for differentiation therapy of acute myeloid leukemia.

Background Aberrant DNA methylation has been firmly established as a factor contributing to the pathogenesis of colorectal cancer (CRC) via its capacity to silence tumour suppressor genes. However, the methylation status of multiple tumour suppressor genes and their roles in promoting CRC metastasis are not well characterised. Methods We explored the methylation and expression profiles of CPEB1 (the gene encoding cytoplasmic polyadenylation element-binding protein 1), a candidate CRC tumour suppressor gene, using The Cancer Genome Atlas (TCGA) database and validated these results in both CRC cell lines and cells from Han Chinese CRC patients (n = 104). The functional role of CPEB1 in CRC was examined in experiments performed in vitro and in vivo. A candidate transcription factor capable of regulating CPEB1 expression was predicted in silico and validated by luciferase reporter, DNA pull-down, and electrophoretic mobility shift assays. Results Hypermethylation and decreased expression of CPEB1 in CRC tumour tissues were revealed by TCGA database. We also identified a significant inverse correlation (Pearson’s R = − 0.43, P CPEB1 expression. We validated these results in CRC samples and two CRC cell lines. We also demonstrated that up-regulation of CPEB1 resulted in significantly decreased tumour growth, migration, invasion, and tumorigenicity and promoted tumour cell apoptosis both in vitro and in vivo. We identified the transcription factors CCAAT enhancer-binding protein beta (CEBPB) and transcription factor CP2 (TFCP2) as critical regulators of CPEB1 expression. Hypermethylation of the CPEB1 promoter resulted in a simultaneous increase in the capacity for TFCP2 binding and a decreased likelihood of CEBPB binding, both of which led to diminished expression of CPEB1. Conclusions Our results identified a novel tumour-suppressive role of CPEB1 in CRC and found that hypermethylation of the CPEB1 promoter may lead to diminished expression due to decreased chromatin accessibility and transcription factor binding. Collectively, these results suggest a potential role for CPEB1 in the diagnosis and treatment of CRC.

This study explored circulating miRNAs and target genes associated with metabolic syndrome (MetS) and cardiometabolic risk in obese patients. Small-RNA sequencing was used to assess the peripheral blood miRNome of 12 obese subjects (6 MetS and 6 non-MetS). Differentially expressed miRNAs and target genes were further analyzed by qPCR in a larger sample of obese patients (48 MetS and 32 non-MetS). miRNA:mRNA interactions were studied using in silico tools. miRNome analysis identified 10 downregulated miRNAs in MetS compared to non-Met patients (p &lt, 0.05). In silico studies revealed three miRNAs (miR-155, miR-181a, and let-7a) and their predictive targets (CCAAT/enhancer-binding protein beta—CEBPB, KRAS proto-oncogene, GTPase—KRAS and suppressor of cytokine signaling 1—SOCS1) with a potential role in the insulin receptor signaling pathway. miR-155 expression was reduced and CEBPB mRNA levels were increased in MetS patients (p &lt, 0.05), and these effects were correlated with the number of MetS diagnostic criteria (p &lt, 0.05). Increased HOMA-IR (&gt, 7.6) was associated with low miR-155 levels, high CEBPB expression, and serum hsCRP (p &lt, 0.05). miR-155 was negatively correlated with CEBPB, HOMA-IR, and plasma fibrinogen, and positively correlated with serum adiponectin (p &lt, 0.05). Downregulation of circulating miR-155 is associated with insulin resistance, poor glycemic control, and increased MetS-related cardiometabolic risk, and these effects are potentially mediated by interaction with CEBPB.

Background and objectives Sepsis is a leading cause of mortality and morbidity in the intensive care unit. Regulatory mechanisms underlying the disease progression and prognosis are largely unknown. The study aimed to identify master regulators of mortality-related modules, providing potential therapeutic target for further translational experiments. Methods The dataset GSE65682 from the Gene Expression Omnibus (GEO) database was utilized for bioinformatic analysis. Consensus weighted gene co-expression netwoek analysis (WGCNA) was performed to identify modules of sepsis. The module most significantly associated with mortality were further analyzed for the identification of master regulators of transcription factors and miRNA. Results A total number of 682 subjects with various causes of sepsis were included for consensus WGCNA analysis, which identified 27 modules. The network was well preserved among different causes of sepsis. Two modules designated as black and light yellow module were found to be associated with mortality outcome. Key regulators of the black and light yellow modules were the transcription factor CEBPB (normalized enrichment score = 5.53) and ETV6 (NES = 6), respectively. The top 5 miRNA regulated the most number of genes were hsa-miR-335-5p (n = 59), hsa-miR-26b-5p (n = 57), hsa-miR-16-5p (n = 44), hsa-miR-17-5p (n = 42), and hsa-miR-124-3p (n = 38). Clustering analysis in 2-dimension space derived from manifold learning identified two subclasses of sepsis, which showed significant association with survival in Cox proportional hazard model (p = 0.018). Conclusions The present study showed that the black and light-yellow modules were significantly associated with mortality outcome. Master regulators of the module included transcription factor CEBPB and ETV6. miRNA-target interactions identified significantly enriched miRNA.

Hepatitis B virus (HBV), a highly persistent pathogen causing hepatocellular carcinoma (HCC), takes full advantage of host machinery, presenting therapeutic targets. Here we aimed to identify novel druggable host cellular factors using the reporter HBV we have recently generated. In an RNAi screen of G protein-coupled receptors (GPCRs), GPCR39 (GPR39) appeared as the top hit to facilitate HBV proliferation. Lentiviral overexpression of active GPR39 proteins and an agonist enhanced HBV replication and transcriptional activities of viral promoters, inducing the expression of CCAAT/enhancer binding protein (CEBP)-&beta, (CEBPB). Meanwhile, GPR39 was uncovered to activate the heat shock response, upregulating the expression of proviral heat shock proteins (HSPs). In addition, glioma-associated oncogene homologue signaling, a recently reported target of GPR39, was suggested to inhibit HBV replication and eventually suppress expression of CEBPB and HSPs. Thus, GPR39 provirally governed intracellular circuits simultaneously affecting the carcinopathogenetic gene functions. GPR39 and the regulated signaling networks would serve as antiviral targets, and strategies with selective inhibitors of GPR39 functions can develop host-targeted antiviral therapies preventing HCC.

Objective Long noncoding RNAs (lncRNAs) are currently considered to have a vital and wide range of biological functions, but the molecular mechanism underlying triglycerides metabolism remains poorly understood. This study aims to identify novel lncRNAs differentially expressed in rat livers with hypertriglyceridemia and elucidated the function role in TG metabolism. Methods Differentially expressions of lncRNAs in rat livers with hypertriglyceridemia were identified by transcriptome sequencing and validated by real-time PCR. The role of lnc19959.2 in triglyceride metabolism was assessed both in vitro and in vivo. RNA pulldown and RIP assays were conducted to evaluate the interactions between lnc19959.2 and its target proteins. ChIP and Dual report assays were performed to detect the interactions between transcription factors and promoters of its target genes. Results We identified a novel lncRNA, and lnc19959.2 was upregulated in rat livers with hypertriglyceridemia. The knockdown of lnc19959.2 has profound TG lowering effects in vitro and in vivo. Subsequently, the genome-wide analysis identified that the knockdown of lnc19959.2 caused the deregulation of many genes during TG homeostasis. Further mechanism studies revealed that lnc19959.2 upregulated ApoA4 expression via ubiquitinated transcription inhibitor factor Purb, while it specifically interacted with hnRNPA2B1 to downregulate the expression of Cpt1a, Tm7sf2, and Gpam, respectively. In the upstream pathway, palmitate acid upregulated CCAAT/Enhancer-Binding Protein Beta (Cebpb) and facilitated its binding to the promoter of lnc19959.2, which resulted in significant promotion of lnc19959.2 transcriptional activity. Conclusions Our findings provide novel insights into a new layer regulatory complexity of an lncRNA modulating triglyceride homeostasis by a novel lncRNA lnc19959.2., Graphical abstract Intracellular high triglyceride status facilitated Cebpb binding to promoter of lnc19959.2, resulted in increasing transcriptional activity of lnc19959.2. On the one hand, Lnc19959.2 up-regulate ApoA4 expression via ubiquitinated transcription inhibitor factor Purb; on the other hand, lnc19959.2-hnRNPA2B1 complex acts on Cpt1a, Tm7sf2 and Gpam in some ways to reduce the efficiency of RNA polymerase II occupy on the target gene promoter. Finally, these processes exacerbate the accumulation of intracellular triglycerides leading to hypertriglyceridemia.Image 1, Highlights • lnc19959.2 was identified as a novel LncRNA in hypertriglyceridemic rat liver. • lnc19959.2 was involved in triglyceride metabolism in vivo and in vitro. • lnc19959.2 upregulated ApoA4 expression via ubiquitinated transcription inhibitor factor Purb. • lnc19959.2 interacted with hnRNPA2B1 and cooperated with RNP II that controls expression of Cpt1a, Tm7sf2 and Gpam.

Supplemental Digital Content is available in the text., Introduction of targeted therapy in the treatment of metastatic cutaneous malignant melanoma (CMM) has improved clinical outcome during the last years. However, only in a subset of the CMM patients, this will lead to long-term effects. CEBPB is a transcription factor that has been implicated in various physiological and pathological processes, including cancer development. We have investigated its prognostic impact on CMM and unexpectedly found that higher CEBPB mRNA levels correlated with a longer overall survival. Furthermore, in a small cohort of patients with metastatic CMM treated with BRAF-inhibitors, higher levels of CEBPB mRNA expression in the tumor cells prior treatment correlated to a longer progression-free survival. We have characterized an overlapping antisense transcript, CEBPB-AS1, with the aim to investigate the regulation of CEBPB expression in CMM and its impact on BRAF-inhibitor sensitivity. We demonstrated that silencing of CEBPB-AS1 resulted in epigenetic modifications in the CEBPB promoter and in increased CEBPB mRNA and protein levels, inhibited proliferation and partially resensitized BRAF-inhibitor resistant CMM cells to this drug-induced apoptosis. Our data suggest that targeting CEBPB-AS1 may represent a valuable tool to sensitize CMM cells to the BRAF-inhibitor-based therapies.

Catabolism of the essential amino acid tryptophan is a key metabolic pathway contributing to the immunosuppressive tumor microenvironment and therefore a viable drug target for cancer immunotherapy. In addition to the rate-limiting enzyme indoleamine-2,3-dioxygenase-1 (IDO1), tryptophan catabolism via tryptophan-2,3-dioxygenase (TDO2) is a feature of many tumors, particularly malignant gliomas. The pathways regulating TDO2 in tumors are poorly understood; using unbiased promoter and gene expression analyses, we identify a distinct CCAAT/enhancer-binding protein β (C/EBPβ) binding site in the promoter of TDO2 essential for driving constitutive TDO2 expression in glioblastoma cells. Using The Cancer Genome Atlas (TCGA) data, we find that C/EBPβ expression is correlated with TDO2, and both are enriched in malignant glioma of the mesenchymal subtype and associated with poor patient outcome. We determine that TDO2 expression is sustained mainly by the LAP isoform of CEBPB and interleukin-1β, which activates TDO2 via C/EBPβ in a mitogen-activated protein kinase (MAPK) kinase-dependent fashion. In summary, we provide evidence for a novel regulatory and therapeutically targetable pathway of immunosuppressive tryptophan degradation in a subtype of glioblastoma with a particularly poor prognosis.

The biologically active form of vitamin D3, 1&alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), modulates innate and adaptive immunity via genes regulated by the transcription factor vitamin D receptor (VDR). In order to identify the key vitamin D target genes involved in these processes, transcriptome-wide datasets were compared, which were obtained from a human monocytic cell line (THP-1) and peripheral blood mononuclear cells (PBMCs) treated in vitro by 1,25(OH)2D3, filtered using different approaches, as well as from PBMCs of individuals supplemented with a vitamin D3 bolus. The led to the genes ACVRL1, CAMP, CD14, CD93, CEBPB, FN1, MAPK13, NINJ1, LILRB4, LRRC25, SEMA6B, SRGN, THBD, THEMIS2 and TREM1. Public epigenome- and transcriptome-wide data from THP-1 cells were used to characterize these genes based on the level of their VDR-driven enhancers as well as the level of the dynamics of their mRNA production. Both types of datasets allowed the categorization of the vitamin D target genes into three groups according to their role in (i) acute response to infection, (ii) infection in general and (iii) autoimmunity. In conclusion, 15 genes were identified as major mediators of the action of vitamin D in innate and adaptive immunity and their individual functions are explained based on different gene regulatory scenarios.

MYO18B has been proposed to contribute to the progression of hepatocellular carcinoma (HCC). However, the signals that govern MYO18B transcription are not known. Here we show that, a network of C19MC miRNA-520G, IFN-γ, CEBPB and p53 transcriptional-defects promote MYO18B mRNA expression in HCCs. IFN-γ by itself suppresses MYO18B transcription, but promotes it when miRNA-520G is stably overexpressed. Similarly, CEBPB-liver-enriched activator protein (LAP) isoform overexpression suppresses MYO18B transcription but promotes transcription when the cells are treated with IFN-γ. Furthermore, miR-520G together with mutant-p53 promotes MYO18B transcription. Conversely, bFGF suppresses MYO18B mRNA irrespective of CEBPB, miR-520G overexpression or IFN-γ treatment. Finally high MYO18B expression reflects poor prognosis while high MYL5 or MYO1B expression reflects better survival of HCC patients. Thus, we identified a network of positive and negative regulators of MYO18B mRNA expression which reflects the survival of HCC patients.

Currently, the environmental impact of ubiquitous plastic debris triggered quite some public attention. However, the global impact of microplastic on human health is by and large either unknown or neglected. By looking at the underlying biochemical mechanisms leading to the global health threat microplastic was discovered to carry persistent organic pollutants, such as polycyclic aromatic hydrocarbons (PAH), to marine life. The effect of microplastic-ingestion in the human body remains unfortunately somewhat elusive as of yet. For this reason, we screened for compounds binding to the human estrogen receptor α (ERα) and identified the PAH compounds indeno[1,2,3-cd]pyrene (Indpy) and picene (Pice) with a high binding affinity. We applied next generation sequencing to analyze the differentially expressed genes in MCF-7 cells after treatment with Indpy and Pice. We found 8 upregulated genes: ABCC5, CCNG2, CYP1A1, DDIT4, IER3, RUNX2, STC2, and SLC7A5 and 14 downregulated genes: ADORA1, CEBPB, CELSR2, CTSD, CXCL12, KRT19, PGR, PKIB, RARA, RET, SEMA3B, SIAH2, TFAP2C, and XBP1 induced by both ligands and associated with ESR1-regulation. The altered gene expression may influence cell proliferation and metastasis, favoring cancer development with a poor response to therapy. In addition, we confirmed the binding of Indpy and Pice to ERα using molecular docking and microscale thermophoresis. ERα activation was measured with ESR1-overexpressing HEK293 (HEK-ESR1) cells and confirmed for Indpy. In conclusion, we showed an ESR1-mediated influence of the PAH compounds Indpy and Pice on the gene expression pattern of MCF-7 cells, possibly also promoting breast cancer development in patients.

Background. Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by chronic progressive stenosis or occlusion of the bilateral internal carotid artery (ICA), the anterior cerebral artery (ACA), and the middle cerebral artery (MCA). MMD is secondary to the formation of an abnormal vascular network at the base of the skull. However, the etiology and pathogenesis of MMD remain poorly understood. Methods. A competing endogenous RNA (ceRNA) network was constructed by analyzing sample-matched messenger RNA (mRNA), long non-coding RNA (lncRNA), and microRNA (miRNA) expression profiles from MMD patients and control samples. Then, a protein-protein interaction (PPI) network was constructed to identify crucial genes associated with MMD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were employed with the DAVID database to investigate the underlying functions of differentially expressed mRNAs (DEmRNAs) involved in the ceRNA network. CMap was used to identify potential small drug molecules. Results. A total of 94 miRNAs, 3649 lncRNAs, and 2294 mRNAs were differentially expressed between MMD patients and control samples. A synergistic ceRNA lncRNA-miRNA-mRNA regulatory network was constructed. Core regulatory miRNAs (miR-107 and miR-423-5p) and key mRNAs (STAT5B, FOSL2, CEBPB, and CXCL16) involved in the ceRNA network were identified. GO and KEGG analyses indicated that the DEmRNAs were involved in the regulation of the immune system and inflammation in MMD. Finally, two potential small molecule drugs, CAY-10415 and indirubin, were identified by CMap as candidate drugs for treating MMD. Conclusions. The present study used bioinformatics analysis of candidate RNAs to identify a series of clearly altered miRNAs, lncRNAs, and mRNAs involved in MMD. Furthermore, a ceRNA lncRNA-miRNA-mRNA regulatory network was constructed, which provides insights into the novel molecular pathogenesis of MMD, thus giving promising clues for clinical therapy.

Background Neonatal sepsis is an inflammatory systemic syndrome, which is a major cause of morbidity and mortality in premature infants. We analyzed the expression profile data of E-MTAB-4785 to reveal the pathogenesis of the disease. Methods The expression profile dataset E-MTAB-4785, which contained 17 sepsis samples and 19 normal samples, was obtained from the ArrayExpress database. The differentially expressed genes (DEGs) were analyzed by the Bayesian testing method in limma package. Based on the DAVID online tool, enrichment analysis was conducted for the DEGs. Using STRING database and Cytoscape software, protein-protein interaction (PPI) network and module analyses were performed. Besides, transcription factor (TF)-DEG regulatory network was also constructed by Cytoscape software. Additionally, miRNA-DEG pairs were searched using miR2Disease and miRWalk 2.0 databases, followed by miRNA-DEG regulatory network was visualized by Cytoscape software. Results A total of 275 DEGs were identified from the sepsis samples in comparison to normal samples. TSPO, MAPK14, and ZAP70 were the hub nodes in the PPI network. Pathway enrichment analysis indicated that CEBPB and MAPK14 were enriched in TNF signaling pathway. Moreover, CEBPB and has-miR-150 might function in neonatal sepsis separately through targeting MAPK14 and BCL11B in the regulatory networks. These genes and miRNA might be novel targets for the clinical treatment of neonatal sepsis. Conclusion TSPO, ZAP70, CEBPB targeting MAPK14, has-miR-150 targeting BCL11B might affect the pathogenesis of neonatal sepsis. However, their roles in neonatal sepsis still needed to be confirmed by further experimental researches.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease. Although some genes and miRNAs related with HCM have been studied, the molecular regulatory mechanisms between miRNAs and transcription factors (TFs) in HCM have not been systematically elucidated. In this study, we proposed a novel method for identifying dysregulated miRNA‐TF feed‐forward loops (FFLs) by integrating sample matched miRNA and gene expression profiles and experimentally verified interactions of TF‐target gene and miRNA‐target gene. We identified 316 dysregulated miRNA‐TF FFLs in HCM, which were confirmed to be closely related with HCM from various perspectives. Subpathway enrichment analysis demonstrated that the method was outperformed by the existing method. Furthermore, we systematically analysed the global architecture and feature of gene regulation by miRNAs and TFs in HCM, and the FFL composed of hsa‐miR‐17‐5p, FASN and STAT3 was inferred to play critical roles in HCM. Additionally, we identified two panels of biomarkers defined by three TFs (CEBPB, HIF1A, and STAT3) and four miRNAs (hsa‐miR‐155‐5p, hsa‐miR‐17‐5p, hsa‐miR‐20a‐5p, and hsa‐miR‐181a‐5p) in a discovery cohort of 126 samples, which could differentiate HCM patients from healthy controls with better performance. Our work provides HCM‐related dysregulated miRNA‐TF FFLs for further experimental study, and provides candidate biomarkers for HCM diagnosis and treatment.

BackgroundAn Immunochip study recently identified the association of a number of new genetic loci with Behcet’s disease (BD).ObjectiveTo confirm the association between new genetic loci reported in an Immunochip study and BD in a Han Chinese population.MethodsA two-stage association study was carried out in 1238 patients with BD and 1458 healthy controls. Twenty-two candidate single nucleotide polymorphisms (SNPs) were selected for genotyping by iPLEXGold genotyping or TaqMan SNP assays and a meta-analysis was performed for significantly associated markers.ResultsThe results showed that four SNPs (LACC1/rs9316059, CEBPB-PTPN1/rs913678, ADO-EGR2/rs224127 and RIPK2/rs10094579) were associated with BD in an allelic association test (rs9316059 T allele: pc=4.95×10−8, OR=0.687; rs913678 C allele: pc=3.01×10−4, OR=1.297; rs224127 A allele: pc=3.77×10−4, OR=1.274; rs10094579 A allele: pc=6.93×10−4, OR=1.302). For four SNPs tested by meta-analysis, the association with BD was strengthened and all exceeded genome-wide significance (rs9316059: p=2.96×10−16; rs913678: p=2.09×10−16; rs224127: p=5.28×10−13; rs10094579: p=9.21×10−11).ConclusionsOur findings confirmed the association of four loci (LACC1, CEBPB-PTPN1, ADO-EGR2 and RIPK2) in Chinese Han patients with BD.

Study question What is the transcriptional network governed by the androgen receptor (AR) in human epididymis epithelial (HEE) cells from the caput region and if the network is tissue-specific, how is this achieved? Summary answer About 200 genes are differentially expressed in the caput HEE cells after AR activation; the AR transcriptional network is tissue-specific and may be mediated in part by distinct AR co-factors including CAAT-enhancer binding protein beta (CEBPB) and runt-related transcription factor 1 (RUNX1). What is known already Little is known about the AR transcriptional program genome wide in HEE cells, nor its co-factors in those cells. AR has been best studied in the prostate gland epithelium and prostate cancer cell lines, due to the important role of this factor in prostate cancer. However AR-associated differentially expressed genes (DEGs) and AR co-factors have not yet been compared between human epididymis and prostate epithelial cells. Study design, size, duration Caput HEE cells from two donors were exposed to the synthetic androgen R1881 at 1 nM for 12-16 h after 72 h of hormone starvation. Participants/materials, setting, methods Chromatin was prepared from R1881-treated and vehicle control HEE cells. AR-associated chromatin was purified by chromatin immunoprecipitation (ChIP) and AR occupancy genome wide was revealed by deep sequencing (ChIP-seq). Two independent biological replicates were performed. Total RNA was prepared from R1881 and control-treated HEE cells and gene expression profiles were documented by RNA-seq. The interaction of the potential novel AR co-factors CEBPB and RUNX1, identified through in-silico motif analysis of AR ChIP-seq data, was examined by ChIP-qPCR after siRNA-mediated depletion of each co-factor individually or simultaneously. Main results and the role of chance The results identify about 200 genes that are differentially expressed (DEGs) in HEE cells after AR activation. Some of these DEGs show occupancy of AR at their promoters or cis-regulatory elements suggesting direct regulation. However, there is little overlap in AR-associated DEGs between HEE and prostate epithelial cells. Inspection of over-represented motifs in AR ChIP-seq peaks identified CEBPB and RUNX1 as potential co-factors, with no evidence for FOXA1, which is an important co-factor in the prostate epithelium. CEBPB and RUNX1 ChIP-seq in HEE cells showed that both these factors often occupied AR-binding sites, though rarely simultaneously. Further analysis at a single AR-regulated locus (FK506-binding protein 5, FKPB5) suggests that CEBPB may be a co-activator. These data suggest a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium. Large scale data AR ChIP-seq and RNA-seq data are deposited at GEO: GSE109063. Limitations, reasons for caution There is substantial donor-to-donor variation in primary HEE cells cultures. We applied stringent statistical tests with a false discovery rate (FDR) of 0.1% for ChIP-seq and standard pipelines for RNA-seq so it is possible that we have missed some AR-regulated genes that are important in caput epididymis function. Wider implications of the findings Our data suggest that a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium. Since this cell layer has a critical role in normal sperm maturation, the results are of broader significance in understanding the mechanisms underlying the maintenance of fertility in men. Study funding/competing interests This work was funded by the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Development: R01 HD068901 (PI: Harris). The authors have no competing interests to declare.

Increased adiposity is a hallmark of obesity and overweight, which affect 2.2 billion people world-wide. Understanding the genetic and molecular mechanisms that underlie obesity-related phenotypes can help to improve treatment options and drug development. Here we perform promoter Capture Hi–C in human adipocytes to investigate interactions between gene promoters and distal elements as a transcription-regulating mechanism contributing to these phenotypes. We find that promoter-interacting elements in human adipocytes are enriched for adipose-related transcription factor motifs, such as PPARG and CEBPB, and contribute to heritability of cis-regulated gene expression. We further intersect these data with published genome-wide association studies for BMI and BMI-related metabolic traits to identify the genes that are under genetic cis regulation in human adipocytes via chromosomal interactions. This integrative genomics approach identifies four cis-eQTL-eGene relationships associated with BMI or obesity-related traits, including rs4776984 and MAP2K5, which we further confirm by EMSA, and highlights 38 additional candidate genes.

Made available in DSpace on 2018-12-11T17:22:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Financiadora de Estudos e Projetos Background/Aims: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)2D3 in human preadipocyte proliferation and differentiation. Methods: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)2D3 on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. Results: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)2D3 treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)2D3 induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)2D3 also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)2D3 induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)2D3 had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. Conclusions: We demonstrated for the first time that 1,25(OH)2D3 induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)2D3 in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals. São Paulo State University (UNESP) School of Medicine Department of Pathology Maastricht University Department of Bioinformatics - BiGCaT NUTRIM School for Nutrition and Translational Research in Metabolism State University of Londrina (UEL) Department of Biochemistry and Biotechnology State University of Londrina (UEL) Department of General Biology Maastricht University Maastricht Centre for Systems Biology (MaCSBio) Centro de Ciências Bio. Dept. de Bio. Geral Lab. de Genética Toxicológica Univ. Estadual de Londrina Rodovia Celso Garcia Cid, PR 445 Km 380, Campus Universitário São Paulo State University (UNESP) School of Medicine Department of Pathology CNPq: 150067/2017-8 CAPES: 9933/2014-00

FAM46C is one of the most recurrently mutated genes in multiple myeloma; however its role in disease pathogenesis has not been determined. Here we demonstrate that wild-type (WT) FAM46C overexpression induces substantial cytotoxicity in multiple myeloma cells. In contrast, FAM46C mutations found in multiple myeloma patients abrogate this cytotoxicity, indicating a survival advantage conferred by the FAM46C mutant phenotype. WT FAM46C overexpression downregulated IRF4, CEBPB, and MYC and upregulated immunoglobulin (Ig) light chain and HSPA5/BIP. Furthermore, pathway analysis suggests that enforced FAM46C expression activated the unfolded protein response pathway and induced mitochondrial dysfunction. CRISPR-mediated depletion of endogenous FAM46C enhanced multiple myeloma cell growth, decreased Ig light chain and HSPA5/BIP expression, activated ERK and antiapoptotic signaling, and conferred relative resistance to dexamethasone and lenalidomide treatments. Genes altered in FAM46C-depleted cells were enriched for signaling pathways regulating estrogen, glucocorticoid, B-cell receptor signaling, and ATM signaling. Together these results implicate FAM46C in myeloma cell growth and survival and identify FAM46C mutation as a contributor to myeloma pathogenesis and disease progression via perturbation in plasma cell differentiation and endoplasmic reticulum homeostasis. Cancer Res; 77(16); 4317–27. ©2017 AACR.

The placenta is the principal organ regulating respiratory, nutritional, endocrine and metabolic functions on behalf of the developing fetus. Changes in gene expression patterns of placenta-specific genes may influence fetal growth. We profiled the expression of 17 genes related to placenta functioning in term placentas (n=677) to identify genes differentially expressed across birth weight categories [small (SGA), appropriate (AGA) and large (LGA) for gestational age]. ABCG2, CEBPB, CRH, GCM1, GPC3, INSL4, PGF and PLAC1 were inversely associated with LGA status, with odds ratios (ORs) and 95% confidence intervals (CI) ranging from GCM1 (OR=0.44, 95% CI: 0.29, 0.70) to CRH (OR=0.73, 95% CI: 0.61, 0.88). NR3C1 was positively associated with LGA status (OR=2.33, 95% CI: 1.43, 3.78). PLAC1 (OR=0.66, 95% CI: 0.47, 0.92) and ABCG2 (OR=0.63, 95% CI: 0.44, 0.91) were additionally inversely associated with SGA status, and PGF was positively associated with SGA status (OR=1.59, 95% CI=1.08, 2.35). General trends were confirmed in an independent cohort (n=306). Given that aberrant fetal growth may have long-lasting effects, our results suggest the potential utility of placental gene expression profiles as potential early markers of disease onset later in life.

Background Invasion and angiogenesis are major hallmarks of glioblastoma (GBM) growth. While invasive tumor cells grow adjacent to blood vessels in normal brain tissue, tumor cells within neovascularized regions exhibit hypoxic stress and promote angiogenesis. The distinct microenvironments likely differentially affect metabolic processes within the tumor cells. Methods In the present study, we analyzed gene expression and metabolic changes in a human GBM xenograft model that displayed invasive and angiogenic phenotypes. In addition, we used glioma patient biopsies to confirm the results from the xenograft model. Results We demonstrate that the angiogenic switch in our xenograft model is linked to a proneural-to-mesenchymal transition that is associated with upregulation of the transcription factors BHLHE40, CEBPB, and STAT3. Metabolic analyses revealed that angiogenic xenografts employed higher rates of glycolysis compared with invasive xenografts. Likewise, patient biopsies exhibited higher expression of the glycolytic enzyme lactate dehydrogenase A and glucose transporter 1 in hypoxic areas compared with the invasive edge and lower-grade tumors. Analysis of the mitochondrial respiratory chain showed reduction of complex I in angiogenic xenografts and hypoxic regions of GBM samples compared with invasive xenografts, nonhypoxic GBM regions, and lower-grade tumors. In vitro hypoxia experiments additionally revealed metabolic adaptation of invasive tumor cells, which increased lactate production under long-term hypoxia. Conclusions The use of glycolysis versus mitochondrial respiration for energy production within human GBM tumors is highly dependent on the specific microenvironment. The metabolic adaptability of GBM cells highlights the difficulty of targeting one specific metabolic pathway for effective therapeutic intervention.

MicroRNAs (miRNAs) are master post-transcriptional regulators of gene expression and their specific footprints reflect disease conditions. Over the last few years, several primary reports have described the deregulation of cell-free miRNAs in Parkinson’s disease (PD), however, results have been rather inconsistent due to preanalytical and analytical challenges. This study integrated the data across twenty-four reports to identify steadily deregulated miRNAs that may assist in the path towards biomarker development and molecular characterization of the underlying pathology. Stringent KEGG pathway analysis of the miRNA targets revealed FoxO, Prolactin, TNF, and ErbB signaling pathways as the most significantly enriched categories while Gene Ontology analysis revealed that the protein targets are mostly associated with transcription. Chromosomal location of the consistently deregulated miRNAs revealed that over a third of them were clustered at the same location at Chr14q32 suggesting that they are co-regulated by specific transcription factors. This genomic region is inherently unstable due to expanded TGG repeats and responsible for human abnormalities. Stringent analysis of transcription factor sites surrounding the deregulated miRNAs revealed that CREB1, CEBPB and MAZ sites existed in approximately half of the miRNAs, including all of the miRNAs located at Chr14q32. Additional studies are now needed to determine the biomarker potential of the consistently deregulated miRNAs in PD and the therapeutic implications of these bioinformatics insights.

Autophagy is the cell self-eating mechanism to maintain cell homeostasis by removing damaged intracellular proteins or organelles. It has also been implicated in the development and differentiation of various cell types including the adipocyte. Several links between adipogenic transcription factors and key autophagy genes has been suggested. In this study, we tried to model the gene expression and their transcriptional regulation during the adipocyte differentiation using high-throughput sequencing datasets of the 3T3-L1 cell model. We applied the gene expression and co-expression analysis to all and the subset of autophagy genes to study the binding, and occupancy patterns of adipogenic factors, co-factors and histone modifications on key autophagy genes. We also analyzed the gene expression of key autophagy genes under different transcription factor knockdown adipocyte cells. We found that a significant percent of the variance in the autophagy gene expression is explained by the differentiation stage of the cell. Adipogenic master regulators, such as CEBPB and PPARG target key autophagy genes directly. In addition, the same factor may also control autophagy gene expression indirectly through autophagy transcription factors such as FOXO1, TFEB or XBP1. Finally, the binding of adipogenic factors is associated with certain patterns of co-factors binding that might modulate the functions. Some of the findings were further confirmed under the knockdown of the adipogenic factors in the differentiating adipocytes. In conclusion, autophagy genes are regulated as part of the transcriptional programs through adipogenic factors either directly or indirectly through autophagy transcription factors during adipogenesis.

This study identified a transcription factor that might bind to the 5&rsquo, regulatory region of the HTR1A and explored the potential effect on 5-HT1A receptor expression. Based on JASPAR predictions, the binding of the transcription factor was demonstrated using the electrophoretic mobility shift assay (EMSA). Vectors over-expressing the transcription factor were co-transfected into HEK-293 and SK-N-SH cells with the recombinant pGL3 vector, and relative fluorescence intensity was measured to determine regulatory activity. Additionally, the qRT-PCR and Western blot were also used to identify whether the transcription factor modulated the endogenous expression of 5-HT1A receptor. The results suggest that the transcription factor CCAA/T enhancer binding protein beta (CEBPB) likely binds to the -1219 to -1209 bp (ATG+1) region of the HTR1A. Two sequences located in the -722 to -372 bp and -119 to +99 bp were also identified. Although the effect of CEBPB on endogenous 5-HT1A receptor expression was not significant, it exhibited the strong inhibition on the relative fluorescence intensity and the mRNA level of HTR1A. CEBPB inhibited the human HTR1A expression by binding to the sequence -1219 - -1209 bp. This is useful and informative for ascertaining the regulation of 5-HT1A receptor and mental diseases.

Background: Sepsis is a serious clinical condition with a poor prognosis, despite improvements in diagnosis and treatment.Therefore, novel biomarkers are necessary that can help with estimating prognosis and improving clinical outcomes of patients with sepsis. Methods: The gene expression profiles GSE54514 and GSE63042 were downloaded from the GEO database. DEGs were screened by t test after logarithmization of raw data; then, the common DEGs between the 2 gene expression profiles were identified by up-regulation and down-regulation intersection. The DEGs were analyzed using bioinformatics, and a protein-protein interaction (PPI) survival network was constructed using STRING. Survival curves were constructed to explore the relationship between core genes and the prognosis of sepsis patients based on GSE54514 data. Results: A total of 688 common DEGs were identified between survivors and non-survivors of sepsis, and 96 genes were involved in survival networks. The crucial genes Signal transducer and activator of transcription 5A (STAT5A), CCAAT/enhancer-binding protein beta (CEBPB), Myc proto-oncogene protein (MYC), and REL-associated protein (RELA) were identified and showed increased expression in sepsis survivors. These crucial genes had a positive correlation with patients’ survival time according to the survival analysis. Conclusions: Our findings indicate that the genes STAT5A, CEBPB, MYC, and RELA may be important in predicting the prognosis of sepsis patients.

Background Colorectal cancer (CRC) is one of the most common forms of cancer worldwide. The tumor microenvironment plays a key role in promoting the occurrence of chemoresistance in solid cancers. Effective targets to overcome resistance are necessary to improve the survival and prognosis of CRC patients. This study aimed to evaluate the molecular mechanisms of the tumor microenvironment that might be involved in chemoresistance in patients with CRC. Methods We evaluated the effects of CCL20 on chemoresistance of CRC by recruitment of regulatory T cells (Tregs) in vitro and in vivo. Results We found that the level of CCL20 derived from tumor cells was significantly higher in Folfox-resistant patients than in Folfox-sensitive patients. The high level of CCL20 was closely associated with chemoresistance and poor survival in CRC patients. Among the drugs in Folfox chemotherapy, we confirmed that 5-FU increased the expression of CCL20 in CRC. Moreover, CCL20 derived from 5-FU-resistant CRC cells promoted recruitment of Tregs. Tregs further enhanced the chemoresistance of CRC cells to 5-FU. FOXO1/CEBPB/NF-κB signaling was activated in CRC cells after 5-FU treatment and was required for CCL20 upregulation mediated by 5-FU. Furthermore, CCL20 blockade suppressed tumor progression and restored 5-FU sensitivity in CRC. Lastly, the expression of these signaling molecules mediating chemoresistance was closely correlated with poor survival of CRC patients. Conclusions CRC cell-secreted CCL20 can recruit Tregs to promote chemoresistance via FOXO1/CEBPB/NF-κB signaling, indicating that the FOXO1/CEBPB/NF-κB/CCL20 axis might provide a promising target for CRC treatment. Electronic supplementary material The online version of this article (10.1186/s40425-019-0701-2) contains supplementary material, which is available to authorized users.

Introduction: Growth differentiation factor 15 (GDF15), a newly identified member of transforming growth factor (GDF) superfamily, is upregulated in ovarian (OV) cancer. Upregulated GDF15 positively correlates with poor prognosis of OV cancer. Thus, elucidation of the mechanism underlying GDF15 overexpression is important. Method and Results: PROMO and JASPAR prediction software were used to find transcription factors for GDF15 expression. Data from TCGA database were analyzed to find long noncoding RNAs (lncRNAs) that were also abnormally expressed in OV cancer and had associations with GDF15 expression. Transcription factor CEBPB was predicted as an important regulator of GDF15, confirmed by luciferase reporter assay. However, CEBPB expression was not significantly changed in OV cancer. Data from TCGA database showed that lncRNA GAS5 is downregulated in OV cancer and its expression is negatively correlated with GDF15 expression. RPISeq showed high affinity of GAS5 to CEBPB and this was confirmed by RNA-binding protein immunoprecipitation assay. GAS5 overexpression increased its binding to CEBPB and consequently downregulated GDF15. GAS5 overexpression and GDF15 knockdown decreased viability and increased apoptosis of OV cancer cells, but CEBPB overexpression had opposite effects. However, simultaneous GAS5 and CEBPB overexpression or CEBPB overexpression together with GDF15 knockdown had no effect on cell viability and apoptosis. Conclusion: GAS5 functions as decoy of CEBPB, blocking transcription-promoting effect of CEBPB on GDF15.

Objective To screen the gene related to osteosarcoma (OS) metastasis and the molecular mechanism. Methods GEO database and R2 chip analysis platform were used to screen genes related to OS metastasis. UCSC gene browser was used to find the transcription factor (TF) of CLEC5A. The mRNA level and protein expression of CLEC5A in OS tissues and normal tissues were determined by RT‐PCR and Western blotting, respectively. OS cell lines MG‐63 were transfected with CEBPB recombinant plasmid. After transfection, the expression of CLEC5A in MG‐63 cells was determined and the cell proliferation situation was determined by clone formation assay. Results Three genes CLEC5A, ALOX5AP, and RNASE3 were obtained, and CLEC5A has the highest correlation with OS. CLEC5A has screened the gene related to OS metastasis, and CEBPB can be taken as TF regulating downstream gene CLEC5A. CEBPB can regulate the downstream CLEC5A as transcription factor. The relative mRNA level and protein expression of CLEC5A in OS tissues were significantly higher than those in normal tissues. CLEC5A can prevent OS metastasis. Transfection of CEBPB increased the expression of CLEC5A in MG‐63 cells and also inhibited the proliferation of OS. Conclusion CEBPB can inhibit the proliferation of OS cells via regulating the expression of CLEC5A.

Background: The eutopic endometrium has been suggested to play a crucial role in the pathogenesis of adenomyosis. However, the specific genes in eutopic endometrium responsible for the pathogenesis of adenomyosis still remain to be elucidated. We aim to identify differentially expressed genes (DEGs) and molecular pathways/networks in eutopic endometrium from adenomyosis patients, and provide a new insight into disease mechanisms at a transcriptome level. Methods: RNA sequencing (RNA-Seq) was performed with 12 eutopic endometrium from adenomyosis and control groups. DEGs in adenomyosis were validated by quantitative real-time PCR (qPCR) and immunochemistry. Functional annotations of the DEGs were analyzed with Ingenuity Pathway Analysis (IPA). Quantitative DNA methylation analysis of CEBPB was performed with MassArray system. Findings: Totally, 373 genes were differentially expressed in the adenomyosis eutopic endometrium compared to matched controls. Bioinformatic analysis indicated that IL-6 signaling and ERK/MAPK signaling was activated in adenomyosis endometrium. We also found that the increased expression and DNA hypomethylation of C/EBPI² were associated with adenomyosis. Interpretation: Our results revealed key pathways and networks in eutopic endometrium of adenomyosis. The study is the first to propose the link between C/EBPI² and adenomyosis and can improve the understanding of the pathogenesis of adenomyosis. Funding Statement: This work is supported by the National Natural Science Foundation of China [NO. 81771551, 81501276 and 81401219], the Shanghai Municipal Commission of Science and Technology Program [NO. 17411972800 and 15411966700], the Shanghai Municipal Commission of Health and Family Planning [NO. 20154Y0039], the Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai [NO. 2018YQ39], the Shanghai Shenkang Hospital Development Center Clinical Science and Technology Innovation Project [NO. SHDC12017123], and the Combined Engineering and Medical Project of Shanghai Jiao Tong University [NO. YG2015MS41 and YG2017MS39]. Declaration of Interests: The authors have declared that no competing interests exist. Ethics Approval Statement: The study protocols were approved by the Ethics Review Committee of IPMCH and conducted according to the Declaration of Helsinki Principles.