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Background: Gastric cancer (GC) is a common tumor found worldwide, and cisplatin is the first-line agent for the treatment of GC. However, the resistance to cisplatin is an obstacle. Here, we aim to explore the biological mechanism of long noncoding RNA regulator of reprogramming (ROR) in the cisplatin resistance of GC. Materials and Methods: ROR, miR-519d-3p, and high mobility group protein A2 (HMGA2) expression in GC tissues and cells were measured by quantitative real-time polymerase chain reaction and Western blot. Cell viability, migration, invasion, and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, transwell assay, and flow cytometry, respectively. The relative protein expression was detected by Western blot. The interactions between miR-519d-3p and ROR, HMGA2 were predicted using miRcode and starBase v2.0 online database, and then verified by dual luciferase reporter assay and RNA immunoprecipitation assay. In addition, the xenograft tumor mouse model was constructed to verify the biological role of ROR in vivo. Results: The levels of ROR, HMGA2 were significantly upregulated, and miR-519d-3p was apparently downregulated in GC tissues and cells. The miRcode and starBase v2.0 online websites and dual luciferase reporter assay validated that miR-519d-3p directly interacted with ROR and HMGA2. Furthermore, ROR knockdown downregulated HMGA2 to restrain cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and cisplatin resistance in GC cells by targeting miR-519d-3p. In addition, the depletion of ROR repressed the xenograft tumor growth in vivo. Conclusion: In conclusion, we first found the ROR/miR-519d-3p/HMGA2 regulatory network to regulate cell proliferation, migration, invasion, EMT, and cisplatin resistance in GC, and this may shed light on the GC tumorigenesis.

Background: Colorectal cancer (CRC) is a significant public problem and the third cause of cancer-induced death all over the world. In addition, long noncoding RNA (lncRNA) has been reported as a vital mediator in human cancer. However, the precise role of lncRNA myocardial infarction associated transcript (MIAT) in CRC is unclear. Methods: The abundance of MIAT, miR-488-3p, and the type 1 insulin-like growth factor receptor (IGF1R) was measured by real-time quantitative polymerase chain reaction assay. The western blot assay was carried out to assess the protein level in CRC samples or control group. The cell activity, abilities of migration and invasion, and glycolysis were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), transwell, and testing glucose consumption and lactate product, correspondingly. The target association between miR-488-3p, MIAT, or IGF1R was predicted and established by bioinformatics tools, dual-luciferase reporter, and RNA pull-down assays, correspondingly. The effects of MIAT silencing in vivo were analyzed by animal experiments. Results: LncRNA MIAT was upregulated in CRC sample and that was positively correlated with IGF1R expression. Loss-of-functional assay suggested that knockdown of MIAT impeded cell activity, migration, invasion, and glycolysis of CRC cells in vivo, along with xenograft growth in vivo. Moreover, silencing of IGF1R inhibited the progression of CRC. Therefore, overexpression of IGF1R could abolish silencing of MIAT-induced effects on CRC cells. Mechanistically, MIAT was a sponge for miR-488-3p, thereby regulating IGF1R expression in CRC. Conclusion: The present study confirmed that the "MIAT/miR-488-3p/IGF1R" pathway was involved in the development of CRC, which may be the target for developing therapeutic approaches for CRC.

Background: Colorectal cancer (CRC) is the third most common malignancy worldwide. Matrine can act as a potential antitumor drug, and its antitumor activities have been tested in various cancers, including CRC. However, the effect of matrine and the related mechanisms on CRC cells remains poorly defined. Materials and Methods: CRC cells were treated with different concentrations of matrine, and then MTT, flow cytometric, and transwell assays were used to assess cell proliferation, apoptosis, invasion, and migration. MiR-10b-5p and Phosphatase and tensin homolog (PTEN) expression levels were measured by quantitative real-time polymerase chain reaction and western blot assay. The binding interaction of miR-10b-5p and PTEN were predicted by TargetScan and verified by a dual-luciferase reporter and RIP assay. The effect of matrine, miR-10b-5p, and PTEN on CRC cell proliferation, apoptosis, migration, and invasion was detected by MTT, flow cytometric, and transwell assays severally. Results: Matrine notably restrained proliferation, invasion, and migration and boosted apoptosis of CRC cells, as well as downregulated miR-10b-5p expression and upregulated PTEN protein level. PTEN was a direct target of miR-10b-5p in CRC cells. MiR-10b-5p knockdown and matrine treatment inhibited cell proliferation, migration, and invasion and induced apoptosis, and reintroduction of si-PTEN partly regained the inhibiting effect. Besides, MiR-10b-5p knockdown and matrine treatment repressed CRC growth in vivo. Conclusion: Matrine could suppress proliferation, migration, and invasion and induce apoptosis of CRC cells via the miR-10b/PTEN pathway, providing the potential molecular mechanism of matrine in blocking CRC progression.

Background: Long non-coding RNA actin filament-associated protein1-antisense RNA 1 (AFAP1-AS1) was confirmed to be associated with tumorigenesis. However, the role of AFAP1-AS1 in breast cancer was...

Background: Breast cancer is one of the most prevalent cancers that often occur in females. Long noncoding RNA differentiation antagonizing nonprotein coding RNA (DANCR) has been involved in the pathogenesis of various tumors, including breast cancer. This study aimed to investigate the role and underlying mechanism of DANCR in breast cancer. Materials and Methods: The level of DANCR was detected in breast cancer tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cell apoptosis was assessed using flow cytometry. Cell migration and invasion were estimated by the Transwell assay. The relationship between DANCR, miR-4319, and vesicle-associated membrane protein-associated protein B (VAPB) was confirmed by bioinformatic analysis and dual-luciferase reporter assay. The level of microRNA-4319 (miR-4319) was tested by qRT-PCR. The expression of VAPB was measured by qRT-PCR or western blot assay. Results: DANCR and VAPB were upregulated, while miR-4319 was downregulated in breast cancer tissues and cells. Knockdown of DANCR hindered proliferation, migration, and invasion and promoted apoptosis of breast cancer cells. DANCR knockdown inhibited breast cancer development through regulating miR-4319. Inhibition of miR-4319 restrained breast cancer cell progression by targeting VAPB. Moreover, DANCR regulated VAPB expression by sponging miR-4319 in breast cancer cells. Conclusion: DANCR facilitated breast cancer cell progression through regulating the miR-4319/VAPB axis, indicating that DANCR might be a potential biomarker and therapeutic target for breast cancer treatment.

Background: Circular RNAs (circRNAs) have recently emerged as crucial regulatory molecules in prostate cancer (PCa), but few researches focus on the effects of circRNAs on PCa radiosensitivity. The issue will be addressed in this study using circRNA Cyclin B2 (circ_CCNB2) as an object. Materials and Methods: All RNA and protein levels were severally examined using quantitative real-time polymerase chain reaction and Western blot. Colony formation assay and flow cytometry were implemented for detecting cell colony capacity and apoptotic cells, respectively. Cellular migration and invasion abilities were evaluated by transwell assay. The combination between potential target molecules was analyzed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The effect of circ_CCNB2 on PCa radiosensitivity in vivo was explored using xenograft models in mice. Results: Circ_CCNB2 was upregulated in irradiation-resistant PCa tissues and cells. Circ_CCNB2 knockdown had promoted effect on the radiosensitivity of irradiation-resistant PCa cells by inhibiting autophagy. Besides, circ_CCNB2 could directly sponge miR-30b-5p, and the promotion of circ_CCNB2 knockdown on PCa radiosensitivity was achieved by elevating miR-30b-5p. MiR-30b-5p enhanced the radiosensitivity of irradiation-resistant PCa cells through repressing the expression of its target kinesin family member 18A (KIF18A). Furthermore, circ_CCNB2 regulated the KIF18A level through targeting miR-30b-5p. Circ_CCNB2 downregulation facilitated PCa radiosensitivity in vivo through inhibiting autophagy by miR-30b-5p/KIF18A. Conclusions: In this study, knockdown of circ_CCNB2 was shown to promote PCa radiosensitivity through autophagy repression by miR-30b-5p/KIF18A axis, developing a molecular resistance mechanism of PCa radiotherapy and a feasible strategy to increase radiosensitivity.

Background Prostate cancer (PCa) is a heavy health-threat for human worldwide. Recent studies revealed that microRNAs (miRNAs) are associated with the progression of human cancers, including PCa. However, no study has been performed to investigate the role of microNA-4286 (miR-4286) on PCa. Methods Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to analyze the expression level of miR-4286 in PCa cells. Connection of miR-4286 and spalt like transcription factor 1 (SALL1) was analyzed with bioinformatic analysis tool, dual-luciferase activity reporter assay, and western blot. Effects of miR-4286 and SALL1 on PCa cell behaviors were examined in vitro. Results We showed miR-4286 expression was significantly increased in PCa cells compared with normal cell line. Knockdown of miR-4286 could inhibit PCa cell proliferation but promote cell apoptosis via targeting SALL1. Conclusions Our study suggested that miR-4286 overexpression represents a tumor promoter role in PCa.

Brain EGR1 (early growth response protein 1) overexpression aggravates focal ischemic brain injury, but its role in intracerebral hemorrhage (ICH) induced cerebral injury remains obscure. In this study, a rat ICH model was established by injecting type VII collagenase into the brain, and EGR1 knockdown reversed the increase of hematoma area, neurological function score, brain water content, blood–brain barrier (BBB) permeability, inflammation, p300 and retinoid a X receptor-α (RXRα) protein levels, as well as RXRα acetylation level induced by ICH. EGR1 expression was up-regulated in primary brain microvascular endothelial cells (BMECs), neurons, and astrocytes after ICH induction, and the up-regulation was most significant in BMECs. We also found that EGR1 promoted RXRα acetylation level by regulating p300 in BMECs. Subsequently, the BMECs were treated with OGD (oxygen glucose deprivation) plus hemin to simulate ICH condition. And silencing EGR1 rescued the upregulation of cell inflammation and the reduction of cell viability and TEER (transendothelial electric resistance) caused by OGD plus hemin via p300-mediated RXRα acetylation. Furthermore, the STAT3/NF-κB pathway was activated after treatment with OGD plus hemin, which was suppressed by silencing EGR1. And treatment with Stattic (an inhibitor of STAT3) restrained the effect of OGD plus hemin on NF-κB pathway activity, inflammation, cell viability and TEER. In conclusion, EGR1 increased RXRα acetylation level by regulating p300, thereby aggravating brain damage in ICH rat model and dysfunction in BMECs, which might through the STAT3/NF-κB pathway.

Colorectal cancer (CRC) is the third main cause of cancer-relevant deaths worldwide, and its incidence has increased in recent decades. Previous studies have indicated that certain long noncoding RNAs (lncRNAs) have regulatory roles in tumor occurrence and progression. Often, lncRNAs are competitive endogenous RNAs that sponge microRNAs to up-regulate mRNAs. Here, we examined the role of a novel lncRNA gamma-butyrobetaine hydroxylase 1 antisense RNA 1 (BBOX1-AS1) in CRC. We observed that BBOX1-AS1 is overexpressed in CRC cell lines, and BBOX1-AS1 knockdown enhances cell proliferation, migration and invasion while reducing cell apoptosis. miR-361-3p is present at a low level in CRC and is negatively modified by BBOX1-AS1. Moreover, miR-361-3p was validated to be targeted by BBOX1-AS1. Src homology 2 B adaptor protein 1 (SH2B1) was notably upregulated in CRC cell lines and was identified as a downstream gene of miR-361-3p. In addition, we found that miR-361-3p amplification can suppress the expression of SH2B1. Finally, data from rescue assays suggested that overexpression of SH2B1 counteracted BBOX1-AS1 silencing-mediated inhibition of CRC progression. In conclusion, BBOX1-AS1 promotes CRC progression by sponging hsa-miR-361-3p and up-regulating SH2B1.

AIMS: Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis.METHODS & RESULTS: Myeloid specific PHD knockout (PHDko) mice were obtained via bone marrow transplantation (PHD1ko, PHD3ko) or conditional knockdown through lysozyme M-driven Cre recombinase (PHD2cko). Mice were fed high cholesterol diet for 6-12 weeks to induce atherosclerosis. Aortic root plaque size was significantly augmented 2.6-fold in PHD2cko, and 1.4-fold in PHD3ko compared to controls, but was unchanged in PHD1ko mice. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the HIF1α/BNIP3 axis. Bulk and single cell RNA data of PHD2cko bone-marrow-derived macrophages (BMDM) and plaque macrophages, respectively, showed enhanced HIF1α/BNIP3 signaling, which was validated in vitro by siRNA silencing. Human plaque BNIP3 mRNA was positively associated with plaque necrotic core size, suggesting similar pro-apoptotic effects in human. Further, PHD2cko plaques displayed enhanced fibrosis, while macrophage collagen breakdown by matrix metalloproteinases, collagen production and proliferation were unaltered. Instead, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. In silico analysis of macrophage-fibroblast communication predicted SPP1 (osteopontin) signaling as regulator, which was corroborated by enhanced plaque SPP1 protein in vivo. Increased SPP1 mRNA expression upon PHD2cko was preferentially observed in foamy plaque macrophages expressing "triggering receptor expressed on myeloid cells-2" (TREM2hi) evidenced by single-cell RNA, but not in neutrophils. This confirmed enhanced fibrotic signaling by PHD2cko macrophages to fibroblasts, in vitro as well as in vivo.CONCLUSION: Myeloid PHD2cko and PHD3ko enhanced atherosclerotic plaque growth and macrophage apoptosis, while PHD2cko macrophages further activated collagen secretion by fibroblasts in vitro, likely via paracrine SPP1 signaling through TREM2hi macrophages.TRANSLATIONAL OUTLOOK: This study shows that myeloid PHD isoforms PHD2 and PHD3 worsen plaque characteristics and phenotype, such as plaque size, macrophage accumulation, apoptosis, and collagen accumulation in mice. We show both direct effects on macrophages and paracrine effects of macrophage PHD2 loss on vessel wall fibroblast populations. Broad spectrum-PHD inhibitors, e.g. Roxadustat, are currently being prescribed to chronic kidney disease patients, who are already at risk for cardiovascular disease. When considering this study and the pro-fibrotic and pro-apoptotic effects we report, broad PHD inhibition may therefore be sub-optimal and more targeted PHD inhibition of PHD1 should be considered.

Background: Circular RNAs (circRNAs) are crucial regulators in human cancers, including nonsmall cell lung cancer (NSCLC). In this study, we aim to explore the biological functions and molecular mechanisms of circ_0074027 in NSCLC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of circ_0074027, paired like homeodomain 1 (PITX1) mRNA, microRNA-335-5p (miR-335-5p), and cullin 4B (CUL4B) mRNA. The feature of circ_0074027 was analyzed by RNase R digestion assay. Flow cytometry analysis was adopted to analyze cell cycle and cell apoptosis. Cell counting kit-8 (CCK-8) assay and colony formation assay were performed to assess cell proliferation. Western blot assay was conducted to measure protein levels. Dual-luciferase reporter and RNA pull-down assays were carried out to verify the relationships among circ_0074027, miR-335-5p, and CUL4B. The murine xenograft model was established to investigate the role of circ_0074027 in vivo. Results: High expression of circ_0074027 was found in NSCLC tissues and cells. Circ_0074027 knockdown suppressed cell viability, cell cycle process, and colony formation and promoted apoptosis in NSCLC cells in vitro and inhibited tumor growth in vivo. Circ_0074027 acted as a sponge of miR-335-5p. The effect of circ_0074027 knockdown on NSCLC progression was weakened by miR-335-5p inhibition. Moreover, CUL4B was a target gene of miR-335-5p. CUL4B overexpression reversed the inhibitory effects on cell viability, cell cycle process, and colony formation and the promotional effect on cell apoptosis caused by miR-335-5p in NSCLC. Conclusion: Circ_0074027 facilitated NSCLC cell progression through regulating miR-335-5p/CUL4B axis.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly malignant breast cancer subtype with a poor prognosis. The cell cycle regulator cyclin A2 (CCNA2) plays a role in tumor development. Herein, we explored the role of CCNA2 in TNBC. METHODS: We analyzed CCNA2 expression in 15 pairs of TNBC and adjacent tissues and assessed the relationship between CCNA2 expression using the tissue microarray cohort. Furthermore, we used two TNBC cohort datasets to analyze the correlation between CCNA2 and E2F transcription factor 1 (E2F1) and a luciferase reporter to explore their association. Through rescue experiments, we analyzed the effects of E2F1 knockdown on CCNA2 expression and cellular behavior. RESULTS: We found that CCNA2 expression in TNBC was significantly higher than that in adjacent tissues with similar observations in MDA-MB-231 and MDA-MB-468 cells. E2F1 was highly correlated with CCNA2 as observed through bioinformatics analysis (R= 0.80, P< 0.001) and through TNBC tissue verification analysis (R= 0.53, P< 0.001). We determined that E2F1 binds the +677 position within the CCNA2 promoter. Moreover, CCNA2 overexpression increased cell proliferation, invasion, and migration owing to E2F1 upregulation in TNBC. CONCLUSION: Our data indicate that E2F1 promotes TNBC proliferation and invasion by upregulating CCNA2 expression. E2F1 and CCNA2 are potential candidates that may be targeted for effective TNBC treatment.

Background Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. Multiple intronic and distal regulatory elements are responsible for regulating differential CFTR expression across tissues. Methods Drawing on published data, 18 high-priority genomic regions were identified and interrogated for CFTR-enhancer function using CRISPR/dCas9-based epigenome editing tools. Each region was evaluated by dCas9p300 and dCas9KRAB for its ability to enhance or repress CFTR expression, respectively. Results Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9p300 mediates a significant increase in CFTR mRNA levels when targeted to the promoter and a region 44 kb upstream of the transcriptional start site in a CFTR-low expressing cell line. Multiple gRNAs targeting the promoter induced a robust increase in CFTR protein levels. In contrast, dCas9KRAB-mediated repression is much more robust with 10 of the 18 evaluated genomic regions inducing CFTR protein knockdown. To evaluate the therapeutic efficacy of modulating CFTR gene regulation, dCas9p300 was used to induce elevated levels of CFTR from the endogenous locus in ΔF508/ΔF508 human bronchial epithelial cells. Ussing chamber studies demonstrated a synergistic increase in ion transport in response to CRISPR-induced expression of ΔF508 CFTR mRNA along with VX809 treatment. Conclusions CRISPR/dCas9-based epigenome-editing provides a previously unexplored tool for interrogating CFTR enhancer function. Here, we demonstrate that therapeutic interventions that increase the expression of CFTR may improve the efficacy of CFTR modulators. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.

Ribosomal protein L3-like (RPL3L) is a poorly characterized ribosomal protein that is exclusively expressed in skeletal and cardiac muscle. RPL3L is also downregulated in Duchenne muscular dystrophy (DMD), suggesting that it may play an important role in muscle biology. In this study, we investigated the role of RPL3L in skeletal muscle of healthy C57 and dystrophic mdx mice. We show that RPL3L is developmentally regulated and that intramuscular adeno-associated virus (AAV)-mediated RPL3L knockdown in the tibialis anterior of C57 and mdx mice results in increased specific force with improved resistance to eccentric contraction induced muscle damage in dystrophic muscles. The mechanism by which RPL3L knockdown improves muscle function remains unclear. Histological observations showed a significant increase in muscle length and decrease in muscle cross-sectional area after RPL3L inhibition suggesting that this ribosomal protein may play a role in myofiber morphology. The endogenous downregulation of RPL3L in DMD may be a protective mechanism that attempts to improve skeletal muscle function and counteract the dystrophic phenotype.

Background Increased physical activity is a common feature of anorexia nervosa (AN). Although high activity levels are associated with greater risk of developing AN, particularly when combined with dieting, most individuals who diet and exercise maintain a healthy body weight. It is unclear why some individuals develop AN while most do not. A rodent model of resilience and vulnerability to AN would be valuable to research. Dopamine, which is believed to play a crucial role in AN, regulates both reward and activity and may modulate vulnerability. Methods Adolescent and young adult female C57BL/6N mice were tested in the activity-based anorexia (ABA) model, with an extended period of food restriction in adult mice. ABA was also tested in dopamine transporter knockdown mice and wild-type littermates. Mice that adapted to conditions and maintained a stable body weight were characterized as resilient. Results In adults, vulnerable and resilient phenotypes emerged in both the ABA and food-restricted mice without wheels. Vulnerable mice exhibited a pronounced increase in running throughout the light cycle, which dramatically peaked prior to requiring removal from the experiment. Resilient mice exhibited an adaptive decrease in total running, appropriate food anticipatory activity, and increased consumption, thereby achieving stable body weight. Hyperdopaminergia accelerated progression of the vulnerable phenotype. Conclusions Our demonstration of distinct resilient and vulnerable phenotypes in mouse ABA significantly advances the utility of the model for identifying genes and neural substrates mediating AN risk and resilience. Modulation of dopamine may play a central role in the underlying circuit.

Background Pristimerin, a natural flavonoid compound, has potential anti-tumor activities. These activities have been illustrated in various cancer cell lines, including MDA-MB-231 cells. MDA-MB-231 cells are a representative mesenchymal subtype of triple negative breast cancer (MES-TNBC) cell line. Currently, the main treatment for patients with advanced MES-TNBC is cytotoxic chemotherapy. We tried to examine the role and effect of pristimerin on epithelial–-mesenchymal transition (EMT) in MDA-MB-231 cells. Material and methods The effects of pristimerin on the proliferation of MDA-MB-231 cells were investigated by cloning formation growth assay. In vitro transwell and adhesion assays were performed for cell invasion and adhesion. The expression levels of EMT markers in E-cadherin and N-cadherin were examined by western blotting. We also established overexpressed- and silenced-integrin β3 cell lines to evaluate the role of integrin β3 in mediating the EMT reversion events in MDA-MB-231 cells. Results Pristimerin inhibited cell proliferation, and its inhibitory effect was dose-dependent. We demonstrated that pristimerin reserved EMT by upregulating E-cadherin and downregulating N-cadherin expression. Meanwhile, we revealed that pristimerin inhibited mRNA and protein expression of integrin β3, which is a key heterodimeric transmembrane receptor associated with EMT. These inhibitory effects and reversion of EMT were enhanced when integrin β3 was knockdown in MDA-MB-231 cells, while the overexpression of integrin β3 attenuated these effects. In vivo studies using xenograft mouse model demonstrated that pristimerin inhibited tumor growth. Conclusions Our findings provide important insights into the effects of pristimerin on inhibiting cancer progression and EMT reversion by suppression of integrin β3.

Background Long non-coding RNA (lncRNA) is implicated in the progression of multiple cancers. This study aimed to explore the expression characteristics, biological function and molecular mechanism of lncRNA HAGLROS expression in NSCLC. Methods Quantitative real-time polymerase chain reaction (RT-PCR) was adopted to detect HAGLROS expression in NSCLC tissues and normal lung tissues. Survival curve was plotted by Kaplan–Meier method. Gain-of-function and loss-of-function models were respectively established to investigate the biological functions of HAGLROS, miR-100 and SMARCA5. MTT and Transwell assays were carried out to monitor the changes in proliferation, migration and invasion of NSCLC cells. Bioinformatics analysis and dual-luciferase reporter assay were used to verify the binding sites between HAGLROS and miR-100. Western blot was performed to determine the regulatory effects of HAGLROS and miR-100 on SMARCA5 protein expression. Results Up-regulated HAGLROS expression was observed in NSCLC tissues and cell lines. Over-expressed HAGLROS promoted the malignant phenotypes of NSCLC cells; conversely, HAGLROS knockdown repressed the malignant phenotypes of NSCLC cells. HAGLROS repressed miR-100 expression to promote SMARCA5 expression in NSCLC cells, and miR-100 overexpression or SMARCA5 knockdown counteracted the oncogenic functions of HAGLROS. Conclusions These results conclude that HAGLROS is a tumor promoter in NSCLC, and it regulates the malignant phenotypes of NSCLC cells via miR-100/SMARCA5 axis.

Background: Evidence from previous investigations points to a rising trend in the incidence of colorectal cancer (CRC) worldwide. The mortality resulting from this cancer is high. Unlike nonsmall cell lung cancer for which LINC01123 has been investigated, there are few reports on how this long noncoding RNA (lncRNA) regulates CRC. Materials and Methods: The authors evaluated the expression of LINC01123 in CRC tissues by quantitative real-time polymerase chain reaction. Its impact on cancer cell behavior was analyzed with cell counting kit-8 (CCK-8), colony formation, and Transwell invasion assays. To establish the mechanisms of LINC01123 in CRC they carried out RIP and luciferase reporter assays. Results: The results show that LINC01123 expression is abnormally elevated in CRC tissues and cell lines. High LINC01123 expression closely correlates with poor prognosis, advanced TNM stage, and lymph-node metastasis. The authors also show that knockdown of LINC01123 inhibits proliferation and invasion in CRC cells. In mechanism, it is revealed that LINC01123 may function as competitive endogenous RNA (ceRNA) against miR-625-5p to promote LIM and SH3 protein 1 (LASP1) expression. Conclusions: The data indicate that high LINC01123 exerts its oncogenic roles by regulating the miR-625-5p/LASP1 axis in CRC progression.

Mammalian Eps15 homology domain 1 (EHD1) participates in the development of non-small cell lung cancer (NSCLC). However, its role in mediating aerobic glycolysis remains unclear. Herein, microarray analysis revealed that EHD1 expression was significantly correlated with the glycolysis/gluconeogenesis pathway. Clinically, EHD1 expression was positively correlated with the maximum standard uptake value (SUVmax) in 18F-FDG PET/CT scans. Additionally, EHD1 knockdown inhibited aerobic glycolysis and proliferation in vitro and in vivo. Furthermore, Wnt/β-catenin signaling was identified as a critical EHD1-regulated pathway. Co-IP, native gel electrophoresis, and immunoblotting showed that EHD1 contributed to 14-3-3 dimerization via 14-3-3ζ and subsequent activation of β-catenin/c-Myc signaling. Analysis of the EHD1 regulatory region via ENCODE revealed the potential for c-Myc recruitment, leading to transcriptional activation of EHD1 and formation of an EHD1/14-3-3ζ/β-catenin/c-Myc positive feedback circuit. Notably, blocking this circuit with a Wnt/β-catenin inhibitor dramatically inhibited tumor growth in vivo. The positive correlations among EHD1, 14-3-3ζ, c-Myc, and LDHA were further confirmed in NSCLC tissues. Collectively, our study demonstrated that EHD1 activates a 14-3-3ζ/β-catenin/c-Myc regulatory circuit that synergistically promotes aerobic glycolysis and may constitute a promising therapeutic target for NSCLC.

BACKGROUND: Role of aquaporin-8 (AQP8) in cervical cancer has not been fully elucidated. OBJECTIVE: We aim to explore the impacts of AQP8 on viability, apoptosis and metastasis in cervical cancer cells. METHODS: AQP8 protein expression in cervical carcinoma specimens and cell lines was detected by IHC and western blot analysis. Lentivirus-mediated transfection was used to upregulate and knockdown AQP8 in cells. Cell viability and apoptosis were assessed by CCK-8 and flow cytometry assays, respectively. Transwell experiments were conducted to investigate cell invasive and migratory capabilities. EMT-related markers were detected by western blot analysis. RESULTS: A strong positive of AQP8 protein expression was observed in cervical cancer tissues. Western blot analysis confirmed overexpression and knockdown of AQP8 in SiHa cells. AQP8-overexpressed SiHa cells displayed an enhanced viability, reduced apoptotic rate, increased invasive and migratory abilities. Knockdown of AQP8 inhibited the viability, promoted the apoptosis, and suppressed invasion and migration. Furthermore, AQP8 overexpression significantly upregulated vimentin and N-cadherin, and downregulated E-cadherin, which were reversed by AQP8 knockdown. CONCLUSIONS: AQP8 increases viability, inhibits apoptosis, and facilitates metastasis in SiHa cells. This may be associated with EMT-related markers regulated by AQP8. AQP8 could serve as a potential marker for cervical cancer progression.

Osteoporosis, fracture, large-scale craniofacial defects and osteonecrosis are hot topics and are still underdiagnosed and undertreated in the clinic. It is urgent to understand the molecular mechanisms corresponding to the regulation of bone formation. CMTM3 (CKLF-like MARVEL transmembrane domain containing 3) connects the classic chemokine to the transmembrane 4 superfamily and plays an important role in intracellular vesicles transport, EGF receptor function maintenance and cancer development. However, its expression and function in bone remain unclear. In this paper, we found that the bone volume/total volume, trabecular number, trabecular thickness and bone surface area/bone volume of Cmtm3 KO mice increased significantly, and trabecular separation and trabecular pattern factor decreased in Cmtm3 KO mice compared with WT mice by microcomputed tomography. Moreover, the bone mineral content, bone mineral density, ultimate force and stiffness were also increased in Cmtm3 KO mice. Using in vitro analysis, we showed that CMTM3 expression decreases during the differentiation of hBMSCs to osteoblasts. Knockdown of CMTM3 promoted ALP and mineralization of hBMSCs and facilitated osteoblastic differentiation with increasing RUNX2 expression. However, overexpression of CMTM3 got the opposite results. These results proved that CMTM3 was essential for osteogenic differentiation. In addition, knockdown of CMTM3 enhanced p-Erk1/2, but had no significant effect on p-Akt or p-STAT3 in hBMSCs and MC3T3-E1 cells. Taken together, our results indicated that Erk1/2 and RUNX2 pathways mediated by CMTM3 were involved in the process of osteogenic differentiation, and CMTM3 might be a new potential target in the treatment of bone formation-related disease.

Background: Asthma is a chronic inflammatory disease characterized by airway inflammation and airway hyperresponsiveness (AHR). IL-33 is considered as one of the most critical molecules in asthma pathogenesis. IL-33 is stored in nucleus and passively released during necrosis. But little is known about whether living cells can release IL-33 and how this process is regulated. Objective: We sought to investigate the role of polymerase I and transcript release factor (PTRF) in IL-33 release and asthma pathogenesis. Methods: Ovalbumin (OVA)-induced asthma model in PTRF+/− mice were employed to dissect the role of PTRF in vivo. Then, further in vitro experiments were carried out to unwind the potential mechanism involved. Results: In OVA asthma model with challenge phase, PTRF+/− mice showed a greater airway hyper-reaction, with an intense airway inflammation and more eosinophils in bronchoalveolar lavage fluid (BALF). Consistently, more acute type 2 immune response in lung and a higher IL-33 level in BALF were found in PTRF+/− mice. In OVA asthma model without challenge phase, airway inflammation and local type 2 immune responses were comparable between control mice and PTRF+/− mice. Knockdown of PTRF in 16HBE led to a significantly increased level of IL-33 in cell culture supernatants in response to LPS or HDM. Immunoprecipitation assay clarified Y158 as the major phosphorylation site of PTRF, which was also critical for the interaction of IL-33 and PTRF. Overexpression of dephosphorylated mutant Y158F of PTRF sequestered IL-33 in nucleus together with PTRF and limited IL-33 extracellular secretion. Conclusion: Partial loss of PTRF led to a greater AHR and potent type 2 immune responses during challenge phase of asthma model, without influencing the sensitization phase. PTRF phosphorylation status determined subcellular location of PTRF and, therefore, regulated IL-33 release.

Purpose NUF2 has been implicated in multiple cancers recently, suggesting NUF2 may play a role in the common tumorigenesis process. In this study, we aim to perform comprehensive meta-analysis of NUF2 expression in the cancer types included in the Cancer Genome Atlas (TCGA). Materials and methods RNA-sequencing data in 31 cancer types in the TCGA data and 11 independent datasets were used to examine NUF2 expression. Silencing NUF2 using targeting shRNAs in hepatocellular carcinoma cell lines was used to evaluate NUF2's role in hepatocellular carcinoma (HCC) in vitro and in vivo. Results NUF2 up-regulation is significantly observed in 23 out of the 31 cancer types in the TCGA datasets and validated in 13 major cancer types using 11 independent datasets. NUF2 overexpression was clinically important as high NUF2 was significantly associated with tumor stages in eight different cancers. High NUF2 was also associated with significantly poorer patient overall survival and disease-free survival in eight and six cancers, respectively. We proceeded to validate NUF2 overexpression and its negative association with overall survival at the protein level in an independent cohort of 40 HCC patients. Compared to the non-targeting controls, NUF2 knockdown cells showed significantly reduced ability to grow, migrate into a scratch wound and invade the 8 μm porous membrane in vitro. Moreover, NUF2 knockdown cells also formed significantly smaller tumors than control cells in mouse xenograft assays in vivo. Conclusion NUF2 up-regulation is a common feature of many cancers. The prognostic potential and functional impact of NUF2 up-regulation warrant further studies.

Background Hepatocyte senescence is a core event that mediates the occurrence and development of alcoholic liver disease. Nuclear factor of activated T-cells 4 (NFATc4) is a key driver of nonalcoholic steatohepatitis. However, little was known about the implication of NFATc4 for alcoholic liver disease. This study was aimed to investigate the role of NFATc4 in hepatocyte senescence and further elucidate the underlying mechanism. Methods Real-time PCR, Western blot, immunofluorescence staining, and enzyme-linked immunosorbent assay were performed to explore the role of NFATc4 in hepatocyte senescence. Results NFATc4 was induced in ethanol-incubated hepatocytes. NFATc4 knockdown recovered cell viability and reduced the release of aspartate transaminase, alanine transaminase, and lactic dehydrogenase from ethanol-incubated hepatocytes. NFATc4 knockdown protected mice from alcoholic liver injury and inflammation. NFATc4 knockdown counteracted ethanol-induced hepatocyte senescence, evidenced by decreased senescence-associated β-galactosidase positivity and reduced p16, p21, HMGA1, and γH2AX, which was validated in in vivo studies. Peroxisome proliferator-activated receptor (PPAR)γ was inhibited by NFATc4 in ethanol-treated hepatocytes. PPARγ deficiency abrogated the inhibitory effects of NFATc4 knockdown on hepatocyte senescence, oxidative stress, and hepatic steatosis in mice with alcoholic liver disease. Conclusions This work discovered that ethanol enhanced NFATc4 expression, which further triggered hepatocyte senescence via repression of PPARγ.

Cutaneous squamous cell carcinomas (SCCs) are frequent heterogeneous tumors arising from sun-exposed regions of the skin and characterized by complex pathogenesis. HOPX is a member of the homeodomain-containing superfamily of proteins holding an atypical homeodomain unable to bind to DNA. First discovered in the heart as a regulator of cardiac development, in the skin, HOPX modulates the terminal differentiation of keratinocytes. There is a particular interest in studying HOPX in squamous skin carcinogenesis because it has the atypical structure and the functional duality as an oncogene and a tumor suppressor gene, reported in different malignancies. In this study, we analyzed the effects of HOPX knockdown and overexpression on SCC tumorigenicity in vitro and in vivo. Our data show that HOPX knockdown in SCC cells inhibits their proliferative and invasive activity through the acceleration of apoptosis. We established that methylation of two alternative HOPX promoters leads to differential expression of HOPX transcripts in normal keratinocytes and SCC cells. Importantly, we report that HOPX acts as an oncogene in the pathogenesis of SCC probably through the activation of the second alternative promoter and the modulation of apoptosis.

The C (Cys) 2H (His) 2-type transcription factor is one of the most important transcription factors in plants and plays a regulatory role in the physiological responses of rice to abiotic stresses. A novel rice C2H2-type zinc finger protein, abscisic acid (ABA)-drought-reactive oxygen species (ROS) 3 (OsADR3), was found to confer drought stress tolerance by enhancing antioxidant defense and regulating OsGPX1. Overexpression of OsADR3 in rice increased tolerance to drought stress by increasing ROS scavenging ability and ABA sensitivity. In contrast, CRISPR/Cas9-mediated knockout of osadr3 increased the sensitivity of rice to drought and oxidative stress. An exogenous ROS-scavenging reagent restored the drought-stress tolerance of osadr3-CRISPR plants. Global transcriptome analysis suggested that OsADR3 increased the expression of OsGPX1 under drought stress. Electrophoretic mobility shift, yeast one-hybrid, and dual-luciferase reporter assays revealed that OsADR3 modified the expression of OsGPX1 by directly binding to its promoter. Knockdown of OsGPX1 repressed ROS scavenging ability under drought stress in OsADR3-overexpression plants. These findings suggest that OsADR3 plays a positive regulatory role in drought-stress tolerance by inducing antioxidant defense and associated with the ABA signaling pathway in rice.

Aims Pathological cardiac hypertrophy induced by activation of the renin–angiotensin–aldosterone system (RAAS) is one of the leading causes of heart failure. However, in current clinical practice, the strategy for targeting the RAAS is not sufficient to reverse hypertrophy. Here, we investigated the effect of prostaglandin E1 (PGE1) on angiotensin II (AngII)-induced cardiac hypertrophy and potential molecular mechanisms underlying the effect. Methods and results Adult male C57 mice were continuously infused with AngII or saline and treated daily with PGE1 or vehicle for two weeks. Neonatal rat cardiomyocytes were cultured to detect AngII-induced hypertrophic responses. We found that PGE1 ameliorated AngII-induced cardiac hypertrophy both in vivo and in vitro. The RNA sequencing (RNA-seq) and expression pattern analysis results suggest that Netrin-1 (Ntn1) is the specific target gene of PGE1. The protective effect of PGE1 was eliminated after knockdown of Ntn1. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the PGE1-mediated signaling pathway changes are associated with the mitogen-activated protein kinase (MAPK) pathway. PGE1 suppressed AngII-induced activation of the MAPK signaling pathway, and such an effect was attenuated by Ntn1 knockdown. Blockade of MAPK signaling rescued the phenotype of cardiomyocytes caused by Ntn1 knockdown, indicating that MAPK signaling may act as the downstream effector of Ntn1. Furthermore, inhibition of the E-prostanoid (EP) 3 receptor, as opposed to the EP1, EP2, or EP4 receptor, in cardiomyocytes reversed the effect of PGE1, and activation of EP3 by sulprostone, a specific agonist, mimicked the effect of PGE1. Conclusion In conclusion, PGE1 ameliorates AngII-induced cardiac hypertrophy through activation of the EP3 receptor and upregulation of Ntn1, which inhibits the downstream MAPK signaling pathway. Thus, targeting EP3, as well as the Ntn1–MAPK axis, may represent a novel approach for treating pathological cardiac hypertrophy.

Medulloblastoma, the most common malignant brain tumor in children, consists of four molecular subgroups WNT, SHH, Group 3, and Group 4. Group 3 has the worst survival rate among the four subgroups and is characterized by the expression of retina-specific genes. CRX, the master regulator of the photoreceptor differentiation, is aberrantly expressed in Group 3 medulloblastomas. CRX expression increased the proliferation, anchorage-independent growth, invasion potential, and tumorigenicity of medulloblastoma cells indicating the oncogenic role of CRX in medulloblastoma pathogenesis. CRX knockdown resulted in the downregulation of expression of several retina-specific genes like IMPG2, PDC, RCVRN. and Group 3 specific genes like GABRA5, MYC, PROM1. Thus, CRX plays a major role not only in the expression of retina-specific genes but also in defining Group 3 identity. Increased expression of several pro-apoptotic genes upon CRX knockdown suggests that CRX could protect Group 3 medulloblastoma cells from cell death. Several negative regulators of the TGF-β signaling pathway like SMAD7, PMEPA1, KLF2 were upregulated upon the CRX knockdown. Western blot analysis showed a decrease in the levels of (Phospho)-SMAD2, total levels of SMAD2, SMAD4, and an increase in the levels of SMAD7 indicating inhibition of the TGF-β signaling pathway upon CRX knockdown. Copy number variations in several genes involved in the TGF-β signaling pathway occur in a subset of Group 3 tumors. Autocrine TGF-β/activin signaling has recently been reported to be active in a subset of Group 3 medulloblastomas. CRX knockdown resulting in the inhibition of the TGF-β/activin signaling pathway demonstrates an interaction between the two Group 3 specific oncogenic pathways and suggests simultaneous targeting of both CRX and TGF-β signaling as a possible therapeutic strategy.

Myocardial injury is a severe complication of sepsis and contributes substantially to the death of critically ill patients. Long non-coding RNAs (lncRNAs) participate in the pathogenesis of sepsis-induced myocardial injury. In this study, we investigated the role of lncRNA X-inactive specific transcript (XIST) in septic myocardial injury and explored its mechanism. Lipopolysaccharide (LPS)-stimulated H9C2 cells and rats subjected to cecal ligation and puncture (CLP) were used as the in vitro and in vivo models. After exposure to LPS, XIST and c-Fos levels were upregulated, but miR-150-5p was downregulated in H9C2 cardiomyocytes and myocardial tissues. XIST affected viability, apoptosis, and pyroptosis in LPS-challenged H9C2 cells. Moreover, XIST knockdown attenuated LPS-induced injury in H9C2 cells by targeting the miR-150-5p/c-Fos axis. c-Fos could bound to the promoter of the TXNIP/XIST gene and enhanced TXNIP/XIST expression. Silencing of XIST improved cardiac function and survival rate and reduced apoptosis and pyroptosis by regulating the miR-150-5p/c-Fos axis in septic rats in vivo. Taken together, our data show that XIST/miR-150-5p/c-Fos axis affected septic myocardial injury, which may indicate a novel therapeutic strategy for sepsis-induced myocardial injury.

Neuropathic pain is a devastating disease and exists tolerance to current available analgesics. MicroRNAs were reported to be involved in the regulation of neuropathic pain, but the biological role of miR-216a-5p in neuropathic pain remains unclear. In this study, we constructed a CCI rat model of neuropathic pain. Our results showed that the expression of miR-216a-5p was downregulated in CCI rats, and mechanical allodynia and thermal hyperalgesia in CCI rats were improved by miR-216a-5p overexpression, suggesting that miR-216a-5p overexpression alleviated neuropathic pain. Moreover, ELISA showed that miR-216a-5p overexpression inhibited concentration and mRNA expression of IL-6, TNF-α and IL-1β as well as suppressed microglial infiltration, indicating that miR-216a-5p overexpression inhibited neuroinflammation. Besides, we found that miR-216a-5p upregulation inactivated the Wnt/β-catenin signaling pathway. Furthermore, KDM3A was the downstream target of miR-216a-5p and KDM3A knockdown attenuated neuropathic pain. Finally, through rescue assay, we found that KDM3A countervailed miR-216a-5p mediated regulation of neuropathic pain via the Wnt/β-catenin signaling pathway. To sum up, our study confirmed that miR-216a-5p alleviated neuropathic pain in rats by targeting KDM3A and inactivating the Wnt/β-catenin signaling pathway, which may open a new and useful way for treatment of neuropathic pain.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr −/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-β1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600–1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor β1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphor-AKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and −2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis.

Background and aims The dynamic N6-methyladenosine (m6 A) mRNA modification is essential for acute stress response and cancer progression. Sublethal heat stress from insufficient radiofrequency ablation (IRFA) has been confirmed to promote HCC progression; however, whether m6 A machinery is involved in IRFA-induced HCC recurrence remains open for study. Approach and results Using an IRFA HCC orthotopic mouse model, we detected a higher level of m6 A reader YTH N6-methyladenosine RNA binding protein 1-3 (YTHDF1) in the sublethal-heat-exposed transitional zone close to the ablation center than that in the farther area. In addition, we validated the increased m6 A modification and elevated YTHDF1 protein level in sublethal-heat-treated HCC cell lines, HCC patient-derived xenograft (PDX) mouse model, and patients' HCC tissues. Functionally, gain-of-function/loss-of-function assays showed that YTHDF1 promotes HCC cell viability and metastasis. Knockdown of YTHDF1 drastically restrains the tumor metastasis evoked by sublethal heat treatment in tail vein injection lung metastasis and orthotopic HCC mouse models. Mechanistically, we found that sublethal heat treatment increases epidermal factor growth receptor (EGFR) m6 A modification in the vicinity of the 5' untranslated region and promotes its binding with YTHDF1, which enhances the translation of EGFR mRNA. The sublethal-heat-induced up-regulation of EGFR level was further confirmed in the IRFA HCC PDX mouse model and patients' tissues. Combination of YTHDF1 silencing and EGFR inhibition suppressed the malignancies of HCC cells synergically. Conclusions The m6 A-YTHDF1-EGFR axis promotes HCC progression after IRFA, supporting the rationale for targeting m6 A machinery combined with EGFR inhibitors to suppress HCC metastasis after RFA.

Myocardial infarction (MI)-induced the activation of NLRP3 inflammasome has been well known to aggravate myocardial injury and cardiac dysfunction by causing inflammation and pyroptosis in the heart. Circular RNAs (circRNAs) have been demonstrated to play critical roles in cardiovascular diseases. However, the functions and mechanisms of circRNAs in modulating cardiac inflammatory response and cardiomyocyte pyroptosis remain largely unknown. We revealed that circHelz, a novel circRNA transcribed from the helicase with zinc finger (Helz) gene, was significantly upregulated in both the ischemic myocardium of MI mouse and neonatal mouse ventricular cardiomyocytes (NMVCs) exposed to hypoxia. Overexpression of circHelz caused cardiomyocyte injury in NMVCs by activating the NLRP3 inflammasome and inducing pyroptosis, while circHelz silencing reduced these effects induced by hypoxia. Furthermore, knockdown of circHelz remarkably attenuated NLRP3 expression, decreased myocardial infarct size, pyroptosis, inflammation, and increased cardiac function in vivo after MI. Overexpression of miR-133a-3p in cardiomyocytes greatly prevented pyroptosis in the presence of hypoxia or circHelz by targeting NLRP3 in NMVCs. Mechanistically, circHelz functioned as an endogenous sponge for miR-133a-3p via suppressing its activity. Overall, our results demonstrate that circHelz causes myocardial injury by triggering the NLRP3 inflammasome-mediated pro-inflammatory response and subsequent pyroptosis in cardiomyocytes by inhibiting miR-133a-3p function. Therefore, interfering with circHelz/miR-133a-3p/NLRP3 axis might be a promising therapeutic approach for ischemic cardiac diseases.

Genome editing is an addition, deletion, or replacement of a gene to remove or initiating explicit and preferred characters in the genome. Utilizing gene-editing tools like CRISPR-Cas9 technology could be accomplished either by gene-based methodology or protein-based technology that has been under scrutiny for the protracted time wherein physical techniques, viral and non-viral strategies have been utilized together. Transplanting ex vivo CRISPR edited cells empowers screening of single guide RNAs with high-throughput and CRISPR based screening in organoids transplantation to validate cancer cells including colorectal carcinoma in various phases of its development and treatment.CRISPR knockout screens have recognized genes that drive interest in colon cancer to develop hallmarks, especially in some cancer cell lines with single guide RNA, to disclose drug resistance mechanisms. One advantage of this method is to deal with CRISPR knockout genomic screening, which disrupts gene expression, rather than the partial knockdown that is mostly done with RNA interference and CRISPR/Cas technology. This technique is used to treat different forms of cancer because of its proficient editing of the target gene, along with the CRISPR/Cas system. Latest research has shown that the CRISPR/Cas gene-editing technique has theoretically reformed the expression in colorectal carcinoma of long non-coding RNA. For the next decade CRISPR/Cas9 technology will positively fuel the development of more in vivo gene editing clinical trials in colon cancer and will have an enormous impact on molecular medicine.

Background A strong relationship between long intergenic non-protein coding RNA 511 (LINC00511) and glioma has been previously reported but the mechanism of LINC00511 in glioma is yet to be determined. This study examined the mechanism of LINC00511 in glioma. Methods The expression of LINC00511 in glioma was determined by bioinformatics analysis and real-time quantitative PCR (RT-qPCR) analysis. The target relationship between genes was predicted by starBase, TargetScan, and was verified by dual-luciferase. Subsequently, siRNA targeting LINC00511 (siLINC00511) and miR-15a-5p mimic were transfected into glioma cells to examine the effect on biological characteristics using cell counting kit-8, clone formation, flow cytometry, wound-healing, and transwell. MiR-15a-5p inhibitor and AEBP1 were used for in vitro rescue experiments, and tumorigenesis assay and immunohistochemical assays were performed for in vivo experiments. Epithelial-mesenchymal transition (EMT) and p65 phosphorylation were examined by Western blot. Results LINC00511 was predicted and verified to be up-regulated in glioma. SiLINC00511 suppressed cell viability, proliferation, migration and invasion, accelerated apoptosis of glioma cells. Mechanically, siLINC00511 promoted E-cadherin expression but suppressed N-cadherin and Snail expressions. MiR-15a-5p bound to LINC00511, and miR-15a-5p inhibitor partially reversed the effect and regulation of siLINC00511 on glioma cells. AEBP1, a target gene of miR-15a-5p, could activate p65 phosphorylation to promote EMT protein expression and partially reverse the inhibitory effect of miR-15a-5p mimic on the malignant phenotype of glioma cells. SiLINC00511 inhibited tumor growth, down-regulated miR-15a-5p expression and up-regulated AEBP1 and Ki67 expressions in vivo. Conclusion LINC00511 knockdown inhibits glioma cell progression via miR-15a-5p/AEBP1 axis.

Long noncoding RNAs (lncRNAs) are emerging as crucial contributors to the development of hepatocellular carcinoma (HCC) and are involved in the stemness regulation of liver cancer stem cells (LCSCs). However, cancer cell stemness-associated lncRNAs and their relevance in prediction of clinical prognosis remain largely unexplored. In this study, through the transcriptome-wide screen, we identified a total of 136 LCSC-associated lncRNAs. We evaluated the prognostic value of these lncRNAs and optimally established an 11-lncRNA (including AC008622.2, AC015908.3, AC020915.2, AC025176.1, AC026356.2, AC099850.3, CYTOR, DDX11-AS1, HTR2A-AS1, LINC02870, and SNHG3) prognostic risk model. Multivariate analysis revealed that the risk score is an independent prognostic predictor for HCC patients, which outperforms the traditional clinical pathological factors. Gene set enrichment analysis suggested that the high-risk score reflects the alteration of pathways involved in cell cycle, oxidative phosphorylation, and metabolism. Furthermore, functional studies on SNHG12, the leading candidate of the risk lncRNAs, revealed that knockdown of SNHG12 reduces the abilities of HCC cells stemness, proliferation, migration, and invasion. In summary, we constructed a prognostic risk model based on 11 LCSC-associated lncRNAs, which might be a promising prognostic predictor for HCC patients and highlight the involvement of lncRNAs in LCSC-associated treatment strategy in clinical practice.

The deficiency of dead cell clearance is a prominent pathogenic factor in systemic lupus erythematosus (SLE). In this study, the overexpression of miR-210-5p resulted in the accumulation of secondary necrotic cells (SNECs) in macrophages through the reduction of protein degradation. The upreguation of miR-210-5p inhibited NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and SNEC clearance. miR-210-5p overexpression suppressed Sp1 and HSCARG expression, and the knockdown of SP1 and HSCARG inhibited NOX expression and superoxide production in macrophages. Furthermore, patients with active SLE expressed a higher level of miR-210-5p and lower expression of SP1 and HSCARG in peripheral blood mononuclear cells. In summary, our findings indicate that the upregulation of miR-210-5p increases the accumulation of SNECs through a decrease in the Sp1-and HSCARG-mediated NOX activity and ROS generation in macrophages. Our results also suggest that targeting miR-210-5p may have therapeutic potential for SLE.

Background Dysfunction in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) leads to bone loss/osteoporosis. The catenin beta interacting protein 1 (CTNNBIP1) is an inhibitor of Wnt/β-catenin signaling, whose role in osteogenesis remains elusive. This study aimed to reveal the effects of miR-486–3p/CTNNBIP1 in osteogenesis. Methods Bone marrow samples from healthy individuals and osteoporosis patients and mice with sham or ovariectomy (OVX) surgeries were collected. Levels of CTNNBIP1 and miR-486–3p were assessed. A dual-luciferase reporter assay was used to confirm the interactions between CTNNBIP1 and miR-486–3p. MiR-486–3p mimics/inhibitor or CTNNBIP1 overexpression lentiviruses were transfected to human BMSCs (hBMSCs) and an osteogenic assay was performed. Alizarin red S (ARS) and Alkaline phosphatase (ALP) intensity and expression of osteogenic genes Runx2, Alp, Cola1 and Bglap were measured. Key proteins in the Wnt/β-catenin pathway including active β-catenin, Bcl-2, and Cyclin D1 were assessed. Results CTNNBIP1 was upregulated while miR-486–3p was downregulated in osteoporosis patients and OVX mice. CTNNBIP1 was confirmed as a target of miR-486–3p. MiR-486–3p overexpression promoted, while miR-486–3p knockdown suppressed, osteogenic differentiation and Wnt/β-catenin signaling. Rescue experiments confirmed the negative effects of CTNNBIP1 overexpression on osteoblastic differentiation and that miR-486–3p mimics could reverse canonical Wnt signaling. Conclusion This study demonstrated that miR-486–3p targets CTNNBIP1, thus activating the Wnt/β-catenin signaling pathway to promote osteogenesis of BMSCs.

Objective To identify targets and discover drugs for ovarian endometriosis (OE) Design A basic study based on a data-driven hypothesis and experimental validation Setting Center for Reproductive Medicine Patient(s)/Animal(s) Fourteen patients with OE and 7 healthy donors were recruited, and 15 female C57/BL6 mice were involved. Intervention(s) Samples of OE lesions and normal endometrium were obtained. The ITPR1-knockdowned ectopic human endometrial stromal cells (HESCs) were subjected to ribonucleic acid (RNA) sequencing, cell-counting kit-8 (CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EdU) staining, and flow cytometry. Camptothecin was administered to HESCs and in an OE mouse model. Main Outcome Measure(s) ITPR1 expression in OE lesions and normal endometrium, cell proliferation and apoptosis of HESCs with ITPR1 knockdown or camptothecin treatment, and autograft volume in the OE mouse model Result(s) Two significant OE-relevant gene modules were identified and involved the PI3K/Akt and aging-relevant pathways. Fifteen hub genes were identified and confirmed, among which the most significant gene, ITPR1, was robustly elevated in OE lesions. RNA sequencing revealed that ITPR1 was highly relevant to cell proliferation and apoptosis, which was further confirmed by CCK-8 assay, EdU staining, and flow cytometry analysis. ITPR1 knockdown inhibited cell proliferation and induced HESC apoptosis. The candidate drugs targeting these modules were screened, among which camptothecin and irinotecan were identified as promising drugs. Both compounds suppressed HESC proliferation and induced apoptosis; ITPR1 expression was suppressed by camptothecin. The therapeutic effect of camptothecin was also validated in the OE mouse model. Conclusion(s) This study identified the therapeutic targets and promising drugs for OE and shed light on the use of camptothecin in OE treatment.

This article was designed to explore the effects and mechanisms of miR-185-5p on the replication of hepatitis C virus (HCV). Quantitative reverse transcription PCR (qRT-PCR) was performed for detecting the abundance of miR-185-5p and HCV RNA in HCV-infected primary hepatocytes and Huh7.5 cells. Dual-luciferase reporter gene assay was used for exploring the interaction between miR-185-5p and GALNT8. Western blot analyzed protein expression of GALNT8, NS3, and NS5A. miR-185-5p was remarkably downregulated in HCV-infected primary hepatocytes and Huh7.5 cells. miR-185-5p upregulation inhibited HCV RNA expression, while its inhibition promoted HCV replication. miR-185-5p induced accumulation of NS3 and NS5A in the cells. Dual-luciferase reporter gene assay verified the targeted relationship between miR-185-5p and GALNT8. In addition, the effects of overexpressing or knocking down miR-185-5p on HCV replication could be correspondingly eliminated by the overexpression or knockdown of GALNT8. miR-185-5p may target GALNT8 in JFH1-infected Huh7.5 cells and then inhibit HCV replication. miR-185-5p may be a potential target for treating HCV.

Background and aims In clinical and experimental NASH, the origin of the scar-forming myofibroblast is the HSC. We used foz/foz mice on a Western diet to characterize in detail the phenotypic changes of HSCs in a NASH model. Approach and results We examined the single-cell expression profiles (scRNA sequencing) of HSCs purified from the normal livers of foz/foz mice on a chow diet, in NASH with fibrosis of foz/foz mice on a Western diet, and in livers during regression of NASH after switching back to a chow diet. Selected genes were analyzed using immunohistochemistry, quantitative real-time PCR, and short hairpin RNA knockdown in primary mouse HSCs. Our analysis of the normal liver identified two distinct clusters of quiescent HSCs that correspond to their acinar position of either pericentral vein or periportal vein. The NASH livers had four distinct HSC clusters, including one representing the classic fibrogenic myofibroblast. The three other HSC clusters consisted of a proliferating cluster, an intermediate activated cluster, and an immune and inflammatory cluster. The livers with NASH regression had one cluster of inactivated HSCs, which was similar to, but distinct from, the quiescent HSCs. Conclusions Analysis of single-cell RNA sequencing in combination with an interrogation of previous studies revealed an unanticipated heterogeneity of HSC phenotypes under normal and injured states.

circRNAs have been suggested to modulate NSCLC tumorigenesis and drug resistance. Whether circSNX6 affects NSCLC remains unclear. In this study, we aim to investigate the role of circSNX6 in drug resistance of NSCLC exposed to cisplatin. RT-qPCR method was used to investigate expression levels of circSNX6, miR-137 and CXCL12. MTT, cell colony formation and TUNEL assays were utilized to assess cell viability, proliferation, apoptosis, respectively. Xenograft assay was conducted to examine in vivo tumor growth. circSNX6 overexpression caused enhanced cell viability and proliferation of H1299 and Calu-1, while it inhibited apoptosis under cisplatin treatment. miR-137 inhibitor greatly rescued cell viability, proliferation and apoptosis of circSNX6 knockdown H1299 cells. miR-137 mimic increased ROS generation, as well as reduced GSH and SOD levels, whereas miR-137 inhibitor exerted opposing effect. circSNX6 knockdown also enhanced ROS generation, as well as decreased GSH and SOD levels. CXCL12 partially restored miR-137 mimic-modulated cell viability, proliferation and apoptosis. Herein, our group proposes circSNX6 as key regulator for drug resistance of NSCLC. The findings provide solid groundings for understanding of NSCLC pathogenesis and development of therapeutics.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a crucial regulator of neuronal development, neuronal survival, axonal regeneration, and synaptic plasticity. In this study we examined the potential role of PTEN in cognitive function in a mouse model of perioperative neurocognitive disorder (PND). Adult male C57BL/6J mice received intracerebroventricular injections of small interfering RNA (siRNA) against PTEN or control siRNA 3 days prior to exploratory laparotomy (n = 8 per group). A group of healthy mice not undergoing surgery included as additional control. Barnes maze and fear conditioning tests were conducted 7 days after surgery. Mice were then sacrificed to examine the expression of PTEN, AMP-activated protein kinase (AMPK), ionized calcium binding adaptor molecule (Iba)-1, B-cell lymphoma (Bcl)-2, Bcl2-associated X protein (Bax), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the hippocampus. The microglial activation was examined by immunohistochemistry using Iba-1 as a microglia maker. Nissl and terminal transferase deoxyuridine triphosphate nick-end labeling (TUNEL) staining were used to measure cell death and apoptosis. In comparison to the healthy controls, surgically treated mice had longer latency to identify the target box in both training and testing sessions in the Barnes maze test and shorter freezing time in the fear conditioning test. Surgically treated mice had increased expression of PTEN, AMPK, Bax, IL-1β, and TNF-α, as well as increasing number of activated microglia and apoptosis neurons in the hippocampus. PTEN knockdown significantly attenuated the behavioral deficits in Barnes maze and fear conditioning tests, as well as over-expression of PTEN, AMPK, Bax, IL-1β, and TNF-α induced by surgery. PTEN knockdown could attenuate cognitive deficits induced by trauma, likely through inhibiting the activation of microglia.

Fatty acid binding protein 5 (FABP5) has been reported to play an important role in various cancers. We found that high FABP5 expression was associated with poor histological differentiation and vascular invasion. High FABP5 expression indicated a poor prognosis. Downregulation of FABP5 suppressed cell proliferation, cell migration and invasion, and induced cell apoptosis. Bioinformatic analysis revealed that the Hippo signaling pathway was related to FABP5. We found that overexpression of yes-associated protein 1 (YAP1) could partially reverse the effect of FABP5 knockdown on growth and apoptosis. The FABP5 inhibitor SBFI-26 suppressed the proliferation and promoted the apoptosis of gastric cancer (GC) cells and interfered with the Hippo signaling pathway by inhibiting YAP1. Our data suggested that FABP5 might act as a potential target associated with the Hippo signaling pathway for GC treatment.

Long non-coding RNAs (lncRNAs) are essential drivers or suppressors in human hepatocellular carcinoma (HCC) by participating in controlling transcription, translation, mRNA stability, and protein degradation protein-protein interaction. TM4SF1-AS1 is recently identified as a tumor-promoting factor in lung cancer. Nevertheless, its function in HCC and related molecular mechanisms remain unknown. Here, our data indicated that either hypoxia or hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (DMOG) induced the upregulation of TM4SF1-AS1 in HCC cells. HIF-1α knockdown rather than HIF-2α silencing remarkably abrogated hypoxia-upregulated TM4SF1-AS1 expression. Furthermore, we confirmed the elevated expression of TM4SF1-AS1 in HCC tissue samples and cell lines. The silencing of TM4SF1-AS1 prominently inhibited the proliferative, migratory, and invasive abilities of HCC cells. TM4SF1-AS1 depletion significantly blocked hypoxia-enhanced Hep3B cell proliferation and mobility. Interfering TM4SF1-AS1 remarkably reduced TM4SF1 mRNA and protein levels in HCC cells. But TM4SF1-AS1 knockdown did not impact the stability of TM4SF1 mRNA. Hypoxia enhanced the expression of TM4SF1 mRNA, which was subsequently decreased by TM4SF1-AS1 knockdown in HCC cells. We confirmed the positive correlation between TM4SF1 mRNA and TM4SF1-AS1 expression in HCC specimens. Finally, TM4SF1 prominently reversed the inhibitory role of TM4SF1-AS1 depletion in Hep3B cells. In summary, hypoxia-responsive TM4SF1-AS1 was overexpressed in human HCC and contributed to the malignant behaviors of tumor cells by enhancing TM4SF1-AS1 expression.

Anti-angiogenesis serves as an effective tumor therapy approach. In a previous study, we found that β3-endonexin expressed in vascular endothelial cells was involved in promoting proliferation and angiogenesis partially by facilitating VEGF expression. However, it still remains unclear if β3-endonexin in vascular endothelial cells also employs other mechanisms in regulating angiogenesis. In this study, we utilized a β3-endonexin mutant (M2) carrying a defective nuclear localization sequence to disrupt its nuclear localization and evaluated its ability to promote HUVEC proliferation and formation of tube-like vascular structures. In addition, we performed yeast 2-hybrid assay to identify potential functional effectors of β3-endonexin. We found that both wild type β3-endonexin and the M2 mutant could localize to centrosomes in HUVECs and both were able to promote HUVEC proliferation and formation of vascular structures. However, the M2 mutant failed to promote VEGF expression in HUVECs. Further, we found that both wild type β3-endonexin and the M2 mutant were capable of binding to ninein, a centrosomal protein with a proangiogenic effect. Knockdown of ninein in HUVECs impeded centrosome localization of wild type β3-endonexin and the M2 mutant and inhibited HUVEC proliferation and formation of vascular structures. Taken together, these findings suggest that β3-endonexin interacts with centrosome ninein and contributes to HUVEC proliferation and formation of vascular structures.

Chronic myeloid leukemia (CML) is characterized by the formation of the BCR-ABL fusion gene. The BCR-ABL protein leads to an increased level of reactive oxygen species, which is a major cause of endogenous DNA double-strand breaks (DSBs). CML cells are prone to rely on a highly mutagenic alternative end-joining (Alt-EJ) pathway to cope with enhanced DSBs, which aggravates chromosomal instability. Hence, targeting dysregulated DNA repair proteins provides new insights into cancer treatment. In this study, we discovered the abnormal upregulation of Flap endonuclease 1 (FEN1) in CML, as well as FEN1's participation in the error-prone Alt-EJ repair pathway and its interplay with DNA Ligase1 and proliferating cell nuclear antigen in DSB repair. Knockdown of FEN1 by shRNA not only inhibited the proliferation and induced apoptosis but also enhanced the efficacy of imatinib (IM) in drug-resistant CML cell K562/G01. Moreover, excessive DSB accumulation was detected after FEN1 inhibition. In summary, our results demonstrated that FEN1 is a promising therapeutic target in CML treatment. This work extends the understanding of regulating abnormal DSB repair for cancer treatment.

Vascular calcification is very commonly observed in patients with chronic kidney disease (CKD), but there is no efficient therapy available. Oxidative stress plays critical roles in the progression of vascular calcification. Celastrol (Cel), a natural constituent derived from Chinese herbals, exhibits anti-oxidative stress activity. Here, we investigated the effect of celastrol on vascular calcification using vascular smooth muscle cells (VSMCs), arterial rings and CKD rats. Alizarin red staining and gene expression analysis showed that Cel dose-dependently inhibited rat VSMC calcification and osteogenic differentiation. Similarly, ex vivo study revealed that Cel inhibited calcification of rat and human arterial rings. In addition, micro-computed tomography, alizarin red staining and calcium content analysis confirmed that Cel inhibited aortic calcification in CKD rats. Interestingly, Cel treatment increased the mRNA and protein levels of heme oxygenase-1 (HMOX-1), and reduced the levels of reactive oxygen species (ROS) in VSMCs. Furthermore, both pharmacological inhibition of HMOX-1 and knockdown of HMOX-1 by siRNA independently counteracted the inhibitory effect of Cel on vascular calcification. Moreover, knockdown of HMOX-1 prevented Cel treatment-mediated reduction in ROS levels. Finally, Cel treatment reduced Vitamin D3-induced aortic calcification in mice and this effect was blocked by HMOX-1 inhibitor ZnPP9. Collectively, our results suggest that up-regulation of HMOX-1 is required for the inhibitory effect of Cel on vascular calcification. Modulation of HMOX-1 may provide a novel strategy for the treatment of vascular calcification in CKD.

Background The effects of PSENEN mutations in patients with acne inversa (AI) are poorly understood. Hyperproliferation of follicular keratinocytes and resulting occlusion may constitute the initial pathophysiology. Objective To investigate the effects of PSENEN knockdown on γ-secretase subunits, biological behaviors, and related signaling pathways in keratinocytes. Methods HaCaT cells were divided into an experimental group (PSENEN knock down), a negative control group, and a blank control group. Whole transcriptome sequencing was used to measure differences in mRNA expression of the whole genome; real-time PCR and Western blotting were performed to determine the interference efficiency and the effects of interference on the components of γ-secretase and related molecules. CCK-8 was used to measure cell proliferation, and flow cytometry was used to measure apoptosis and the cell cycle. Results A comparison of five healthy controls with three patients with PSENEN mutation (c.66delG, c.279delC, c.229_230insCACC) revealed decreased expression of mRNA and protein in skin lesions of the experimental group. In this group, expression of the other components of γ-secretase presenilin C-terminal fragment decreased, expression of immature nicastrin increased, expression of mature nicastrin decreased, and expression of anterior pharynx defective-1 remained unchanged. KEGG analysis revealed that differentially expressed molecules were enriched in m-TOR signaling pathways. Subsequent verification confirmed that differences in PI3K-AKT-mTOR signaling pathway molecules, cell proliferation, apoptosis, cell cycle and the expression levels of Ki-67, KRT1, and IVL between the groups were not statistically significant. Conclusions PSENEN mutations alone may be insufficient to cause the development of AI, or they may only induce a mild phenotype of AI.