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Hepatocellular carcinoma (HCC) is a common cancer with high morbidity and mortality. As we all know, the alteration of DNA methylation has a crucial impact on the occurrence of HCC. However, the mechanism of the effect of DNA methylation in different regions on gene expression is still unclear. Here, by computing and analyzing the distribution of differential methylation in 12 different regions in HCC tissues and adjacent normal tissues, not only the hypermethylation of CpG islands and global hypomethylation were found, but also a stable distribution pattern of differential methylation in HCC was found. Then the correlations between DNA methylations in different regions and gene expressions were calculated, and the diversity of correlations in different regions was determined. The key genes of differential methylation and differential expression related to the survival of HCC patients were obtained by using Cox regression analysis, a four-gene prognostic risk scoring model was constructed, and the prognostic performance was well verified. The regions of the differentially methylated CpG sites corresponding to the four key genes were located and their influences on the expression were analyzed. The results indicate that the promoter, first exon, 5'UTR, sixth exon, N_Shore, and S_Shore hypomethylation promotes the expression of key oncogenes, which together lead to the occurrence of HCC. These results might help to study the role of DNA methylation in HCC and provide potential biomarkers for the diagnosis of HCC.

Background Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs) present unique molecular signatures, but the tumorigenesis of EBVaGCs and the role EBV plays during this process remain poorly understood. Methods We applied whole-exome sequencing, EBV genome sequencing, and whole-genome bisulfite sequencing to multiple samples (n = 123) derived from the same patients (n = 25), which covered saliva samples and different histological stages from morphologically normal epithelial tissues to dysplasia and EBVaGCs. We compared the genomic landscape between EBVaGCs and their precursor lesions and traced the clonal evolution for each patient. We also analyzed genome sequences of EBV from samples of different histological types. Finally, the key molecular events promoting the tumor evolution were demonstrated by MTT, IC50, and colony formation assay in vitro experiments and in vivo xenograft experiments. Results Our analysis revealed increasing mutational burden and EBV load from normal tissues and low-grade dysplasia (LD) to high-grade dysplasia (HD) and EBVaGCs, and oncogenic amplifications occurred late in EBVaGCs. Interestingly, within each patient, EBVaGCs and HDs were monoclonal and harbored single-strain-originated EBV, but saliva or normal tissues/LDs had different EBV strains from that in EBVaGCs. Compared with precursor lesions, tumor cells showed incremental methylation in promotor regions, whereas EBV presented consistent hypermethylation. Dominant alterations targeting the PI3K-Akt and Wnt pathways were found in EBV-infected cells. The combinational inhibition of these two pathways in EBV-positive tumor cells confirmed their synergistic function. Conclusions We portrayed the (epi) genomic evolution process of EBVaGCs, revealed the extensive genomic diversity of EBV between tumors and normal tissue sites, and demonstrated the synergistic activation of the PI3K and Wnt pathways in EBVaGCs, offering a new potential treatment strategy for this disease.

We previously reported that cyclin D1 silencing interferes with RAD51 accumulation and increases the sensitivity of BRCA1 wild-type ovarian cancer cells to olaparib. However, the mechanisms associated with cyclin D1 overexpression in ovarian cancer are not fully understood. TargetScan predicted the potential binding sites for microRNA-20b (miR-20b) and the 3′-untranslated region of cyclin D1 mRNA; thus, we used luciferase reporter assay to verify those binding sites. The Kaplan-Meier method and log-rank test were used to examine the relationship between miR-20b and progression-free survival of ovarian cancer patients in The Cancer Genome Atlas ( n = 367) dataset. In vitro experiments were performed to evaluate the effects of miR-20b on cyclin D1 expression, cell cycle and response to olaparib. A peritoneal cavity metastasis model of ovarian cancer was established to determine the effect of miR-20b on the sensitivity of olaparib. Immunohistochemistry was performed to evaluate molecular mechanisms. In this work, we demonstrated that miR-20b down-regulates cyclin D1, increases the sensitivity of ovarian cancer cells to olaparib, reduces the expression of RAD51, and induces cell cycle arrest in G0/G1 phase. Ovarian cancer patients with higher expression of miR-20b had significantly longer progression-free survival. These results indicate that miR-20b may be a potential clinical indicator for the sensitivity of ovarian cancer to olaparib and the survival of ovarian cancer patients. Our findings suggest that miR-20b may have therapeutic value in combination with olaparib treatment for ovarian cancer.

Recent years have seen the emergence of conditionally activated diagnostics and therapeutics that leverage protease-cleavable peptide linkers to enhance their specificity for cancer. However, due to a lack of methods to measure and localize protease activity directly within the tissue microenvironment, the design of protease-activated agents has been necessarily empirical, yielding suboptimal results when translated to patients. To address the need for spatially resolved protease activity profiling in cancer, we developed a new class of in situ probes that can be applied to fresh-frozen tissue sections in a manner analogous to immunofluorescence staining. These activatable zymography probes (AZP) detected dysregulated protease activity in human prostate cancer biopsy samples, enabling disease classification. AZPs were leveraged within a generalizable framework to design conditional cancer diagnostics and therapeutics and showcased in the Hi-Myc mouse model of prostate cancer, which models features of early pathogenesis. Multiplexed screening against barcoded substrates yielded a peptide, S16, that was robustly and specifically cleaved by tumor-associated metalloproteinases in the Hi-Myc model. In situ labeling with an AZP incorporating S16 revealed a potential role of metalloproteinase dysregulation in proliferative, premalignant Hi-Myc prostatic glands. Systemic administration of an in vivo imaging probe incorporating S16 perfectly classified diseased and healthy prostates, supporting the relevance of ex vivo activity assays to in vivo translation. We envision AZPs will enable new insights into the biology of protease dysregulation in cancer and accelerate the development of conditional diagnostics and therapeutics for multiple cancer types. Significance: Visualization of protease activity within the native tissue context using AZPs provides new biological insights into protease dysregulation in cancer and guides the design of conditional diagnostics and therapeutics.

As RNA-binding proteins, cytoplasmic polyadenylation element binding proteins (CPEBs) have drawn increasing attention for their function of controlling gene expression related to malignant transformation via post-transcriptional regulation. However, the contribution of CPEB3 to malignant development in cancers is poorly understood. In this study, we explored the clinical, biological, and mechanical role of CPEB3 in colorectal cancer progression. We showed that colorectal cancer tissues exhibited dampened CPEB3 expression which was closely associated with poor prognosis in patients with colorectal cancer (47 vs. 62 months, P = 0.035, n=99). Down-regulation CPEB3 promoted proliferation, migration, and invasion in colorectal cancer cells and vice versa. Mechanistically, CPEB3 performed as an RNA binding protein binding to 3ʹUTR of JAK1 mRNA to inhibit JAK/STAT pathways in colorectal cancer cells. Knockdown of CPEB3 induced active JAK-STAT signaling, thereby triggering the proliferation and metastasis capacity of colorectal cancer cells. These results suggest that CPEB3 functions as a tumor suppressor in colorectal cancer through its post-transcriptional regulation of JAK/STAT signaling. Implications: This study identified a novel role of the RNA binding protein CPEB3 in inhibiting cell proliferation and migration as well as the underlining mechanisms in colorectal cancer cells.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy with a complex tumor ecosystem. How the interplay between tumor cells, EBV, and the microenvironment contributes to NPC progression and immune evasion remains unclear. Here we performed single-cell RNA sequencing on ~104,000 cells from 19 EBV(+) NPCs and 7 nonmalignant nasopharyngeal biopsies, simultaneously profiling the transcriptomes of malignant cells, EBV, stromal and immune cells. Overall, we identified global upregulation of interferon responses in the multicellular ecosystem of NPC. Notably, an epithelial–immune dual feature of malignant cells was discovered and associated with poor prognosis. Functional experiments revealed that tumor cells with this dual feature exhibited a higher capacity for tumorigenesis. Further characterization of the cellular components of the tumor microenvironment (TME) and their interactions with tumor cells revealed that the dual feature of tumor cells was positively correlated with the expression of co-inhibitory receptors on CD8(+) tumor-infiltrating T cells. In addition, tumor cells with the dual feature were found to repress IFN-γ production by T cells, demonstrating their capacity for immune suppression. Our results provide new insights into the multicellular ecosystem of NPC and offer important clinical implications.

Liver resection for hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) offers a chance of cure, although survival is often limited. The actual 3-year survival and its associated prognostic factors have not been reported. A nationwide database of HCC patients with PVTT who underwent liver resection with ‘curative’ intent was analyzed. The clinicopathologic characteristics, the perioperative, and survival outcomes for the actual long-term survivors were compared with the non-long-term survivors (patients who died within 3 years of surgery). Univariable and multivariable regression analyses were performed to identify predictive factors associated with long-term survival outcomes. The study included 1590 patients with an actuarial 3-year survival of 16.6%, while the actual 3-year survival rate was 11.7%. There were 171 patients who survived for at least 3 years after surgery and 1290 who died within 3 years of surgery. Multivariable regression analysis revealed that total bilirubin > 17.1 μmol/l, AFP > 400 ng/ml, types of hepatectomy, extent of PVTT, intraoperative blood loss > 400 ml, tumor diameter > 5 cm, tumor encapsulation, R0 resection, liver cirrhosis, adjuvant TACE, postoperative early recurrence (

BackgroundUneven distribution of health resources is higher in Tibet than in other regions. The development of core professional capability for general practitioners (GPs) is the main goal of continuing medical education (CME) training programs.AimThis study aimed to explore the needs of CME for GPs and provide advice for the development of policy, practice, and CME curriculums.MethodsWe conducted a cross-sectional online survey among GPs in Tibet Autonomous Region, China. We designed an online questionnaire including the demographic section, training contents, and training formats about CME.ResultsA total of 108 questionnaires were included in this study. Notably, 79 (73.15%) were women and 56 participants (51.85%) were working in primary care settings. We developed a curriculum priority: first-choice, major alternatives, and secondary considerations. The topics identified as first-choice for CME were related to “cardiovascular disease” (85.19%), “respiratory disease” (81.48%), and “digestive disease” (80.56%). Major alternatives included two essential knowledge and eight clinical skill items. We rated 10 items as secondary considerations. Only 39.81% ranked mental health as an essential priority; bedside teaching (51.85%) was the first choice.ConclusionWe presented priority areas identified in this study to focus on CME for GPs in Tibet. The 23 topics may reflect the features of general practice, which increasingly require common disease management skills, while a demand-oriented curriculum and staged training plans should be adopted. CME programs should be adapted dynamically to respond to evolving needs.

Using the China Health and Retirement Longitudinal Study, this paper studies the impact of abnormal hot temperature on residents' demand for commercial health insurance. The results show that for every 1°F rise in abnormal temperature, the probability of people buying commercial health insurance increased by 6%. Furthermore, the abnormal hot temperature has a more significant impact on the commercial health insurance demand of women, residents in the South and residents in the East. Channel analysis shows that abnormal hot temperature affects the demand for commercial health insurance through two channels: increasing residents' concern about climate risk and affecting health. This paper provides evidence for actively promoting sustainable development and improving the construction of medical security system.

Cisplatin-based chemotherapy remains the primary treatment for unresectable and metastatic muscle-invasive bladder cancers (MIBCs). However, tumors frequently develop chemoresistance. Here, we established a primary and orthotopic MIBC mouse model with gene-edited organoids to recapitulate the full course of chemotherapy in patients. We found that partial squamous differentiation, called semi-squamatization, is associated with acquired chemoresistance in both mice and human MIBCs. Multi-omics analyses showed that cathepsin H (CTSH) is correlated with chemoresistance and semi-squamatization. Cathepsin inhibition by E64 treatment induces full squamous differentiation and pyroptosis, and thus specifically restrains chemoresistant MIBCs. Mechanistically, E64 treatment activates the tumor necrosis factor pathway, which is required for the terminal differentiation and pyroptosis of chemoresistant MIBC cells. Our study revealed that semi-squamatization is a type of lineage plasticity associated with chemoresistance, suggesting that differentiation via targeting of CTSH is a potential therapeutic strategy for the treatment of chemoresistant MIBCs.

Distant metastasis remains the major cause for treatment failure in patients with nasopharyngeal carcinoma (NPC). Thus, it is necessary to investigate the underlying regulation mechanisms and potential biomarkers for NPC metastasis. Nogo-B (neurite outgrowth inhibitor B), encoded by reticulon-4, has been shown to be associated with the progression and advanced stage of several cancer types. However, the relationship between Nogo-B and NPC remains unknown. In this study, we found that higher expression of Nogo-B was detected in NPC cells and tissues. Higher expression of Nogo-B was statistically relevant to N stage, M stage, and poor prognosis in NPC patients. Further functional investigations indicated that Nogo-B overexpression could increase the migration, invasion, and metastasis ability of NPC cells in vitro and in vivo. Mechanistically, Nogo-B promoted epithelial-mesenchymal transition (EMT) and enhanced the invasive potency by interacting directly with its receptor NgR3 in NPC. Additionally, overexpression of Nogo-B could upregulate the protein levels of p-RhoA, SRF, and MRTFA. A positive relationship was found between the expression of Nogo-B and the p-RhoA in NPC patients as well as in mouse lung xenografts. Nogo-Bhigh p-RhoAhigh expression was significantly associated with N stage, M stage, and poor prognosis in NPC patients. Notably, CCG-1423, an inhibitor of the RhoA-SRF-MRTFA pathway, could reverse the invasive potency of Nogo-B and NgR3 in NPC cell lines, and decrease the expression of N-Cadherin, indicating that CCG-1423 may be a potential target drug of NPC. Taken together, our findings reveal that Nogo-B enhances the migration and invasion potency of NPC cells via EMT by binding to its receptor NgR3 to regulate the RhoA-SRF-MRTFA pathway. These findings could provide a novel insight into understanding the metastasis mechanism and targeted therapy of advanced NPC.

Delphinidin is a type of anthocyanin monomer with antioxidant, anti-inflammatory, and anti-tumor effects. However, the biological mechanisms underlying its anti-breast cancer activity have not been thoroughly studied. We further studied the effect of delphinidin on breast cancer cells through comprehensive network pharmacology, cellular and molecular experiments. We acquired the know therapeutic targets of delphinidin and obtained differentially expressed genes (DEGs) of breast cancer using RTCGA. We used topological analysis to screen out the 106 core targets of delphinium anti-breast cancer and performed functional analysis. These genes were mainly enriched in the pathways in cancer, Progesterone-mediated oocyte maturation and cell cycle. Then, by taking the intersection of the three analyzed data sets, important core targets (EGFR, TOP2A and PTGS2) were obtained and molecular-docking was performed to validate the result. Additionally, In Vitro experiments, MCF-7 and BT-474 cell proliferation was inhibited in a dose-dependent manner by delphinidin and the expressions of EGFR, TOP2A and PTGS were reduced. Moreover, delphinidin influenced cell cycle, the expressions of cdk1 and cyclin B1 were reduced. Furthermore, delphinidin induced apoptosis by activating the MAPK-Signaling pathway. Collectively, our findings suggested that delphinidin may offer effective approaches in breast cancer prevention and therapy.Supplemental data for this article is available online at http://dx.doi.org/10.1080/01635581.2021.2012582.

Localized scleroderma (LSc) is a disease characterized by the excessive deposition of collagen and thereby thickening of the dermis. In recent years, studies reported that LSc demonstrated compromised skin barrier related to the progression of the disease. However, human studies examining epidermis in scleroderma are still sparse and lack systematic research. This study aims to investigate the structural and functional changes in the LSc epidermis and further explore the underlying mechanisms, providing a new angle to treat LSc in the clinic.A total of 136 skin sites, including lesion and non-lesion control, from 27 LSc patients were analyzed. Ultrasonic testing, trans-epidermal water loss (TEWL), and epidermal hydration were assessed to investigate the structural and functional alternations; correlations between these parameters were analyzed. To explore the underlying mechanism, skin-fibrosis mouse model and cellular model by bleomycin (BLM) were deployed.The epidermal thickness was markedly increased, with a significant decline of hydration (dryness) in the LSc lesion skin. Epidermal hydration presented a negative correlation with the thickness. TEWL was not altered. The mouse model validated these morphological changes in the epidermis and indicated that interleukin-6 (IL-6) was significantly elevated. Furthermore, cellular study demonstrated that increased phosphorylation of p38 in keratinocyte promoted the secretion of IL-6, stimulating cell proliferation.This study characterized the epidermal alterations in LSc patients, suggesting that keratinocyte-derived abnormal IL-6 secretion can lead to the thickening of the epidermis, promoting dryness. The topical application of moisturizer may largely relieve dryness and related pruritus, thus improve the quality of life in LSc patients. Key Points • Epidermal thickness was increased in LSc lesion skin with declined hydration level. • Skin fibrosis mouse model validated the epidermal alteration in LSc patient. • p38-dependent IL-6 overexpression in keratinocyte result in epidermal thickening.

Microvascular invasion (MVI) predicts poor prognosis in patients with hepatocellular carcinoma (HCC). HCC patients with hypercoagulability are prone to develop thrombosis; however, the relationship between preoperative coagulability state, as reflected by the international normalized ratio (INR) level, and MVI remains unclear. From January 2009 to December 2012, HCC patients who underwent R0 liver resection (LR) from four cancer centers entered into this study. The overall survival (OS) and recurrence-free survival (RFS) rates were compared using the Kaplan–Meier method and Cox regression analysis. Of the 2509 HCC patients who were included into this study, 1104 were found to have MVI in the resected specimens. These patients were divided into the low (n = 151), normal (n = 796), and high (n = 157) INR subgroups based on the preoperative INR levels. The low INR subgroup had a significantly higher incidence of MVI than the normal or high INR subgroups (61.6% vs. 41.6% vs. 44.6%; p

BackgroundA major current challenge is to exploit tertiary lymphoid structures (TLSs) to promote the lymphocyte infiltration, activation and differentiation by tumor antigens to increase antitumor immune responses. The mechanisms that underlie the role of TLS formation in the adaptive immune responses against nasopharyngeal carcinoma (NPC) remain largely unknown.MethodsCell populations and the corresponding markers were identified by single-cell RNA sequencing and fluorescence-activated cell sorting analysis. In vitro differentiation experiments were used to simulate the generation, regulation and function of the Th-CXCL13 cell subset in the tumor microenvironment of NPC. These were followed by histological evaluation of the colocalization of tumor-associated B cells (TABs) and Th-CXCL13 cells within TLSs, and statistical analysis of the relationship between the cells in TLSs and overall survival.ResultsA PD-1+CXCR5−CD4+ Th-CXCL13 cell subset was identified in NPC. This subset was a major source of CXCL13, representing the majority of the CD4+ T cells at levels comparable with Th1 and Tfh cells present in the TLSs. Monocytes activated by toll-like receptor 4 agonists served as the antigen-presenting cells that most efficiently triggered the expansion of Th-CXCL13 cells. Transforming growth factor beta 1 (TGF-β1) stimulation and activation of Sox4 were critical for the induction and polarization of Th-CXCL13 cells in this process. The potential functional contributions of TABs recruited by Th-CXCL13 cells which induced plasma cell differentiation and immunoglobulin production via interleukin-21 and CD84 interactions in the TLSs demonstrated improved survival.ConclusionsInduction of Th-CXCL13 cells links innate inflammation to immune privilege in tumor-associated TLSs and might predict better survival.

N 6‐methyladenosine (m6A) modification of mRNA mediates diverse cellular and viral functions. Infection with Epstein–Barr virus (EBV) is causally associated with nasopharyngeal carcinoma (NPC), 10% of gastric carcinoma, and various B‐cell lymphomas, in which the viral latent and lytic phases both play vital roles. Here, we show that EBV transcripts exhibit differential m6A modification in human NPC biopsies, patient‐derived xenograft tissues, and cells at different EBV infection stages. m6A‐modified EBV transcripts are recognized and destabilized by the YTHDF1 protein, which leads to the m6A‐dependent suppression of EBV infection and replication. Mechanistically, YTHDF1 hastens viral RNA decapping and mediates RNA decay by recruiting RNA degradation complexes, including ZAP, DDX17, and DCP2, thereby post‐transcriptionally downregulating the expression of EBV genes. Taken together, our results reveal the critical roles of m6A modifications and their reader YTHDF1 in EBV replication. These findings contribute novel targets for the treatment of EBV‐associated cancers., The m6A reader YTHDF1 promotes viral RNA degradation by recruiting RNA decay complexes, including ZAP, DDX17, and DCP2. YTHDF1 suppresses EBV lytic replication to regulate EBV infection status.

Interferon-stimulated gene 15 (ISG15) is known to be involved in tumor progression. We previously reported that ISG15 expressed on nasopharyngeal carcinoma (NPC) cells and related to poor prognosis of patients with NPC. We further observed that ISG15 can be secreted by NPC cell and expressed on the macrophages in situ. However, the role of ISG15 in tumor-associated macrophages (TAMs) remains poorly understood. In the present study, we found that ISG15 treatment induces macrophages with M2-like phenotype, and the enhancement of NPC cell migration and tumorigenicity. Mechanically, ISG15-induced M2-like phenotype is dependent on the interaction with its receptor, LFA-1, and engagement of SRC family kinase (SFK) signal, and the subsequent secretion of CCL18. Blocking LFA-1, or SRC signal with small molecular inhibitors, or neutralizing with anti-CCL18 antibody can impede the activation of LFA-1-SFK-CCL18 axis in ISG15-treated macrophages. Clinically, ISG15+CD163+TAMs related to impaired survival of patients and advanced tumor stage of NPC. Furthermore, we found ISG15+CD163+macrophages inhibited antitumor CD8+cells responses in NPC. Together, our findings suggested tumor cell-secreted ISG15, which acted as a tumor microenvironmental factor, induces M2-like phenotype, promoting tumor progression and suppression of cytotoxic T lymphocyte response.

Rasal2 has diametric effects on progression of oestrogen receptor-positive (ER+) and -negative (ER-) breast cancers. The relevant causes are unknown. It is also unknown whether the effects of Rasal2 are mediated by an exosome-transport process.Exosomes were purified from breast cancer cells and identified by transmission electron microscopy and flow cytometry analysis. In vivo and in vitro experiments were conducted to investigate the role of Rasal2 in exosome-mediated breast cancer progression. Western blot analysis was performed to detect Rasal2 and p-Rasal2 (phosphorylated Rasal2) expression in ER+/ER- breast cancer cells and in exosomes, cancer tissues and blood of patients with ER+ or ER- breast cancer.Phosphorylation of Rasal2 at Serine 237 promoted tumour growth in both ER+ and ER- tumour cells and tissues. The functions of both p-Rasal2 and non-p-Rasal2 (non-phosphorylated-Rasal2) in the modulation of breast cancer progression are exosome-mediated. p-Rasal2 expression in ER+ breast cancer cells and exosomes, cancer tissues and blood was significantly lower than in ER- tumour cells and patients.p-Rasal2 facilitates tumour progression in both ER+ and ER- breast cancers. The ratio of p-Rasal2/non-p-Rasal2 in ER+ and ER- breast cancers is one of the factors deciding the role of Rasal2 (or total Rasal2) as a suppressor in ER+ breast cancers or as a promoter in ER- breast cancers. Targeting the phosphorylation of Rasal2 machinery may therefore be useful as a therapy to restrain breast cancer progression by reducing p-Rasal2/non-p-Rasal2 ratio, especially in ER- breast cancers. FUND: NSFC and Hong Kong Research Grants Council.

In order to explore the pollution characteristics and health risks of heavy metals in dust from roads around non-ferrous smelting activities in different regions, dust samples from urban roads, non-ferrous smelting industry park roads, and tunnel roads were collected from the Mengzi area of Yunnan Province. The dust samples were suspended on Teflon filters by re-suspension equipment to obtain PM

Metabolic reprogramming plays important roles in development and progression of nasopharyngeal carcinoma (NPC), but the underlying mechanism has not been completely defined. In this work, we found INSL5 was elevated in NPC tumor tissue and the plasma of NPC patients. Plasma INSL5 could serve as a novel diagnostic marker for NPC, especially for serum VCA‐IgA‐negative patients. Moreover, higher plasma INSL5 level was associated with poor disease outcome. Functionally, INSL5 overexpression increased, whereas knockdown of its receptor GPCR142 or inhibition of INSL5 reduced cell proliferation, colony formation, and cell invasion in vitro and tumorigenicity in vivo. Mechanistically, INSL5 enhanced phosphorylation and nuclear translocation of STAT5 and promoted glycolytic gene expression, leading to induced glycolysis in cancer cells. Pharmaceutical inhibition of glycolysis by 2‐DG or blockade of INSL5 by a neutralizing antibody reversed INSL5‐induced proliferation and invasion, indicating that INSL5 can be a potential therapeutic target in NPC. In conclusion, INSL5 enhances NPC progression by regulating cancer cell metabolic reprogramming and is a potential diagnostic and prognostic marker as well as a therapeutic target for NPC., This study reveals that INSL5 promotes tumor progression by regulating cancer cell metabolic reprogramming. INSL5 is a potential diagnostic and prognostic marker as well as a therapeutic target for nasopharyngeal carcinoma (NPC).

Microvascular invasion (MVI) is a risk factor for postoperative survival outcomes for hepatocellular carcinoma (HCC) after liver resection (LR). This study aims to investigate the actual long-term survival and its associated prognostic factors after LR for HCC patients with MVI.This study was conducted on HCC patients with MVI who underwent LR from January 2009 to December 2012 at five major hospitals in China. The patients were divided into the 'long-term survivor group' and the 'short-term survivor group'. The clinicopathologic characteristics, perioperative data and survival outcomes were compared between these two groups. Univariate and multivariate regression analyses were performed to identify predictive factors associated with long-term survival outcomes.The study included 1517 patients with an actual 5-year survival rate of 33.3%. Multivariate regression analysis revealed that HBV DNA 10One-third of HCC patients with MVI reached the long-term survival milestone of 5 years after resection. Anatomical hepatectomy, controlling intraoperative blood loss, a wide resection margin, and postoperative adjuvant TACE should be considered for patients to achieve better long-term survival outcomes.

Background Crosstalk between cancer cells and tumor-associated macrophages (TAMs) mediates tumor progression in colorectal cancer (CRC). Cytoplasmic polyadenylation element binding protein 3 (CPEB3) has been shown to exhibit tumor-suppressive role in CRC. Methods The expression of CPEB3, CD68, CD86 and CD163 was determined in CRC tissues. SW480 or HCT116 cells overexpressing CPEB3 and LoVo or RKO cells with CPEB3 knockdown were constructed. Stably transfected CRC cells were co-cultured with THP-1 macrophages to determine the malignant phenotype of CRC cells, macrophage polarization, and secretory signals. The inhibition of CPEB3 on tumor progression and M2-like TAM polarization was confirmed in nude mice. Results Decreased CPEB3 expression in CRC was associated with fewer CD86+ TAMs and more CD163+ TAMs. CPEB3 knockdown in CRC cells increased the number of CD163+ TAMs and the expression of IL1RA, IL-6, IL-4 and IL-10 in TAM supernatants. TAMs enhanced CRC cell proliferation and invasion via IL-6, and then activated the IL-6R/STAT3 pathway in CRC cells. However, CPEB3 reduced the IL-6R protein levels by directly binding to IL-6R mRNA, leading to decreased phosphorylated-STAT3 expression in CRC cells. CCL2 was significantly increased in CPEB3 knockdown cells, while CCL2 antibody treatment rescued the effect of CPEB3 knockdown in promoting CD163+ TAM polarization. Eventually, we confirmed that CPEB3 inhibits tumor progression and M2-like TAM polarization in vivo. Conclusions CPEB3 is involved in the crosstalk between CRC cells and TAMs by targeting IL-6R/STAT3 signaling.

CTCF is overexpressed in several cancers and plays crucial roles in regulating aggressiveness, but little is known about whether CTCF drives colorectal cancer progression. Here, we identified a tumor-promoting role for CTCF in colorectal cancer. Our study demonstrated that CTCF was upregulated in colorectal cancer specimens compared with adjacent noncancerous colorectal tissues. The overexpression of CTCF promoted colorectal cancer cell proliferation and tumor growth, while the opposite effects were observed in CTCF knockdown cells. Increased GLI1, Shh, PTCH1, and PTCH2 levels were observed in CTCF-overexpressing cells using western blot analyses. CCK-8 and apoptosis assays revealed that 5-fluorouracil chemosensitivity was negatively associated with CTCF expression. Furthermore, we identified that P53 is a direct transcriptional target gene of CTCF in colorectal cancer. Western blot and nuclear extract assays showed that inhibition of P53 can counteract Hedgehog signaling pathway repression induced by CTCF knockdown. In conclusion, we uncovered a crucial role for CTCF regulation that possibly involves the P53-Hedgehog axis and highlighted the clinical utility of colorectal cancer-specific potential therapeutic target as disease progression or clinical response biomarkers.

BACKGROUND Regular aerobic exercises could improve global cognition in older adults with mild cognitive impairment (MCI), such as aerobic dance a type of commonly practiced aerobic exercises. However, its effects remain debatable in improving the cognitive function in patients with MCI. OBJECTIVE The aim of this systematic review and meta-analysis is to evaluate the effects of aerobic dance on cognitive function among older adults with MCI. METHODS We searched articles in the MEDLINE, PubMed, Embase, and The Cochrane Library databases from inception to 28 February 2019, with the following criteria: 1) randomized controlled trials; 2) older adults with MCI; 3) aerobic dance intervention. RESULTS Five studies of 842 participants were identified. This meta-analysis showed that aerobic dance can significantly improve global cognition (Mini-Mental State Examination: MD = 1.43; 95% CI:[0.59, 2.27]; p = 0.0009; Alzheimer's Disease Assessment Scale-Cognitive Subscale: MD=-2.30; 95% CI:[-3.60, -1.00]; p = 0.0005), and delayed recall ability (SMD = 0.46;95% CI: [0.30, 0.62]; p

Background Transforming acidic coiled-coil protein 2 (TACC2) is a member of TACC family proteins which is mainly involved in the stabilization of spindles and regulation of microtubule dynamics through interactions with molecules involved in centrosomes/microtubules. TACC2 is involved in tumorigenesis of variety of cancers but the clinical significance of TACC2 protein in hepatocellular carcinoma (HCC) is still unclear. Objective This study aims to investigate the expression of TACC2 in HCC and determine if clinical significance and prognostic relevance exists. Methods We performed quantitative PCR (qPCR) and western blot to examine TACC2 mRNA and protein expression in paired HCC tissues and matched adjacent non-cancerous tissues. Immunohistochemistry was performed in 106 postoperative HCC samples. Results There was higher expression of TACC2 protein and mRNA in HCC tissue. Immunohistochemistry analysis showed high expression of TACC2 in HCC tissue and was significantly associated with the capsular extension, tumor recurrence and shortened overall and disease free survival. The Cox regression analysis suggested that a high expression of TACC2 was an independent prognostic factor for HCC patients. Conclusion This finding suggests that TACC2 may be a useful tool as a candidate biomarker to predict the recurrence and prognosis of HCC.

Purpose The purpose of this study was to identify novel plasma biomarkers for distinguishing nasopharyngeal carcinoma (NPC) patients from healthy individuals who have positive Epstein-Barr virus (EBV) viral capsid antigen (VCA-IgA). Materials and methods One hundred seventy-four plasma cytokines were analyzed by a Cytokine Array in eight healthy individuals with positive EBV VCA-IgA and eight patients with NPC. Real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry were employed to detect the expression levels of macrophage migration inhibitory factor (MIF) and CC chemokine ligand 3 (CCL3) in NPC cell lines and tumor tissues. Plasma MIF and CCL3 were measured by ELISA in 138 NPC patients, 127 EBV VCA-IgA negative (VN) and 100 EBV VCA-IgA positive healthy donors (VP). Plasma EBV VCA-IgA was determined by immunoenzymatic techniques. Results Thirty-four of the 174 cytokines varied significantly between the VP and NPC group. Plasma MIF and CCL3 were significantly elevated in NPC patients compared with VN and VP. Combination of MIF and CCL3 could be used for the differential diagnosis of NPC from VN cohort (area under the curve [AUC], 0.913; sensitivity, 90.00%; specificity, 80.30%), and combination of MIF, CCL3, and VCA-IgA could be used for the differential diagnosis of NPC from VP cohort (AUC, 0.920; sensitivity, 90.00%; specificity, 84.00%), from (VN+VP) cohort (AUC, 0.961; sensitivity, 90.00%; specificity, 92.00%). Overexpressions of MIF and CCL3 were observed in NPC plasma, NPC cell lines and NPC tissues. Conclusion Plasma MIF, CCL3, and VCA-IgA combination significantly improves the diagnostic specificity of NPC in high-risk individuals.

Adipocyte fatty acid‐binding protein (FABP4) is abundant in macrophage and adipocyte. It is known to be involved in lipid metabolism. The role of FABP4 has been reported in various cancers, such as non‐small cell lung cancer, breast cancer, ovarian cancer, and prostatic cancer. However, its role remains unclear in hepatocellular carcinoma (HCC). In our study, we investigated the expression of FABP4 at both mRNA and protein levels, and by examining 175 cases of patients with cancer of the liver tissue microarray, the significance between the expression of FABP4 and clinical characteristics had been discussed. We found that FABP4 was lowly expressed in HCC tissues compared to the corresponding tissue adjacent, and the expression of FABP4 was significantly associated with the tumor size, PVTT, recurrence‐free survival and overall survival. Moreover, multivariate Cox regression analysis indicated that the expression of FABP4, Alb, AFP, HBsAg, and PVTT were independent risk factors for overall survival, and the expression of FABP4, AFP, GGT, tumor size, and encapsulation were independent risk factors for HCC recurrence. In addition, we revealed that FABP4 suppressed HCC cell proliferation and invasion in vitro. Moreover, overexpression of FABP4 led to inhibit tumor growth and decreased tumor volume in vivo. These phenotypes were associated with altered expression of Snail and p‐STAT3. Our studies thus suggest that FABP4 could be a potential target for HCC chemotherapy.

Cleavage of transfer (t)RNA and ribosomal (r)RNA are critical and conserved steps of translational control for cells to overcome varied environmental stresses. However, enzymes that are responsible for this event have not been fully identified in high eukaryotes. Here, we report a mammalian tRNA/rRNA-targeting endoribonuclease: SLFN13, a member of the Schlafen family. Structural study reveals a unique pseudo-dimeric U-pillow-shaped architecture of the SLFN13 N′-domain that may clamp base-paired RNAs. SLFN13 is able to digest tRNAs and rRNAs in vitro, and the endonucleolytic cleavage dissevers 11 nucleotides from the 3′-terminus of tRNA at the acceptor stem. The cytoplasmically localised SLFN13 inhibits protein synthesis in 293T cells. Moreover, SLFN13 restricts HIV replication in a nucleolytic activity-dependent manner. According to these observations, we term SLFN13 RNase S13. Our study provides insights into the modulation of translational machinery in high eukaryotes, and sheds light on the functional mechanisms of the Schlafen family., Translation inhibition is a strategy for organisms to overcome various environmental stresses including viral infections. Here the authors show that a tRNA/rRNA-targeting RNase Schlafen13 inhibits protein synthesis by directly digesting cytoplasmic tRNA and rRNA with the ability to restrict viral propagation.

Background: Nasopharyngeal carcinomas (NPCs) are malignant tumors originating from the lining epithelium of the nasopharynx. Fusion genes have been confirmed to play important roles in the occurrence and development of various malignant tumors, but the role of fusion genes in NPC is poorly understood. We aimed to explore new fusion genes that promote the occurrence and development of NPC. Methods: RNA-seq was used to search for interchromosomal translocations in 18 NPC tissues. Polymerase chain reaction (PCR) and Sanger sequencing were applied to verify the presence of BCL6-SPECC1L (BS); quantitative PCR (qPCR) and Western blotting were used to measure the expression level of BCL-6 in NPC cells; MTT and in vivo tumorigenesis assays were applied to evaluate the cell proliferation ability; immunofluorescence assays were used to determine the cellular localization of BCL6 and BS; and a luciferase reporter assay was performed to evaluate the ability of BCL6 and BS to inhibit transcription. Results: BS was present in 5.34% (11/206) of primary NPC biopsies and 2.13% (1/47) of head and neck cancer biopsies. The expression of BCL6 was downregulated in NPC, and silencing of endogenous BCL6 promoted NPC cell proliferation in vitro. Overexpression of BCL6 but not BS inhibited the growth of NPC cells in vivo and in vitro. Mechanistically, BCL6 localized in the nucleus can inhibit the G1/S transition to suppress the growth of NPC cells. However, after the fusion of BCL6 and SPECC1L, the product cannot localize to the nucleus, and the transcriptional inhibitory function of BCL6 is abolished, eventually abolishing its tumor suppressor effect and leading to the development of NPC. Conclusion: BS is a novel fusion gene in NPC that may play an important role in the occurrence and development of this cancer. The clinical significance of the BS fusion gene needs further elucidation.

Lymph node metastasis (N classification) is one of the most important prognostic factors of nasopharyngeal carcinoma (NPC), and nerve involvement is associated with the transition of the N category in NPC patients. Although the nervous system has been reported to participate in many types of cancer progression, its functions in NPC progression remains unknown. Through analysis of gene profiling data, we demonstrate an enrichment of genes associated with neuronal development and differentiation in NPC tissues and cell lines. Among these genes, Nogo receptor 3 (NgR3), which was originally identified in the nervous system and plays a role in nerve development and regeneration, was inappropriately overexpressed in NPC cells and tissues. Immunohistochemical analysis demonstrated that the overexpression of NgR3 was correlated with poor prognosis in NPC patients. Overexpression of NgR3 promoted, and knocking down NgR3 inhibited, NPC cell migration and invasion in vitro and metastasis in vivo. The ability of NgR3 to promote cell migration was triggered by the downregulation of E-cadherin and enhanced cytoskeletal rearrangement and cell polarity, which were correlated with the activation of focal adhesion kinase (FAK). Collectively, NgR3 is a novel indicator of poor outcomes in NPC patients and plays an important role in driving the progression of NPC. These results suggest a potential link between the nervous system and NPC progression.Genes involved in the neuronal biological process are enriched in nasopharyngeal carcinoma. Overexpression of NgR3 correlates with poor prognosis of nasopharyngeal carcinoma. NgR3 promotes NPC cell migration by downregulating E-cadherin. NgR3 promotes NPC cell polarity and enhances the formation of NPC cell pseudopodia by activating FAK/Src pathway.

Epstein-Barr virus (EBV) is causally associated with nasopharyngeal carcinoma, 10% of gastric carcinoma and various B cell lymphomas 1 . EBV infects both B cells and epithelial cells 2 . Recently, we reported that epidermal growth factor and Neuropilin 1 markedly enhanced EBV entry into nasopharyngeal epithelial cells 3 . However, knowledge of how EBV infects epithelial cells remains incomplete. To understand the mechanisms through which EBV infects epithelial cells, we integrated microarray and RNA interference screen analyses and found that Ephrin receptor A2 (EphA2) is important for EBV entry into the epithelial cells. EphA2 short interfering RNA knockdown or CRISPR-Cas9 knockout markedly reduced EBV epithelial cell infection, which was mostly restored by EphA2 complementary DNA rescue. EphA2 overexpression increased epithelial cell EBV infection. Soluble EphA2 protein, antibodies against EphA2, soluble EphA2 ligand EphrinA1, or the EphA2 inhibitor 2,5-dimethylpyrrolyl benzoic acid efficiently blocked EBV epithelial cell infection. Mechanistically, EphA2 interacted with EBV entry proteins gH/gL and gB to facilitate EBV internalization and fusion. The EphA2 Ephrin-binding domain and fibronectin type III repeats domain were essential for EphA2-mediated EBV infection, while the intracellular domain was dispensable. This is distinct from Kaposi's sarcoma-associated herpesvirus infection through EphA2 4 . Taken together, our results identify EphA2 as a critical player for EBV epithelial cell entry.

Oral inhalation (OI) of nano-chemotherapeutics holds great potentials in the treatment of lung cancers as it enables direct targeting of drugs to lung tissues, spatial and temporal control of drug release, and decrease in drug-associated systemic and local lung toxicity. Therefore, the design of chemistry of the nanocarriers and their OI formulations for chemotherapeutics delivery to the peripheral lungs and extrapulmonary tissues of relevance such as lymph nodes, may thus afford new opportunities for treating such relevant diseases. In this work we investigated the effect of polyethylene glycol 1000Da (PEG1000) density and doxorubicin (DOX) payload on the interaction of poly(amidoamine) dendrimer (PAMAM) with an in vitro pulmonary epithelium model (Calu-3). DOX, which was conjugated to the PAMAM through a pH-labile bond, showed a strong time-dependent cell kill against Calu-3 cells due to sustained DOX release. The conjugation of DOX to PEGylated PAMAM dendrimers significantly enhances DOX transport across pulmonary epithelium compared to free drug, with the rate of transport increasing as PEGylation degree increases. Transient interaction of PEGylated dendrimers with cellular junctions of the polarized epithelium as probed by a reduction in transepithelial electrical resistance, faster mucus diffusion, along with reduced cellular internalization compared to the non-PEGylated counterpart promotes transport across the epithelial barrier. A cosolvent free method was developed to formulate PEGylated PAMAM-DOX conjugates in pressurized metered-dose inhalers. The resulting aerosol formulations show a very high final particle fractions (>82%). We further demonstrate that aerodynamic particle size distribution of the nanoconjugates can be tweaked with the addition of a biodegradable lactide-based copolymer, which may help tune lung deposition of PAMAM-DOX conjugates to a specific pulmonary area. The combined results suggest that conjugation to PAMAM dendrimers and their surface modification with PEG1000 can be utilized to modulate the transport of DOX across pulmonary epithelium, and also to easily formulate the conjugates in propellant-based inhalers for pulmonary administration of anticancer therapeutics.

AIM To identify whether chitinase 3-like 1 (CHI3L1) serves as a suitable biomarker for the prognosis of esophageal squamous cell carcinoma (ESCC) and to analyze this protein’s cellular source. METHODS An ELISA was conducted to detect the concentration of CHI3L1 in the serum of 150 ESCC patients diagnosed between January 2001 and February 2005. The prognostic relevance of CHI3L1 was evaluated by a Kaplan-Meier and Cox regression analysis. The immunohistochemistry was reanalyzed, and fluorescent staining was utilized to explore the cellular origins of CHI3L1. We stimulated monocyte-derived macrophages (MDMs) with either IL-6 or the supernatant of the ESCC cell line Eca-109 and later investigated the level of CHI3L1 by qPCR and ELISA. RESULTS The level of serum CHI3L1 was higher in older patients (≥ 60) than in patients under the age of 60 (P = 0.001). The patients with higher levels of CHI3L1 had a significantly shorter overall survival, whereas the traditional markers, carcinoembryonic antigen and squamous cell carcinoma antigen, were less effective (P > 0.05). A multivariate Cox analysis (P = 0.001) indicated that CHI3L1 was an independent prognostic factor for ESCC patients. Peritumoral macrophages in ESCC exhibited high levels of CHI3L1. Interleukin-6 (IL-6) and the supernatant of Eca-109 containing IL-6 stimulated MDMs to secrete CHI3L1. The serum concentration of CHI3L1 in the ESCC patients showed a weak correlation with the laboratory inflammatory parameters neutrophil (NEU, P = 0.045), neutrophil/lymphocyte rate (NLR, P = 0.016), and C-reactive protein (CRP, P < 0.001). CONCLUSION Our study first established a connection between the pretreated CHI3L1 and patients with ESCC, and the serum CHI3L1 was primarily secreted by ESCC-surrounded macrophages.

Pulmonary administration of polymer drug conjugates is of great potential clinical significance for treating lung cancer as such regimen significantly increases local drug concentrations while decreases systemic and local side effects. In this work, we demonstrate that nanoparticles prepared with methoxypoly(ethylene glycol) (mPEG)-doxorubicin (DOX) conjugates (mPEG-DOX) that have a pH-sensitive imine bond (Schiff base) can at the same time work as efficient carriers for DOX to kill cancer cells and also as a strategy to directly formulate nanoparticles in propellant-based inhalers. Nanoparticles prepared by precipitation in water had a diameter in the range between 100 and 120 nm. We investigated the effects of molecular weight (MW) of mPEG (1K, 2K, and 5K Da) on the in vitro release kinetics, cellular internalization, and cytotoxicity on in vitro model of lung adenocarcinoma and aerosol characters. It is observed that the DOX released from mPEG-DOX nanoparticles was significantly accelerated in acidic environment, pH 5.5 (endosomal/lysosomal pH) in comparison with pH 7.4 (physiological pH), as designed. Release of DOX from mPEG1K-DOX nanoparticles was significantly greater than those from mPEG2K and mPEG5K counterparts. In vitro cytotoxicity of nanoparticles followed the sequence of mPEG1K-DOXfree DOXmPEG2K-DOX ≫ mPEG5K-DOX, a trend closely following their rate and extent of cellular internalization. mPEG-DOX nanoparticles with mPEG1K and mPEG2K were directly dispersed in hydrofluoroalkane (HFA), while a trace of ethanol was required to disperse mPEG5K-DOX nanoparticles in HFA. These pMDI formulations with high physical stability in HFAs display superior aerosol characteristics conducive to deep lung deposition. The fine particle fractions of these formulations ranged from 40-60%, higher than those of commercial products. Such formulations prepared from nanoparticles of pH-sensitive PEG-drug conjugates may also be envisioned to be extended to formulate other hydrophobic drugs for local delivery with propellant-based inhalers to other pulmonary disorders, thus broadening the impact of the proposed strategy.

Enhancer-promoter interaction (EPI) is an important cis-regulatory mechanism in the regulation of tissue-specific gene expression. However, it still has limitation to precisely identity these interactions so far. In this paper, using diverse genomic features for various regulatory regions, we presented a computational approach to predict EPIs with improved accuracies. Meanwhile, we comprehensively studied more potential regulatory factors that are important to EPIs prediction, such as nucleosome occupancy, enhancer RNA; and found the cell line-specificity and region-specificity of the contributions of diverse regulatory signatures. By adding genomic signatures of segmented regulatory regions, our best accuracies of cross-validation test were about 11%-16% higher than the previous results, indicating the location-specificity of genomic signatures in a regulatory region for predicting EPIs. Additionally, more training samples and related features can provide reliable performances in new cell lines. Consequently, our study provided additional insights into the roles of diverse signature features for predicting long-range EPIs.

Tuberculous spondylitis of vertebral augmentation following percutaneous vertebroplasty or kyphoplasty is rare. We report an unusual case of tuberculous spondylitis diagnosed after percutaneous kyphoplasty (PKP). A 54-year-old woman presented to hospital complaining of back pain following a fall 20 days prior. Radiology showed an acute osteoporotic compression (L3 fracture). The patient denied a history of pulmonary tuberculosis and there were no signs of infection. The patient was discharged from hospital 2 days after undergoing L3 PKP with a dramatic improvement in her back pain. The patient was readmitted 10 months later with a history of recurrent back pain and low-grade fever for 3 months. Imaging examinations showed severe spondylitis at the L2–L3 level, with paravertebral abscess formation and bony destruction of L2 and L3. A positive result of the T-SPOT test preliminarily confirmed the diagnosis of tuberculous spondylitis. The tuberculosis test was positive, and serum C-reactive protein levels and erythrocyte sedimentation were relatively high. Treatment for tuberculous spondylitis was started. She underwent posterior fusion and instrumentation from T12–L5 after markers for infection returned to normal. After surgery, the patient continued antituberculous and anti-osteoporosis treatments. Her low back pain was relieved and low-grade fever and sweating disappeared.

Esophageal squamous dysplasia is believed to be the precursor lesion of esophageal squamous cell carcinoma (ESCC ); however, the genetic evolution from dysplasia to ESCC remains poorly understood. Here, we applied multi-region whole-exome sequencing to samples from two cohorts, 45 ESCC patients with matched dysplasia and carcinoma samples, and 13 tumor-free patients with only dysplasia samples. Our analysis reveals that dysplasia is heavily mutated and harbors most of the driver events reported in ESCC. Moreover, dysplasia is polyclonal, and remarkable heterogeneity is often observed between tumors and their neighboring dysplasia samples. Notably, copy number alterations are prevalent in dysplasia and persist during the ESCC progression, which is distinct from the development of esophageal adenocarcinoma. The sharp contrast in the prevalence of the ‘two-hit’ event on TP53 between the two cohorts suggests that the complete inactivation of TP53 is essential in promoting the development of ESCC., The pathogenesis of oesophageal squamous cell carcinoma is a multi-step process but the genetic determinants behind this progression are unknown. Here the authors use multi-region exome sequencing to comprehensively investigate the genetic evolution of precursor dysplastic lesions and untransformed oesophagus.

The regulation of genes utilizing the RNA interference (RNAi) mechanism via the delivery of synthetic siRNA has great potential in the treatment of a variety of lung diseases. However, the delivery of siRNA to the lungs is challenging due to the poor bioavailability of siRNA when delivered intraveneously, and difficulty in formulating and maintaining the activity of free siRNA when delivered directly to the lungs using inhalation devices. The use of non-viral vectors such as cationic dendrimers can help enhance the stability of siRNA and its delivery to the cell cytosol. Therefore, in this work, we investigate the ability of a triphenylphosphonium (TPP) modified generation 4 poly(amidoamine) (PAMAM) dendrimer (G4NH2-TPP) to enhance the in vitro transfection efficiency of siRNA in a model of the pulmonary epithelium and their aerosol formulations in pressurized metered dose inhalers (pMDIs) and dry powder inhalers (DPIs). Complexes of siRNA and G4NH2-TPP were prepared with varying TPP densities and increasing N/P ratios. The complexation efficiency was modulated by the presence of the TPP on the dendrimer surface, allowing for a looser complexation compared to unmodified dendrimer as determined by gel electrophoresis and polyanion competition assay. An increase in TPP density and N/P ratio led to an increase in the in vitro gene knockdown of stably green fluorescent protein (eGFP) expressing lung alveolar epithelial (A549) cells. G4NH2-12TPP dendriplexes (G4NH2 PAMAM dendrimers containing 12 TPP molecules on the surface complexed with siRNA) at N/P ratio 30 showed the highest in vitro gene knockdown efficiency. To assess the potential of TPP-dendriplexes for pulmonary use, we also developed micron particle technologies for both pMDIs and DPIs and determined their aerosol characteristics utilizing an Andersen Cascade Impactor (ACI). Mannitol microparticles encapsulating 12TPP-dendriplexes were shown to be effective in producing aerosols suitable for deep lung deposition for both pMDI formulations (fine particle fraction of 50-53%) and DPI formulations (fine particle fraction of 39%) with no impact on the in vitro gene knockdown efficiency of the siRNA. This work demonstrates the potential benefits of utilizing TPP-conjugated dendrimers in the formation of dendriplexes for siRNA delivery to the pulmonary epithelium and their aerosol formulation for local delivery to the lungs using portable inhalers.

Nasopharyngeal carcinoma (NPC) is characterized by a high rate of local invasion and early distant metastasis. Increasing evidence indicates that epigenetic abnormalities play important roles in NPC development. However, the epigenetic mechanisms underlying NPC metastasis remain unclear. Here we investigate aberrantly methylated transcription factors in NPC tissues, and we identify the HOP homeobox HOPX as the most significantly hypermethylated gene. Consistently, we find that HOXP expression is downregulated in NPC tissues and NPC cell lines. Restoring HOPX expression suppresses metastasis and enhances chemosensitivity of NPC cells. These effects are mediated by HOPX-mediated epigenetic silencing of SNAIL transcription through the enhancement of histone H3K9 deacetylation in the SNAIL promoter. Moreover, we find that patients with high methylation levels of HOPX exhibit poor clinical outcomes in both the training and validation cohorts. In summary, HOPX acts as a tumour suppressor via the epigenetic regulation of SNAIL transcription, which provides a novel prognostic biomarker for NPC metastasis and therapeutic target for NPC treatment., HOPX is a transcription factor epigenetically silenced in several cancers. Here the authors, by analysing methylation profiles, identify HOPX as a suppressor of metastasis in nasopharyngeal carcinoma: mechanistically HOPX inhibits SNAIL transcription through deacetylation-mediated silencing.

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes.

Although early detection and systemic therapies have improved the diagnosis and clinical cure rate of breast cancer, breast cancer remains the most frequently occurring malignant cancer in women due to a lack of sufficiently effective treatments. Thus, to develop potential targeted therapies and thus benefit more patients, it is helpful to understand how cancer cells work. ZIC family members have been shown to play important roles in neural development and carcinogenesis. In our study, we found that ZIC2 is downregulated in breast cancer tissues at both the mRNA and protein levels. Low expression of ZIC2 was correlated with poor outcome in breast cancer patients and serves as an independent prognostic marker. Furthermore, overexpression of ZIC2 repressed, whereas knockdown of ZIC2 promoted, cell proliferation and colony formation ability in vitro and tumor growth in vivo. Using ChIP-seq and RNA-seq analysis, we screened and identified STAT3 as a potential target for ZIC2. ZIC2 bound to the STAT3 promoter and repressed the promoter activities of STAT3. ZIC2 knockdown induced the expression of STAT3, increasing the level of phosphorylated STAT3. These results suggest that ZIC2 regulates the transcription of STAT3 by directly binding to the STAT3 promoter. Additionally, interfering STAT3 with siRNAs or inhibitors abrogated the oncogenic effects induced by decreased ZIC2. Taken together, our results indicate that ZIC2 serves as a useful prognostic marker in breast cancer and acts as a tumor suppressor by regulating STAT3, implying that STAT3 inhibitors might provide an alternative treatment option for breast cancer patients with ZIC2 downregulation.

Hepatic vein tumour thrombus (HVTT) is a major determinant of survival outcomes for patients with hepatocellular carcinoma (HCC). An Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT model was established to predict the prognosis of patients with HCC and HVTT after liver resection, in order to identify optimal candidates for liver resection.Patients with HCC and HVTT from 15 hospitals in China were included. The EHBH-HVTT model with contour plot was developed using a non-linear model in the training cohort, and subsequently validated in internal and external cohorts.Of 850 patients who met the inclusion criteria, there were 292 patients who had liver resection and 198 who did not in the training cohort, and 124 and 236 in the internal and external validation cohorts respectively. Contour plots for the EHBH-HVTT model were established to predict overall survival (OS) rates of patients visually, based on tumour diameter, number of tumours and portal vein tumour thrombus. This differentiated patients into low- and high-risk groups with distinct long-term prognoses in the liver resection cohort (median OS 34·7 versus 12·0 months; P 0·001), internal validation cohort (32·8 versus 10·4 months; P = 0·002) and external validation cohort (15·2 versus 6·5 months; P = 0·006). On subgroup analysis, the model showed the same efficacy in differentiating patients with HVTT in peripheral and major hepatic veins, the inferior vena cava, or in patients with coexisting portal vein tumour thrombus.The EHBH-HVTT model was accurate in predicting prognosis in patients with HCC and HVTT after liver resection. It identified optimal candidates for liver resection among patients with HCC and HVTT, including tumour thrombus in the inferior vena cava, or coexisting portal vein tumour thrombus.La trombosis tumoral de la vena hepática (hepatic vein tumour thrombus, HVTT) es un determinante importante de los resultados de supervivencia en pacientes con carcinoma hepatocelular (hepatocellular carcinoma, HCC). Se desarrolló el modelo llamado Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT para predecir el pronóstico de los pacientes con HCC y HVTT después de la resección hepática (liver resection, LR), con el fin de identificar los candidatos óptimos para LR entre estos pacientes. MÉTODOS: Se incluyeron pacientes con HCC y HVTT de 15 hospitales en China. El modelo EHBH-HVTT con gráfico de contorno se desarrolló utilizando un modelo no lineal en la cohorte de entrenamiento, siendo posteriormente validado en cohortes internas y externas.De 850 pacientes que cumplieron con los criterios de inclusión, hubo 292 pacientes en el grupo LR y 198 pacientes en el grupo no LR en la cohorte de entrenamiento, y 124 y 236 en las cohortes de validación interna y externa. Los gráficos de contorno del modelo EHBH-HVTT se establecieron para predecir visualmente las tasas de supervivencia global (overall survival, OS) de los pacientes, en función del diámetro del tumor, número de tumores y del trombo tumoral de la vena porta (portal vein tumour thrombus, PVTT). Esto diferenciaba a los pacientes en los grupos de alto y bajo riesgo, con distinto pronóstico a largo plazo en las 3 cohortes (34,7 versus 12,0 meses, 32,8 versus 10,4 meses y 15,2 versus 6,5 meses, P 0,001). En el análisis de subgrupos, el modelo mostró la misma eficacia en la diferenciación de pacientes con HVTT, con trombo tumoral en la vena cava inferior (inferior vena cava tumour thrombus, IVCTT) o en pacientes con PVTT coexistente. CONCLUSIÓN: El modelo EHBH-HVTT fue preciso para la predicción del pronóstico en pacientes con HCC y HVTT después de la LR. Identificó candidatos óptimos para LR en pacientes con HCC y HVTT, incluyendo IVCTT o PVTT coexistente.

Super-enhancers (SEs) are clusters of enhancers marked by extraordinarily high and broad chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) signals for H3K27ac or other transcription factors (TFs). SEs play pivotal roles in development and oncogenesis. Epstein-Barr virus (EBV) super-enhancers (ESEs) are co-occupied by all essential EBV oncogenes and EBV-activated NF-κB subunits. Perturbation of ESEs stops lymphoblastoid cell line (LCL) growth. To further characterize ESEs and identify proteins critical for ESE function, MYC ESEs were cloned upstream of a green fluorescent protein (GFP) reporter. Reporters driven by MYC ESEs 525 kb and 428 kb upstream of MYC (525ESE and 428ESE) had very high activities in LCLs but not in EBV-negative BJAB cells. EBNA2 activated MYC ESE-driven luciferase reporters. CRISPRi targeting 525ESE significantly decreased MYC expression. Genome-wide CRISPR screens identified factors essential for ESE activity. TBP-associated factor (TAF) family proteins, including TAF8, TAF11, and TAF3, were essential for the activity of the integrated 525ESE-driven reporter in LCLs. TAF8 and TAF11 knockout significantly decreased 525ESE activity and MYC transcription. MEF2C was also identified to be essential for 525ESE activity. Depletion of MEF2C decreased 525ESE reporter activity, MYC expression, and LCL growth. MEF2C cDNA resistant to CRIPSR cutting rescued MEF2C knockout and restored 525ESE reporter activity and MYC expression. MEF2C depletion decreased IRF4, EBNA2, and SPI1 binding to 525ESE in LCLs. MEF2C depletion also affected the expression of other ESE target genes, including the ETS1 and BCL2 genes. These data indicated that in addition to EBNA2, TAF family members and MEF2C are essential for ESE activity, MYC expression, and LCL growth. IMPORTANCE SEs play critical roles in cancer development. Since SEs assemble much bigger protein complexes on enhancers than typical enhancers (TEs), they are more sensitive than TEs to perturbations. Understanding the protein composition of SEs that are linked to key oncogenes may identify novel therapeutic targets. A genome-wide CRISPR screen specifically identified proteins essential for MYC ESE activity but not simian virus 40 (SV40) enhancer. These proteins not only were essential for the reporter activity but also were also important for MYC expression and LCL growth. Targeting these proteins may lead to new therapies for EBV-associated cancers.

Epstein-Barr virus (EBV) infection of human primary resting B lymphocytes (RBLs) leads to the establishment of lymphoblastoid cell lines (LCLs) that can grow indefinitely in vitro. EBV transforms RBLs through the expression of viral latency genes, and these genes alter host transcription programs. To globally measure the transcriptome changes during EBV transformation, primary human resting B lymphocytes (RBLs) were infected with B95.8 EBV for 0, 2, 4, 7, 14, 21, and 28 days, and poly(A) plus RNAs were analyzed by transcriptome sequencing (RNA-seq). Analyses of variance (ANOVAs) found 3,669 protein-coding genes that were differentially expressed (false-discovery rate [FDR]

Localized aerosol delivery of gene therapies is a promising treatment of severe pulmonary diseases including lung cancer, cystic fibrosis, COPD and asthma. The administration of drugs by inhalation features multiple benefits including an enhanced patient acceptability and compliance. The application of a spray dried powder formulation has advantages over solutions due to their increased stability and shelf life. Furthermore, optimal sizes of the powder can be obtained by spray drying to allow a deep lung deposition. The present study optimized the parameters involved with spray drying polyplexes formed by polyethylenimine (PEI) and nucleic acids in inert excipients to generate a nano-embedded microparticle (NEM) powder with appropriate aerodynamic diameter. Furthermore, the effects of the excipient matrix used to generate the NEM powder on the biological activity of the nucleic acid and the ability to recover the embedded nanoparticles was investigated. The study showed that bioactivity and nucleic acid integrity was preserved after spray drying, and that polyplexes could be reconstituted from the dry powders made with trehalose but not mannitol as a stabilizer. Scanning electron microscopy (SEM) showed trehalose formulations that formed fused, lightly corrugated spherical particles in the range between 1 and 5 µm, while mannitol formulations had smooth surfaces and consisted of more defined particles. After redispersion of the microparticles in water, polyplex dispersions are obtained that are comparable to the initial formulations before spray drying. Cellular uptake and transfection studies conducted in lung adenocarcinoma cells show that redispersed trehalose particles performed similar to or better than polyplexes that were not spray dried. A method for quantifying polymer and nucleic acid loss following spray drying was developed in order to ensure that equal nucleic acid amounts were used in all in vitro experiments. The results confirm that spray dried NEM formulations containing nucleic acids can be prepared with characteristics known to be optimal for inhalation therapy.