Integrative analysis of chromatin accessibility and transcriptome landscapes in the induction of peritoneal fibrosis by high glucose.

BACKGROUND: Peritoneal fibrosis is the prevailing complication induced by prolonged exposure to high glucose in patients undergoing peritoneal dialysis. METHODS: To elucidate the molecular mechanisms underlying this process, we conducted an integrated analysis of the transcriptome and chromatin accessibility profiles of human peritoneal mesothelial cells (HMrSV5) during high-glucose treatment. RESULTS: Our study identified 2775 differentially expressed genes (DEGs) related to high glucose-triggered pathological changes, including 1164 upregulated and 1611 downregulated genes. Genome-wide DEGs and network analysis revealed enrichment in the epithelial-mesenchymal transition (EMT), inflammatory response, hypoxia, and TGF-beta pathways. The enriched genes included VEGFA, HIF-1α, TGF-ß1, EGF, TWIST2, and SNAI2. Using ATAC-seq, we identified 942 hyper (higher ATAC-seq signal in high glucose-treated HMrSV5 cells than in control cells) and 714 hypo (lower ATAC-seq signal in high glucose-treated HMrSV5 cells versus control cells) peaks with differential accessibility in high glucose-treated HMrSV5 cells versus controls. These differentially accessible regions were positively correlated (R = 0.934) with the nearest DEGs. These genes were associated with 566 up- and 398 downregulated genes, including SNAI2, TGF-ß1, HIF-1α, FGF2, VEGFA, and VEGFC, which are involved in critical pathways identified by transcriptome analysis. Integrated ATAC-seq and RNA-seq analysis also revealed key transcription factors (TFs), such as HIF-1α, ARNTL, ELF1, SMAD3 and XBP1. Importantly, we demonstrated that HIF-1α is involved in the regulation of several key genes associated with EMT and the TGF-beta pathway. Notably, we predicted and experimentally validated that HIF-1α can exacerbate the expression of TGF-ß1 in a high glucose-dependent manner, revealing a novel role of HIF-1α in high glucose-induced pathological changes in human peritoneal mesothelial cells (HPMCs). CONCLUSIONS: In summary, our study provides a comprehensive view of the role of transcriptome deregulation and chromosome accessibility alterations in high glucose-induced pathological fibrotic changes in HPMCs. This analysis identified hub genes, signaling pathways, and key transcription factors involved in peritoneal fibrosis and highlighted the novel glucose-dependent regulation of TGF-ß1 by HIF-1α. This integrated approach has offered a deeper understanding of the pathogenesis of peritoneal fibrosis and has indicated potential therapeutic targets for intervention.

Cellular heterogeneity and transcriptomic profiles during intrahepatic cholangiocarcinoma initiation and progression.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is not fully investigated, and how stromal cells contribute to ICC formation is poorly understood. We aimed to uncover ICC origin, cellular heterogeneity, and critical modulators during ICC initiation/progression, and to decipher how fibroblast and endothelial cells in the stromal compartment favor ICC progression. APPROACH AND RESULTS: We performed single-cell RNA sequencing (scRNA-seq) using AKT/Notch intracellular domain-induced mouse ICC tissues at early, middle, and late stages. We analyzed the transcriptomic landscape, cellular classification and evolution, and intercellular communication during ICC initiation/progression. We confirmed the findings using quantitative real-time PCR, western blotting, immunohistochemistry or immunofluorescence, and gene knockout/knockdown analysis. We identified stress-responding and proliferating subpopulations in late-stage mouse ICC tissues and validated them using human scRNA-seq data sets. By integrating weighted correlation network analysis and protein-protein interaction through least absolute shrinkage and selection operator regression, we identified zinc finger, MIZ-type containing 1 (Zmiz1) and Y box protein 1 (Ybx1) as core transcription factors required by stress-responding and proliferating ICC cells, respectively. Knockout of either one led to the blockade of ICC initiation/progression. Using two other ICC mouse models (YAP/AKT, KRAS/p19) and human ICC scRNA-seq data sets, we confirmed the orchestrating roles of Zmiz1 and Ybx1 in ICC occurrence and development. In addition, hes family bHLH transcription factor 1, cofilin 1, and inhibitor of DNA binding 1 were identified as driver genes for ICC. Moreover, periportal liver sinusoidal endothelial cells could differentiate into tip endothelial cells to promote ICC development, and this was Dll4-Notch4-Efnb2 signaling-dependent. CONCLUSIONS: Stress-responding and ICC proliferating subtypes were identified, and Zmiz1 and Ybx1 were revealed as core transcription factors in these subtypes. Fibroblast-endothelial cell interaction promotes ICC development.

The MicroRNA-199a/214 Cluster Targets E-Cadherin and Claudin-2 and Promotes High Glucose-Induced Peritoneal Fibrosis.

Serum response factor (SRF) was found to be involved in the phenotypic transition and fibrosis of the peritoneal membrane during treatment with peritoneal dialysis (PD), but the exact mechanism remains unclear. SRF regulates microRNAs (miRNAs) that contain the SRF-binding consensus (CArG) element in the promoter region. Therefore, we investigated whether the miR-199a/214 gene cluster, which contains a CArG element in its promoter, is directly regulated by SRF. High-glucose (HG) treatment significantly unregulated the expression of the miR-199a-5p/214-3p gene cluster in human peritoneal mesothelial cells (HPMCs). By chromatin immunoprecipitation and reporter assays, we found that SRF binds to the miR-199a-5p/214-3p gene cluster promoter after HG stimulation. In vitro, in HPMCs, silencing of miR-199a-5p or miR-214-3p inhibited the HG-induced phenotypic transition and cell migration but enhanced cell adhesion, whereas ectopic expression of mimic oligonucleotides had the opposite effects. Both miR-199a-5p and miR-214-3p targeted claudin-2 and E-cadherin mRNAs. In a PD rat model, treatment with an SRF inhibitor silenced miR-199a-5p and miR-214-3p and alleviated HG-PD fluid-induced damage and fibrosis. Overall, this study reveals a novel SRF-miR-199a/miR-214-E-cadherin/claudin-2 axis that mediates damage and fibrosis in PD.

Viral integration promotes SV40T-induced immortalization by disturbing the expression of DNA/chromosome- and ECM-associated functional genes.

Lentivirus containing simian virus 40 large T antigen (SV40T) is routinely used to induce cell immortalization. However, the roles of viral integration itself in this progress is still controversial. Here, we transformed primary mouse embryonic fibroblasts (MEFs) with SV40T lentivirus and studied the roles of viral integration in the immortalization using RNA sequencing (RNA-seq) and whole genome sequencing (WGS). During the immortalization, differentially expressed genes (DGEs) are enriched in viral infection and several diverse activities. However, DEGs between immortalized and aging cells are significantly enriched in DNA/chromosome- and extracellular matrix (ECM)-associated activities. Gene regulatory network (GRN) analysis shows that although p53 is a key regulatory factor, many other transcription factors also play critical roles in the process, like STAT1. Of these DEGs, 32 genes have viral integration in their coding and/or regulatory regions. Our findings suggest that viral integration may promote SV40T-mediated immortalization by disturbing the expression of DNA/chromosome- and ECM-associated genes.

Downregulation of TMEM220 promotes tumor progression in Hepatocellular Carcinoma.

During the process of long-term carcinogenesis, cells accumulate many mutations. Deregulated genes expression causes profound changes in cell proliferation, which is one of the hallmarks of HCC. A comprehensive understanding of these changes will contribute to the molecular mechanism of HCC progression. Through clinical sample analysis, we found that TMEM220 is downregulated in tumor and lower levels of TMEM220 is associated with poor prognosis in HCC patients. Through overexpressing TMEM220 in HCC cell lines, we found that the proliferation of cancer cells was significantly slowed down and metastasis was significantly reduced. For further study of its molecular mechanism, we performed a reverse-phase protein array (RPPA). The results suggest that phenotypic changes caused by TMEM220 in HCC cells might be associated with FOXO and PI3K-Akt pathways. Mechanism studies showed that overexpression of TMEM220 could regulate ß-catenin and FOXO3 transcriptional activity by altering their subcellular localization, affecting the expression of downstream gene p21 and SNAIL, and ultimately reducing the progression of HCC. Altogether, our study proposes a working model in which upregulation of TMEM220 expression alters the genes expression involved in cell proliferation, thereby inhibiting HCC progression, which suggests that TMEM220 might serve as a clinical biomarker.

Integrating bulk and single-cell RNA sequencing reveals cellular heterogeneity and immune infiltration in hepatocellular carcinoma.

Efficacy of immunotherapy in hepatocellular carcinoma (HCC) is blocked by its high degree of inter- and intra-tumor heterogeneity and immunosuppressive tumor microenvironment. However, the correlation between tumor heterogeneity and immunosuppressive microenvironment in HCC has not been well addressed. Here, we endeavored to dissect inter- and intra-tumor heterogeneity in HCC and uncover how they contribute to the immunosuppressive microenvironment. We performed consensus molecular subtyping with non-negative matrix factorization (NMF) clustering to stratify the inter-heterogeneity profile of HCC tumors. We grouped HCC tumors from the Cancer Genome Atlas (TCGA) patients into three subtypes (S1, S2 and S3), where S1 was characterized as a 'hot tumor' profile with high expression level of T cell genes and rate of immune scores. S2 was characterized as a 'cold tumor' profile with the highest tumor purity score, and S3 as an 'immunosuppressed tumor' profile with the poorest prognosis and a high expression level of immunosuppressive genes such as cytotoxic T-lymphocyte-associated protein-4, TIGIT, and PDCD1. Moreover, we combined weighted gene co-expression network analysis and single-cell regulatory network inference and clustering (SCENIC) in the single-cell dataset of the S3-like subtype (CS3) and identified a transcription factor, BATF, which could upregulate immunosuppressive genes. Finally, we identified a cell interaction network in which a myeloid-derived suppressor cell-like macrophage subtype could promote the formation of immunosuppressive T-cells.

MEIS2C and MEIS2D promote tumor progression via Wnt/ß-catenin and hippo/YAP signaling in hepatocellular carcinoma.

BACKGROUND: MEIS2 has been identified as one of the key transcription factors in the gene regulatory network in the development and pathogenesis of human cancers. Our study aims to identify the regulatory mechanisms of MEIS2 in hepatocellular carcinoma (HCC), which could be targeted to develop new therapeutic strategies. METHODS: The variation of MEIS2 levels were assayed in a cohort of HCC patients. The proliferation, clone-formation, migration, and invasion abilities of HCC cells were measured to analyze the effects of MEIS2C and MEIS2D (MEIS2C/D) knockdown with small hairpin RNAs in vitro and in vivo. Chromatin immunoprecipitation (ChIP) was performed to identify MEIS2 binding site. Immunoprecipitation and immunofluorescence assays were employed to detect proteins regulated by MEIS2. RESULTS: The expression of MEIS2C/D was increased in the HCC specimens when compared with the adjacent noncancerous liver (ANL) tissues. Moreover, MEIS2C/D expression negatively correlated with the prognosis of HCC patients. On the other hand, knockdown of MEIS2C/D could inhibit proliferation and diminish migration and invasion of hepatoma cells in vitro and in vivo. Mechanistically, MESI2C activated Wnt/ß-catenin pathway in cooperation with Parafibromin (CDC73), while MEIS2D suppressed Hippo pathway by promoting YAP nuclear translocation via miR-1307-3p/LATS1 axis. Notably, CDC73 could directly either interact with MEIS2C/ß-catenin or MEIS2D/YAP complex, depending on its tyrosine-phosphorylation status. CONCLUSIONS: Our studies indicate that MEISC/D promote HCC development via Wnt/ß-catenin and Hippo/YAP signaling pathways, highlighting the complex molecular network of MEIS2C/D in HCC pathogenesis. These results suggest that MEISC/D may serve as a potential novel therapeutic target for HCC.

Whole-genome sequencing reveals novel tandem-duplication hotspots and a prognostic mutational signature in gastric cancer.

Genome-wide analysis of genomic signatures might reveal novel mechanisms for gastric cancer (GC) tumorigenesis. Here, we analysis structural variations (SVs) and mutational signatures via whole-genome sequencing of 168 GCs. Our data demonstrates diverse models of complex SVs operative in GC, which lead to high-level amplification of oncogenes. We find varying proportion of tandem-duplications (TDs) among individuals and identify 24 TD hotspots involving well-established cancer genes such as CCND1, ERBB2 and MYC. Specifically, we nominate a novel hotspot involving the super-enhancer of ZFP36L2 presents in approximately 10% GCs from different cohorts, the oncogenic role of which is further confirmed by experimental data. In addition, our data reveal a mutational signature, specifically occurring in noncoding region, significantly enriched in tumors with cadherin 1 mutations, and associated with poor prognoses. Collectively, our data suggest that TDs might serve as an important mechanism for cancer gene activation and provide a novel signature for stratification.

Forkhead box C1 promotes colorectal cancer metastasis through transactivating ITGA7 and FGFR4 expression.

Metastatic colorectal cancer (CRC) is one of the most common causes of cancer death worldwide; however, the molecular mechanism underlying CRC metastasis remains unknown. Using an integrated approach, we identified forkhead box C1 (FOXC1) as a novel regulator of CRC metastasis. Elevated expression of FOXC1 is significantly correlated with metastasis, recurrence and reduced survival. FOXC1 overexpression promotes CRC invasion and lung metastasis, whereas FOXC1 knockdown has the opposite effect. In addition, FOXC1 directly binds its target genes integrin α7 (ITGA7) and fibroblast growth factor receptor 4 (FGFR4) and activates their expression. Genetic epistasis analysis confirmed that ITGA7 and FGFR4 act downstream of FOXC1. Furthermore, pharmaceutical inhibition of FGFR4 can reverse CRC metastasis mediated by FOXC1 overexpression. These results suggest that FOXC1 is a prognostic biomarker in CRC patients and targeting the FGFR4 signaling pathway may provide a promising strategy for the treatment of FOXC1-driven CRC metastasis.

Biased random walk model for the prioritization of drug resistance associated proteins.

Multi-drug resistance is the main cause of treatment failure in cancer patients. How to identify molecules underlying drug resistance from multi-omics data remains a great challenge. Here, we introduce a data biased strategy, ProteinRank, to prioritize drug-resistance associated proteins in cancer cells. First, we identified differentially expressed proteins in Adriamycin and Vincristine resistant gastric cancer cells compared to their parental cells using iTRAQ combined with LC-MS/MS experiments, and then mapped them to human protein-protein interaction network; second, we applied ProteinRank to analyze the whole network and rank proteins similar to known drug resistance related proteins. Cross validations demonstrated a better performance of ProteinRank compared to the method without usage of MS data. Further validations confirmed the altered expressions or activities of several top ranked proteins. Functional study showed PIM3 or CAV1 silencing was sufficient to reverse the drug resistance phenotype. These results indicated ProteinRank could prioritize key proteins related to drug resistance in gastric cancer and provided important clues for cancer research.

Nuclear expression of Twist promotes lymphatic metastasis in esophageal squamous cell carcinoma.

Twist-1 protein (also called Twist) has been suggested to be involved in tumor epithelial-mesenchymal transition (EMT) related progression, however, the mechanism by which twist promotes lymph node metastasis is not fully understood. In the present study, we found that nuclear twist expression is clearly correlated with lymph node (LN) metastasis as determined by immunohistochemistry (IHC). A highly invasive EC109 cell subline, EC109-P, was established by repeated in vitro transwell isolations for the cell model. Immunofluorescence (IF) assay demonstrated that nuclear twist expression was markedly higher in the highly invasive EC109-P cell line when compared with EC109 and EC9706 cells. Based on our cell model, the function and mechanism by which twist regulates LN metastasis in ESCC was investigated. The results showed that the overexpression of Twist could significantly increase the invasion and VEGF-C expression of EC9706 cells, whereas the knockdown of twist expression results in the opposite effects. This finding was further strengthened by the results of the analysis of co-expression of twist and VEGF-C by IHC in ESCC clinical samples. In summary, our study indicates that nuclear twist plays an important role in ESCC lymphatic metastasis by increasing the expression of VEGF-C. The combination of twist and VEGF-C detection could be a reliable prediction of LN metastasis in ESCC.