N6-methyladenosine modification of ITGA6 mRNA promotes the development and progression of bladder cancer.

BACKGROUND: Accumulating evidence has revealed the critical roles of N6-methyladenosine (m6A) modification of mRNA in various cancers. However, the biological function and regulation of m6A in bladder cancer (BC) are not yet fully understood. METHODS: We performed cell phenotype analysis and established in vivo mouse xenograft models to assess the effects of m6A-modified ITGA6 on BC growth and progression. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation and luciferase reporter and mutagenesis assays were used to define the mechanism of m6A-modified ITGA6. Immunohistochemical analysis was performed to assess the correlation between METTL3 and ITGA6 expression in bladder cancer patients. FINDINGS: We show that the m6A writer METTL3 and eraser ALKBH5 altered cell adhesion by regulating ITGA6 expression in bladder cancer cells. Moreover, upregulation of ITGA6 is correlated with the increase in METTL3 expression in human BC tissues, and higher expression of ITGA6 in patients indicates a lower survival rate. Mechanistically, m6A is highly enriched within the ITGA6 transcripts, and increased m6A methylations of the ITGA6 mRNA 3'UTR promotes the translation of ITGA6 mRNA via binding of the m6A readers YTHDF1 and YTHDF3. Inhibition of ITGA6 results in decreased growth and progression of bladder cancer cells in vitro and in vivo. Furthermore, overexpression of ITGA6 in METTL3-depleted cells partially restores the BC adhesion, migration and invasion phenotypes. INTERPRETATION: Our results demonstrate an oncogenic role of m6A-modified ITGA6 and show its regulatory mechanisms in BC development and progression, thus identifying a potential therapeutic target for BC. FUND: This work was supported by National Natural Science Foundation of China (81772699, 81472999).

Dynamic m6A mRNA methylation reveals the role of METTL3-m6A-CDCP1 signaling axis in chemical carcinogenesis.

N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNAs. Despite its functional importance in various physiological events, the role of m6A in chemical carcinogenesis remains largely unknown. Here we profiled the dynamic m6A mRNA modification during cellular transformation induced by chemical carcinogens and identified a subset of cell transformation-related, concordantly modulated m6A sites. Notably, the increased m6A in 3'-UTR mRNA of oncogene CDCP1 was found in malignant transformed cells. Mechanistically, the m6A methyltransferase METTL3 and demethylases ALKBH5 mediate the m6A modification in 3'-UTR of CDCP1 mRNA. METTL3 and m6A reader YTHDF1 preferentially recognize m6A residues on CPCP1 3'-UTR and promote CDCP1 translation. We further showed that METTL3 and CDCP1 are upregulated in the bladder cancer patient samples and the expression of METTL3 and CDCP1 is correlated with the progression status of the bladder cancers. Inhibition of the METTL3-m6A-CDCP1 axis resulted in decreased growth and progression of chemical-transformed cells and bladder cancer cells. Most importantly, METTL3-m6A-CDCP1 axis has synergistic effect with chemical carcinogens in promoting malignant transformation of uroepithelial cells and bladder cancer tumorigenesis in vitro and in vivo. Taken together, our results identify dynamic m6A modification in chemical-induced malignant transformation and provide insight into critical roles of the METTL3-m6A-CDCP1 axis in chemical carcinogenesis.

Comprehensive analysis of differential circular RNA expression in a mouse model of colitis-induced colon carcinoma.

Circular RNAs (circRNAs) have received increasing attention for their involvement in the pathogenesis of cancer; however, the characterization and function of circRNAs in colitis-induced colon carcinoma remains largely unknown. A colitis-induced colon carcinoma model was established in mice treated with azoxymethane-dextran sodium sulfate (AOM-DSS), and the circRNA profile was screened by next generation sequencing. Bioinformatic tools, including Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and network analysis were used to predict the functions of differentially expressed circRNAs and potentially coexpressed target genes. Among the detected candidate 3069 circRNA genes, 126 circRNAs were upregulated, and 108 circRNAs were down regulated in colon tissues from AOM/DSS mice compared to those from control mice. A total of six of these candidate circRNAs were validated by RT-PCR. GO analysis revealed that numerous target genes including most microRNAs were involved in the Ras-Raf-MAPK pathway, actin cytoskeleton, focal adhesion, and additional biological processes. Our study revealed a comprehensive expression and functional profile for differentially expressed circRNAs in AOM/DSS induced colon carcinogenesis, indicating possible involvement of these dysregulated circRNAs in the development of colitis-induced colon carcinoma. The mmu-circ-001226/mmu-circ-000287-miRNA-mRNA network may provide a potential mechanism for colitis-associated colorectal cancer.

A novel regulatory circuit of miR­152 and DNMT1 in human bladder cancer.

Downregulation of microRNA­152 (miR­152) has been observed in various types of human malignancies, including Bladder cancer (BC). However, the role of miR­152 in the development and progression of BC is still unclear. In our previous study, we identified a functional crosstalk between miR­152 and DNA methyltransferase 1 (DNMT1) involved in Nis­induced malignant transformation. In the present study, we found that the expression of miR­152 was specifically downregulated in BC cells and tissues via the DNA hypermethylation of the miR­152 promoter. The overexpression of miR­152 in BC cells resulted in a reduction of DNMT1, whereas the inhibition of the expression of miR­152 induced an elevated level of DNMT1. Further studies revealed that miR­152 directly downregulated the expression of DNMT1 by targeting the 3'­UTR of its transcript in BC cells. In addition, ectopic expression of miR­152 in BC cells significantly inhibited cell proliferation, whereas the inhibition of miR­152 expression led to increased cell proliferation. These findings indicated a novel regulatory circuit of miR­152/DNMT1 in BC, and more importantly, the combination of miR­152 and DNMT1 may function as promising therapeutic modalities and early biomarkers for BC.

Long non-coding RNA DBCCR1-003 regulate the expression of DBCCR1 via DNMT1 in bladder cancer.

BACKGROUND: Many long non coding RNAs have been identified as key modulators in cancer development. A lncRNA, DBCCR1-003, derived from the locus of tumor suppressor gene DBCCR1 (deleted in bladder cancer chromosome region 1), has unknown function. In the present study, we explored function and molecular mechanism of DBCCR1-003 in bladder cancer (BC) development. METHODS: We evaluated the expression levels of DBCCR1-003 in tissues and cells with western blot and quantitative real-time polymerase chain reaction. Multiple approaches including chromatin immunoprecipitation assay and RNA immunoprecipitation were used to confirm the direct binding of DBCCR1-003 to DNMT1. The recombinant vector overexpressing DBCCR1-003 was constructed. Cell proliferation assay, colony formation assay and flow cytometric analysis were employed to measure the role of DBCCR1-003 in regulation of cell proliferation, cycle and apoptosis. RESULTS: Firstly we detected the expression of DBCCR1-003, DBCCR1, DNMT1 (DNA methyltransferase 1) and DNA methylation in the promoter of DBCCR1. We found low expression of DBCCR1-003, same as DBCCR1, while high expression of DNMT1 and hypermethylation of DBCCR1 gene promoter in BC tissues and T24 cells line. Further studies revealed that treatment of DNMT inhibitor, 5-aza-2-deoxycytidine(DAC), or overexpression of DBCCR1-003 led to increased DBCCR1 expression by reversion of promoter hypermethylation and DNMT1 binding to DBCCR1 promoter in T24 cells. Importantly, RNA immunoprecipitation (RIP) showed that DBCCR1-003 physically associates with DNMT1. The binding of them was increased with the inhibition of DBCCR1 promoter methylation, indicating that DBCCR1-003 may bind to DNMT1 and prevent DNMT1-mediated the methylation of DBCCR1. Furthermore, overexpression of DBCCR1-003 resulted in significant inhibition of T24 cells growth through the inducing G0/G1 arrest and apoptosis. CONCLUSIONS: Taken together, these findings demonstrated that a novel tumor suppressor DBCCR1-003 regulates the expression of DBCCR1 via binding to DNMT1 and preventing DNMT1-mediated the methylation of DBCCR1 in BC. LncRNA DBCCR1-003 may serve as a novel biomarker and therapeutic target for BC in future cancer clinic.

The relationship between promoter methylation of p16 gene and bladder cancer risk: a meta-analysis.

PURPOSE: Many scientific evidences suggested that the methylation of p16INK4a (p16) was associated with bladder cancer, but some existing studies have yielded inconclusive results about the relationship between p16 promoter methylation and pathological features or the tumor grade of bladder cancer. This meta-analysis of studies aims to evaluate the clinical and prognostic significance of p16 methylation in bladder carcinogenesis. METHODS: Studies were systemically searched via PubMed and Google Scholar in English up to Sept 2015 and a total of ten appropriate studies (693 cases and 290 controls) with an average NOS score of 6.8 were included. The quality of the appropriate studies was measured by the Newcastle-Ottawa Scale (NOS) assessment. RESULTS: The meta-analysis results revealed that the methylation state of p16 was statistically significantly associated with an increased risk of bladder cancer (OR=6.71, 95% CI=3.79-11.87) compared to control, and there is no statistically significantly association between the p16 methylation and the tumor pTNM staging (OR=0.59, 95% CI=0.22-1.60) or the tumor grade (OR=1.01, 95% CI=0.52-1.94) in p16 methylated patients compared to unmethylated patients. CONCLUSIONS: our meta-analysis indicates that p16 promoter methylation may be a promising biomarker for the diagnosis of bladder cancer and the inactivation of p16 may be an early event in bladder carcinogenesis. More studies with larger numbers of participants worldwide are needed to further identify the obvious association above.