METTL3/YTHDF2 m6 A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer.

N6-Methyladenosine (m6 A) modification, the most prevalent modification of eukaryotic messenger RNA (mRNA), is involved in the progression of various tumours. However, the specific role of m6 A in bladder cancer (BCa) is still poorly understood. In this study, we demonstrated the tumour-promoting function and specific regulatory mechanism of m6 A axis, consisting of the core 'writer' protein METTL3 and the major reader protein YTHDF2. Depletion of METTL3 impaired cancer proliferation and cancer metastasis in vitro and in vivo. Through transcriptome sequencing, m6 A methylated RNA immunoprecipitation (MeRIP) and RIP, we determined that the METTL3/YTHDF2 m6 A axis directly degraded the mRNAs of the tumour suppressors SETD7 and KLF4, contributing to the progression of BCa. In addition, overexpression of SETD7 and KLF4 revealed a phenotype consistent with that induced by depletion of the m6 A axis. Thus, our findings on the METTL3/YTHDF2/SETD7/KLF4 m6 A axis provide the insight into the underlying mechanism of carcinogenesis and highlight potential therapeutic targets for BCa.

YTHDF2 mediates the mRNA degradation of the tumor suppressors to induce AKT phosphorylation in N6-methyladenosine-dependent way in prostate cancer.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant modification in mRNA of humans. Emerging evidence has supported the fact that m6A is comprehensively involved in various diseases especially cancers. As a crucial reader, YTHDF2 usually mediates the degradation of m6A-modified mRNAs in m6A-dependent way. However, the function and mechanisms of m6A especially YTHDF2 in prostate cancer (PCa) still remain elusive. METHODS: To investigate the functions and mechanisms of YTHDF2 in PCa, in vitro, in vivo biofunctional assays and epigenetics experiments were performed. Endogenous expression silencing of YTHDF2 and METTL3 was established with lentivirus-based shRNA technique. Colony formation, flow cytometry and trans-well assays were performed for cell function identifications. Subcutaneous xenografts and metastatic mice models were combined with in vivo imaging system to investigate the phenotypes when knocking down YTHDF2 and METTL3. m6A RNA immunoprecipitation (MeRIP) sequencing, mRNA sequencing, RIP-RT-qPCR and bioinformatics analysis were mainly used to screen and validate the direct common targets of YTHDF2 and METTL3. In addition, TCGA database was also used to analyze the expression pattern of YTHDF2, METTL3 and the common target LHPP in PCa, and their correlation with clinical prognosis. RESULTS: The upregulated YTHDF2 and METTL3 in PCa predicted a worse overall survival rate. Knocking down YTHDF2 or METTL3 markedly inhibited the proliferation and migration of PCa in vivo and in vitro. LHPP and NKX3-1 were identified as the direct targets of both YTHDF2 and METTL3. YTHDF2 directly bound to the m6A modification sites of LHPP and NKX3-1 to mediate the mRNA degradation. Knock-down of YTHDF2 or METTL3 significantly induced the expression of LHPP and NKX3-1 at both mRNA and protein level with inhibited phosphorylated AKT. Overexpression of LHPP and NKX3-1 presented the consistent phenotypes and AKT phosphorylation inhibition with knock-down of YTHDF2 or METTL3. Phosphorylated AKT was consequently confirmed as the downstream of METTL3/YTHDF2/LHPP/NKX3-1 to induce tumor proliferation and migration. CONCLUSION: We propose a novel regulatory mechanism in which YTHDF2 mediates the mRNA degradation of the tumor suppressors LHPP and NKX3-1 in m6A-dependent way to regulate AKT phosphorylation-induced tumor progression in prostate cancer. We hope our findings may provide new concepts of PCa biology.

The dual role of N6-methyladenosine modification of RNAs is involved in human cancers.

As the most abundant and reversible RNA modification in eukaryotic cells, m6 A triggers a new layer of epi-transcription. M6 A modification occurs through a methylation process modified by "writers" complexes, reversed by "erasers", and exerts its role depending on various "readers". Emerging evidence shows that there is a strong association between m6 A and human diseases, especially cancers. Herein, we review bi-aspects of m6 A in regulating cancers mediated by the m6 A-associated proteins, which exert vital and specific roles in the development of various cancers. Generally, the m6 A modification performs promotion or inhibition functions (dual role) in tumorigenesis and progression of various cancers, which suggests a new concept in cancer regulations. In addition, m6 A-targeted therapies including competitive antagonists of m6 A-associated proteins may provide a new tumour intervention in the future.

MicroRNA-501-3p inhibits the proliferation of kidney cancer cells by targeting WTAP.

BACKGROUND: Emerging evidence suggests that miR-501-3p plays an important role in the pathogenesis and progression of various carcinomas. However, its role and underlying mechanisms in renal cell carcinoma (RCC) remain to be elucidated. METHODS: Quantitative RT-PCR, western blot, and bioinformatics methods were used to evaluate the expression of miR-501-3p and Wilms' tumor 1-associating protein (WTAP) in RCC cell lines and clinical tissues. The effects of miR-501-3p on the proliferation of RCC cells were investigated using flow cytometric, colony formation, and CCK8 assays. The target gene of miR-501-3p was confirmed by western blotting, qRT-PCR, and dual-luciferase reporter assays. The levels of RNA methylation with N6-methyladenosine (m6 A) following miR-501-3p overexpression or knockdown of its target gene were quantified using a dot-blot assay. RESULTS: miR-501-3p expression was significantly downregulated in human RCC cell lines and tissues. In contrast, its overexpression markedly inhibited cancer cell proliferation in vitro by inducing G1 phase arrest. Moreover, WTAP was verified as a direct target gene of miR-501-3p. WTAP gene knockdown alone efficiently produced the same cancer-inhibiting effects as miR-501-3p overexpression, with the level of m6 A in RCC cells being decreased under both scenarios. The intermolecular interaction between miR-501-3p and WTAP was further substantiated by rescue experiments. CONCLUSION: RCC progression is regulated via the miR-501-3p/WTAP/CDK2 axis and is inhibited by the overexpression of miR-501-3p.

circKDM4C enhances bladder cancer invasion and metastasis through miR-200bc-3p/ZEB1 axis.

Circular RNAs (circRNAs) play essential roles in human bladder cancer (BCa) development, however, unusual expression patterns and functional dysfunction of circRNAs in BCa have not been evaluated. In this study, we validated that circKDM4C (hsa_circ_0001839), derived from the KDM4C gene, is elevated in BCa cell lines as well as tissues. Functionally, overexpression of circKDM4C significantly enhances, and silencing of circKDM4C suppresses migration and invasion capabilities of BCa cells. Mechanistically, circKDM4C can directly interact with miR-200b-3p and miR-200c-3p as a miRNA sponge, which enhances the expression of ZEB1 and promotes mesenchymal phenotype. Conclusively, our findings indicate that circKDM4C may act as a pro-oncogenic factor in BCa invasion and metastasis via the circKDM4C/miR-200bc-3p/ZEB1 axis, which is a potential biomarker or therapeutic target for bladder cancer.

miR-665 inhibits epithelial-to-mesenchymal transition in bladder cancer via the SMAD3/SNAIL axis.

Emerging research indicates that miRNAs can regulate cancer progression by influencing molecular pathways. Here, we studied miR-665, part of the DLK1-DIO3 miRNA cluster, which is downregulated by upstream methylation in bladder cancer. MiR-665 overexpression significantly downregulated the expression of SMAD3, phospho-SMAD3, and SNAIL, reversed epithelial-mesenchymal transition progression, and inhibited the migration of bladder cancer cells. To predict potential targets of miR-665, we used online databases and subsequently determined that miR-665 binds directly to the 3' untranslated region of SMAD3. Moreover, silencing of SMAD3 with small interfering RNAs phenocopied the effect of miR-665 overexpression, and overexpression of SMAD3 restored miR-665-overexpression-induced metastasis. This study revealed the role of the miR-665/SMAD3/SNAIL axis in bladder cancer, as well as the potential of miR-665 as a promising therapeutic target.

CCND1, NOP14 and DNMT3B are involved in miR-502-5p-mediated inhibition of cell migration and proliferation in bladder cancer.

OBJECTIVES: Downregulation of miR-502-5p has emerged as a critical factor in tumour progression in several cancers. Herein, we elucidated the role of miR-502-5p in bladder cancer. MATERIALS AND METHODS: RT-qPCR was performed to examine the expression of miR-502-5p in bladder cancer. And DNA methylation analysis showed that epigenetic mechanisms may contribute to the downregulation of miR-502-5p. Then, wound-healing assay, transwell assay, colony formation assay, CCK8 assay and flow cytometry analysis were applied to evaluate the function of miR-502-5p in bladder cancer cell lines. Western blot was conducted to measure the protein levels of related genes. Furthermore, dual-luciferase reporter assay, in vivo tumorigenesis assay and immunohistochemical staining were also conducted as needed. RESULTS: MiR-502-5p is frequently downregulated in BCa. Meanwhile, hypermethylation of CpG islands contributes to the downregulation of miR-502-5p. Functionally, overexpression of miR-502-5p inhibited cell proliferation and migration in vitro and repressed tumour growth in vivo. CCND1, DNMT3B and NOP14 were identified as direct targets of miR-502-5p. Interestingly, DNMT3B and miR-502-5p established a positive feedback loop in the regulation of bladder cancer. In addition, rescue experiments further validated the direct molecular interaction between miR-502-5p and its targets. CONCLUSIONS: Our study proposed and demonstrated that the miR-502-5p-mediated regulatory network is critical in bladder cancer; this network may be useful in the development of more effective therapies against bladder cancer.

Clinicopathological impacts of c-Met overexpression in bladder cancer: evidence from 1,336 cases.

Background: The clinicopathological impacts of c-Met overexpression in bladder cancer have been investigated in several studies with conflicting results. We performed this systematic review and meta-analysis to assess the pathologic and prognostic roles of c-Met status in bladder cancer patients. Methods: Eligible studies were searched and identified from the PubMed and China National Knowledge Infrastructure (CNKI) databases (up until October 4, 2018). The DerSimonian-Laird random-effects model was used to calculate the pooled risk estimates. Results: Eight studies including 1,336 bladder cancer cases were eventually included in this meta-analysis. We detected a significantly increased risk of poor overall survival (OS) associated with the high expression of c-Met (HR=2.42, 95% CI 1.36-4.32). There was no association between c-Met status and nuclear grade (OR=0.82, 95% CI 0.29-2.31) or tumor stage (OR=1.42, 95% CI 0.41-4.89). Conclusion: This study shows that the overexpression of c-Met in primary cancer tissues is associated with a worse OS in human bladder cancer. However, larger studies using standardized methods and criteria are warranted to verify these findings.

Downregulation of N6-methyladenosine binding YTHDF2 protein mediated by miR-493-3p suppresses prostate cancer by elevating N6-methyladenosine levels.

Recent evidence suggests that m6A modifications regulate the progressions of several types of tumors. YTHDF2, an m6A reader, has been implicated in the regulation of hepatocellular carcinoma (HCC). miR-493-3p has been defined as tumor suppressor that inhibits the progressions of several types of cancers. However, the functions and mechanisms of YTHDF2 and the indirect m6A regulated role of miR-493-3p in prostate cancer (PCa) remains to be elusive. In this study, immuno-histochemical (IHC) staining and chromogenic in situ hybridization (CISH) were performed to find YTHDF2 was frequently upregulated but miR-493-3p was downregulated in both PCa tissues and cell lines (DU-145 and PC3) which was negatively correlated with each other. Knock down of YTHDF2 significantly elevated m6A levels, and inhibited the cell proliferation and migration of DU-145 and PC3 cell lines. The dual-luciferase reporter assay confirmed YTHDF2 as the direct target of miR-493-3p. In addition, forced expression of miR-493-3p consistently elevated the m6A levels and inhibited proliferation and migration with the knock down of YTHDF2. In contrast, overexpression of YTHDF2 and inhibition of miR-493-3p conversely reduced m6A levels. Additionally, the rescue experiments revealed that inhibition of miR-493-3p abrogated the suppression of proliferation and migration induced by si-YTHDF2. To conclude, YTHDF2 and miR-493-3p, as two crucial m6A regulators, are involved in the progression of PCa by indirectly modulating m6A levels. In view of these promising results, YTHDF2 and miR-493-3p may provide new insights into the carcinogenesis and new potential therapeutic targets for PCa.

Secondhand smoking increases bladder cancer risk in nonsmoking population: a meta-analysis.

BACKGROUND: Tobacco smoking has been widely acknowledged to be the most important risk factor for bladder cancer. However, whether secondhand smoking (SHS) increases the risk of bladder cancer still remains uncertain. We conducted a meta-analysis about the risk of bladder cancer and lifetime SHS and childhood SHS. MATERIALS AND METHODS: We searched PubMed, EMBASE, Web of Science, and Chinese National Knowledge Infrastructure (CNKI) up to March 12, 2018, and checked references of the retrieved articles and relevant reviews to include 14 studies. Relative risk (RR) and 95% confidence interval (CI) were used to assess this risk. RESULTS: The pooled RR of 14 eligible studies based on the retrieved articles and relevant reviews illustrated a significantly increased risk of bladder cancer with RR 1.22, 95% CI 1.06-1.4. No heterogeneity or publication bias was found. But we need more evidence to prove a more reliable association between childhood SHS and bladder cancer. CONCLUSION: There was a statistically significant 22% increased risk of bladder cancer for lifetime SHS exposure in nonsmoking patients compared with unexposed nonsmoking population. But the association between childhood SHS exposure compared with unexposed nonsmoking population was unclear. Further research should be conducted to confirm our findings and reveal the potential biological mechanisms.