| Background:N6-methyladenosine(m~6A)is one of the most preventive and abundant modifications on RNA molecules present in eukaryotes.The identification of the fat mass and obesity-associated protein(FTO)as the first m~6A demethylase in 2011 marked that m~6A modification has provoked the interest of researchers once again.Until date,although the function of m~6A in the physiological and biological processes has been investigated,studies on the m~6A level under pathological conditions,especially in m~6A modification of micro RNA,remain limited.Hepatocellular carcinoma(HCC)not only poses a serious threat to human health,but also increases the economic pressure on the global health care system.Prevalence closely mirrors incidence based on the International Agency for Research on Cancer(IARC),which reflects its typically late-stage presentation,limited treatment options.Therefore,it is significance to clarify how m~6A modification affects HCC progression.Research materials and methods:Our study focuses on METTL3,which is significantly up-regulated in HCC cell.We analyzed the relationship between METTL3 expression and the prognosis of HCC patients based on GEPIA website.Differential expression analysis,GO/KEGG and GSEA was used to explore signaling pathways that were regulated by METTL3 in Hep G2.Transfected METTL3 si RNA in HCC cell,CCK8,cloning formation,apoptosis,cell cycle experiments are performed to explore its effect on cell proliferation,cell cycle and apoptosis.To identified potential mi RNAs whose expression were regulated by METTL3,we analyzed the existing data.We used q RT-PCR to study how the expression of mi R-93 is regulated by METTL3.The m~6A-IP-q PCR experiment was used to detect the m~6A level of pri-mi R-93 in HCC cell.Potential targets of mi R-93-5p were predicted by using Targetscan and ENCORI websites and correlation analysis.Dual-luciferase reporter assays proved that CDKN1A was a target of mi R-93-5p.Besides,after transfecting METTL3 si RNA and mi R-93-5p mimics or METTL3 si RNA in HCC cell,q RT-PCR and western blot analyses were conducted to test the m RNA and protein expression of CDKN1A and several function experiments were performed to estimate cell viability,cell cycle and apoptosis in HCC cell differently treated.Research results:Through bioinformatic analysis and q RT-PCR experiments,we found that METTL3 expression was overexpressed and associated with poor prognosis in HCC cell.GSEA analysis based on GSE37001 dataset showed that several signaling pathways involving in cell cycle were suppressed when METTL3 si RNA was transfected in Hep G2.By comparing the existing datasets,we identified 6 mi RNAs whose expression were upregulated in HCC and positively correlated with METTL3expression,including mi R-491-5p,mi R-93-5p,let-7d-3p,mi R-3154,mi R-223-5p,mi R-3657.Combining published studies,we focused on mi R-93-5p.We found that the expression of its precursor was increased by the depletion of METTL3 but the level of mature mi R-93 was reduced in HCC cell.We found that these changes could be rescued when overexpressing METTL3 WT rather than METTL3 mutant whose methyltransferase regions were mutated(aa395-398,DPPW-APPA).The results of dual-luciferase reporter assays and q RT-PCR proved that mi R-93-5p interacted directly with CDKN1A,and the former can negatively regulate the latter.Western blot and q RT-PCR experiments were performed to prove that METTL3 repressed the m RNA and protein expression of CDKN1A by up-regulating mi R-93-5p expression.Function rescue experiments confirmed that METTL3 promotes the proliferation,cell cycle progress and inhibited cell apoptosis by raising mi R-93-5p expression in HCC.Analysis conclusions:1.METTL3 expression is overexpressed and associated with poor prognosis in HCC.2.METTL3 regulates mi R-93 expression in HCC cell through modulating its m~6A methylation.3.METTL3 promotes proliferation and reduces the expression of CDKN1A in HCC cell by increased mi R-93-5p expression. |
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