METTL16对肝癌细胞增殖、迁移和侵袭能力的影响及其作用机制

Effects of METTL16 on proliferation,migration and invasion of hepatocellular carcinoma cells and its mechanism of action

目的探究甲基转移酶样蛋白16(METTL16)对肝癌细胞(HCC)增殖、迁移和侵袭的影响及其潜在的作用机制。方法在HepG2和HCC-LM3细胞中转染METTL16过表达载体及干扰载体,用嘌呤霉素筛选稳定转染细胞,Western blot和实时定量PCR实验评估过表达以及干扰效果;采用CCK-8与Transwell实验检测METTL16对HCC增殖、迁移及侵袭的影响;采用流式细胞术检测METTL16对HCC细胞周期的影响;采用Western blot实验检测扰动METTL16后对肝癌细胞VEGFA-VEGFR2通路相关蛋白表达的影响;采用高通量基因表达数据库(GEO)分析METTL16在人肝癌组织与癌旁组织中的表达水平,通过Log-rank检验比较METTL16高表达和低表达肝癌患者生存期的差异。结果Western blot和实时定量PCR实验显示,在HepG2和HCC-LM3细胞中,METTL16过表达细胞株和干扰细胞株构建成功。CCK-8、Transwell和流式细胞术结果显示,过表达METTL16后,增殖、迁移和侵袭的细胞数目减少,处于G 2/M期的细胞比例上升。敲低METTL16后,增殖、迁移和侵袭的细胞数目增加,处于G 2/M期的细胞比例降低。Western blot检测结果显示过表达METTL16可抑制HepG2细胞中VEGFA-VEGFR2通路相关蛋白VEGFR2、p-AKT、Cyclin B、CDK1的表达,但敲低METTL16后可逆转对上述蛋白的抑制作用。METTL16在人肝癌组织中的表达水平低于癌旁组织,并且METTL16与肝癌患者的生存预后无相关性。结论METTL16可能通过抑制VEGFR2通路抑制肝癌细胞的增殖、迁移和侵袭。

Objective To investigate how the m 6A methylation enzyme Methyltransferase like protein 16(METTL16)exerts its effects on the proliferation,migration and invasion of hepatocellular carcinoma(HCC)cells HepG2 and HCC-LM3,and to further explore the underlying molecular mechanism.Methods The overexpression and RNA interference vectors targeting METTL16 were transfected into HepG2 and HCC-LM3 cells and screened the stable cell lines by purimycin.The expressions of METTL16 were detected by means of qRT-PCR and Western blot assay;in HCC cell lines,Cell counting kit-8(CCK-8),Transwell assays,and flow cytometry were used to observe the effects in the proliferation,migration,invasion and cell cycle after transfection;Western blot assay was used todetect the effect of expression of VEGFA-VEGFR2 pathway-related proteins in hepatocellular carcinoma cells;Gene Expression Omnibus database was used to analyzethe expression levels of METTL16 in human liver cancer tissues and paraneoplastic tissues.Log-rank test was used to compare the clinic pathological characteristics between patients with high and low expression of METTL16 in hepatocellular carcinoma.Results Western blot and real-time quantitative PCR experiments showed that METTL16 overexpressing cell lines and interfering cell lines were successfully constructed in HepG2 and HCC-LM3 cells.CCK-8,Transwell and flow cytometry results showed that overexpression of METTL16 resulted in a decrease in the number of proliferating,migrating and invasive cells,and the number of cells in G 2/M phase proportion increased.Western blot showed that overexpression of METTL16 inhibited the expression of VEGFA-VEGFR2 pathway-related proteins VEGFR2,p-AKT,Cyclin B,and CDK1 in HepG2 cells,but knockdown of METTL16 reversed the inhibition effect on these proteins.Compared to the matched non-tumor liver tissues,METTL16 was downregulated in HCC tissues;however,the levels of METTL16 were not significantly associated with the clinic pathological characteristics of HCC patients.Conclusion METTL16 may inhibit the proliferation,migration and invasion of HCC cells by inhibiting the VEGFR2 pathway.