Objective This study intends to explore the role of Alk B homolog 5(ALKBH5)in proliferation of hepatocellular carcinoma(HCC)cells.Method The expression levels of ALKBH5 and pre-mRNA processing factor 6(PRPF6)in HCC tissues were analyzed via the cancer genome atlas database.Real-time fluorescent quantitative polymerase chain reaction and western-blot were used to determine the mRNA and protein expression levels of ALKBH5 and PRPF6 in HCC cell lines(Huh7 and Hep3B).Fluorescence in situ hybridization determined the subcellular localization of ALKBH5 and PRPF6 in HCC cell lines(Huh 7 and Hep3B).The N6-methyladenosine methylation level of PRPF6 was detected in the immunoprecipitation complex.By up-regulation and knockdown of the gene expression,Huh7 cells and Hep3B cells were transfected respectively and were divided into sh-NC group(negative control transfected cells),sh-ALKBH5 group(short hairpin-ALKBH5 transfected cells)and sh-ALKBH5+ex-PRPF6 group(short hairpin-ALKBH5 transfected+overexpressed pcDNA 3.1-PRPF6 transfected cells).Then,CCK-8 assay and dyeing proliferation test were used to determine the proliferation of the cells in different groups.Tumor xenograft in nude mice checked the biological function of ALKBH5 and PRPF6 in vivo.Besides,the expression levels of AKT/mTOR pathway related proteins in the cells of different groups were detected by western-blot.Result ALKBH5 was up-regulated in HCC cells,which could remove the methylation modification of PRPF6.In vitro and in vivo experiments have showed that ALKBH5 could participate in the malignant proliferation of HCC cells by regulating the expression of PRPF6.Moreover,the knockdown of ALKBH5 could inhibit the phosphorylation of serine-threonine kinase(AKT)and mammalian target of rapamycin(mTOR),while the overexpression of PRPF6 contributed to the phosphorylation of AKT and mTOR.Conclusion ALKBH5 can promote the proliferation of HCC cells,and the related mechanism is achieved through the ALKBH5/PRPF6/AKT/mTOR axis.
关键词
AlkB同源蛋白5/前mRNA加工因子6/N6-甲基腺苷/肝细胞癌/增殖
Key words
Alk B homolog 5/Pre-mRNA processing factor 6/N6-methyladenosine/Hepatocellular carcinoma/Proliferation
维普
万方数据