miR-4698介导GALNT4表达对肝癌细胞增殖和迁移的影响

Effect of miR-4698 mediated GALNT4 expression on proliferation and migration of hepatocellular carcinoma cells

目的检测miR-4698在肝癌组织及细胞系中的相对表达,分析其对肝癌细胞增殖和迁移的影响及分子作用机制。方法采用实时荧光定量PCR(qRT-PCR)法检测肝癌组织和肝癌细胞系中miR-4698的相对表达。在表达最低的肝癌细胞系中,采用脂质体转染法分别转染miR-4698模拟物和对照模拟物,命名为实验组和对照组。qRT-PCR检测转染效率。细胞计数试剂盒(CCK-8)和Transwell迁移实验分别检测过表达miR-4698对肝癌细胞增殖和迁移能力的影响。采用生物信息学和双荧光素酶报告基因实验预测并验证miR-4698与靶基因的结合。qRT-PCR和Western blot分别检测靶基因在mRNA水平和蛋白水平的相对表达。结果与癌旁组织相比,miR-4698在肝癌组织的表达明显降低(P<0.01)。与正常肝细胞系相比,miR-4698在肝癌细胞系的表达明显降低(P<0.05),在Huh7细胞的表达最低(P<0.01)。转染后,与对照组相比,实验组Huh7细胞中miR-4698的表达明显增加(P<0.01)。过表达miR-4698可抑制肝癌Huh7细胞的增殖能力(P<0.05)和迁移能力(P<0.05)。生物信息学显示miR-4698的靶基因是多肽-N-乙酰氨基半乳糖转移酶4(GALNT4),双荧光素酶报告基因证实miR-4698可互补结合GALNT4(P<0.01)。qRT-PCR结果显示,过表达miR-4698可抑制GALNT4基因的表达(P<0.01)。Western blot实验显示,miR-4698过表达后,Huh7细胞中GALNT4蛋白表达降低,细胞增殖相关蛋白Cyclin B和CDK1表达降低,细胞转移相关蛋白N-cadherin和ZEB-2表达降低。结论miR-4698在肝癌组织和细胞系中表达明显下降,过表达miR-4698可通过抑制GALNT4基因的表达,降低肝癌Huh7细胞的增殖和迁移能力。

Objective The relative expression of miR-4698 in liver cancer tissues and cell lines was detected,and its effect on the proliferation and migration of liver cancer cells and its molecular mechanism were analyzed.Methods Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to analyze the relative expression of miR-4698 in liver cancer tissues and liver cancer cell lines.Among the lowest-expressing hepatocellular carcinoma cell lines,miR-4698 mimic and control mimic were transfected with liposome transfection method,and named as experimental group and control group.qRT-PCR was used to detect transfection efficiency.Cell counting kit-8(CCK-8)and transwell migration experiments were used to detect the effects of overexpressing miR-4698 on the proliferation and migration ability of liver cancer cells.Bioinformatics and dual luciferase reporter gene experiments were used to predict and verify the binding of miR-4698 to target gene.qRT-PCR and Western blot were used to detect the relative expression of target gene at mRNA and protein levels,respectively.Results Compared with the adjacent tissues,miR-4698 was significantly lower in the liver cancer(P<0.01).Compared with normal hepatocytes,miR-4698 was significantly lower in hepatoma cell lines(P<0.05),and the lowest in Huh7 cells(P<0.01).After transfection,the expression of miR-4698 in Huh7 cells in experimental group was significantly increased compared with that in the control group(P<0.01).Overexpression of the miR-4698 can inhibit the proliferation and migration of Huh7 cells(P<0.05).Bioinformatics showed that the target gene of miR-4698 was polypeptide-n-acetylgalactosamine transferase 4(GALNT4),and the double luciferase reporter gene confirmed that miR-4698 could bind to GALNT4(P<0.01).qRT-PCR showed that overexpression of miR-4698 could inhibit the expression of GALNT4 gene(P<0.01).Western blot results showed that overexpression of miR-4698 decreased the protein expression of GALNT4,cyclin B and CDK1,and increased the protein expression of N-cadherin and ZEB-2.Conclusions The expression of miR-4698 in liver cancer tissues and cell lines is significantly reduced.Overexpression of miR-4698 can inhibit the expression of GALNT4 gene and reduce the proliferation and migration ability of liver cancer Huh7 cells.