MiR-301b抑制转录因子Klf4影响肝癌细胞迁移

MiR-301b Affects the Migration of Hepatocellular Carcinoma by Inhibiting Klf4

目的:探讨mi R-301b对肝癌细胞迁移能力的影响及其分子机制,为肝癌的分子靶向治疗研究提供新线索。方法:体外培养人肝癌细胞株SK-Hep-1、HCC-LM3和人永生化肝细胞株L02,采用RT-PCR方法检测mi R-301b表达。通过生物信息学软件Targetscan及mi Randa预测mi R-301b的靶基因,筛选出转录因子Klf4基因为mi R-301b的下游靶基因,通过双荧光素酶报告基因实验和Western Blot实验证明其调控作用。通过划痕和Transwell实验探究mi R-301b靶向Klf4基因对肝癌细胞迁移性的影响,Western Blot检测mi R-301b对上皮间质转化标记物E-cadherin、N-cadherin蛋白表达的影响。结果:与正常肝细胞相比,肝癌细胞株中mi R-301b表达水平明显升高。瞬时转染mi R-301b mimic后,实验组mi R-301b的表达显著高于对照组;瞬时转染mi R-301b inhibitor后,实验组mi R-301b的表达显著低于对照组。双荧光素酶报告基因实验显示:mi R-301b直接作用于Klf4基因的3'UTR区,并下调Klf4蛋白的表达,与软件预测结果相符合。划痕实验及Transwell迁移实验显示:mi R-301b通过下调Klf4基因,促进肝癌细胞的迁移。进一步实验显示:过表达mi R-301b显著下调E-cadherin的表达,而上调N-cadherin的表达。结论:mi R-301b在肝癌细胞SK-Hep-1、HCC-LM3中高表达,可能通过抑制靶基因Klf4的表达,促进肝癌的迁移,mi R-301b可能参与了肝癌细胞的上皮间质转化过程。

Objective： To investigate the effect of mi R-301 b on the migration of hepatocellular carcinoma cells and the underlying molecular mechanisms.Methods： Human liver cancer cell lines including SK-Hep-1,HCC-LM3 and human immortalized liver cell line L02 were cultured in vitro.The expression of mi R-301 b was detected by RT-PCR.Through bioinformatics software Targetscan and miRanda,we predicted target genes of mi R-301 b and selected transcription factor Klf4 gene as the downstream target gene of mi R-301 b.Through the dual luciferase reporter gene experiment and Western Blot experiment,we proved its regulating effect.The role of mi R-301 b on the migration of hepatocellular carcinoma cells were verified by wound healing assay and transwell migration assay.The effect of mi R-301 b on the expression of E-cadherin and N-cadherin were investigated by Western Blot.Results： Compared with human immortalized liver cell line L02,the expression of mi R-301 b in HCC cell lines were significantly increased.After being transfected with mi R-301 b mimic,the expression of mi R-301 b was significantly improved than that of the control group.And the expression of mi R-301 b was significantly decreased than that of the control group after being transfected with mi R-301 b inhibitor.The dual-luciferase reporter gene experiment showed that mi R-301 b directly bound with Klf4＇s 3 ＇UTR region and decreased the expression of its protein,which were consistent with the software predicting results.The wound healing and Transwell experiment results showed that mi R-301 b inhibited the migration of hepatocellular carcinoma by downregulating Klf4.Further experiments showed that overexpression of mi R-301 b significantly reduced the level of E-cadherin and increased the level of N-cadherin,which were involved in the process of EMT.Conclusions： mi R-301 b was over-expressed in SK-Hep-1 and HCC-LM3 cells,and promoted the migration ability of HCC cells by inhibiting the expression of Klf4 gene,which may be involved in epithelial mesenchymal transformation progress of HCC cells.