| 董 笑,王 凡,刘 传,宋卫峰,李 琦.MiR-301b抑制转录因子Klf4影响肝癌细胞迁移[J].现代生物医学进展英文版,2018,(10):1806-1811. |
| 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 |
| Received:January 28, 2018 Revised:February 21, 2018 |
| DOI：10.13241/j.cnki.pmb.2018.10.002 |
| 中文关键词: MicroRNA-301b Klf4 肝细胞癌 迁移 |
| 英文关键词: MicroRNA Klf4 Hepatocellular carcinoma Migration |
| 基金项目:国家自然科学基金面上项目（81572310） |
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| 中文摘要: |
| 摘要 目的：探讨miR-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miR-301b表达。通过生物信息学软件Targetscan及miRanda预测miR-301b的靶基因，筛选出转录因子Klf4基因为miR-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miR-301b靶向Klf4基因对肝癌细胞迁移性的影响，Western Blot检测miR-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miR-301b表达水平明显升高。瞬时转染miR-301b mimic后，实验组miR-301b的表达显著高于对照组；瞬时转染miR-301b inhibitor后，实验组miR-301b的表达显著低于对照组。双荧光素酶报告基因实验显示：miR-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：miR-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miR-301b显著下调E-cadherin的表达，而上调N-cadherin的表达。结论：miR-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，miR-301b可能参与了肝癌细胞的上皮间质转化过程。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect of miR-301b 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 miR-301b was detected by RT-PCR. Through bioinformatics software Targetscan and mi- Randa, we predicted target genes of miR-301b and selected transcription factor Klf4 gene as the downstream target gene of miR-301b. Through the dual luciferase reporter gene experiment and Western Blot experiment, we proved its regulating effect. The role of miR-301b on the migration of hepatocellular carcinoma cells were verified by wound healing assay and transwell migration assay. The effect of miR-301b on the expression of E-cadherin and N-cadherin were investigated by Western Blot. Results: Compared with human immortal- ized liver cell line L02, the expression of miR-301b in HCC cell lines were significantly increased. After being transfected with miR-301b mimic, the expression of miR-301b was significantly improved than that of the control group. And the expression of miR-301b was sig- nificantly decreased than that of the control group after being transfected with miR-301b inhibitor. The dual-luciferase reporter gene ex- periment showed that miR-301b directly bound with Klf4's 3 'UTR region and decreased the expression of its protein, which were consis- tent with the software predicting results. The wound healing and Transwell experiment results showed that miR-301b inhibited the migra- tion of hepatocellular carcinoma by downregulating Klf4. Further experiments showed that overexpression of miR-301b significantly re- duced the level of E-cadherin and increased the level of N-cadherin, which were involved in the process of EMT. Conclusion: miR-301b 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. |
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