敲减microRNA-205靶向PTEN抑制食管癌细胞TE1的增殖并促进细胞凋亡
| 作者: |
1宋爽,
1郭海
1 河南医学高等专科学校基础医学部组胚教研室，郑州 450000 |
| 通讯: |
宋爽
Email: songxingrongmed@126.com |
| DOI: | 10.3978/j.issn.2095-6959.2017.04.016 |
摘要
目的：探讨miRNA-205对食管癌细胞株TE1增殖和凋亡能力的影响及其作用机制。方法：实时定量聚合酶链式反应(qRT-PCR)和Western印迹检测正常食管黏膜细胞Het-1A和不同食管癌细胞株(KYSE70，TE12，TE1)中miRNA-205的mRNA及蛋白表达水平。转染miRNA-205抑制剂下调TE1细胞中miRNA-205的表达，采用CCK-8法和流式细胞术检测细胞增殖能力、细胞周期和细胞凋亡情况；Western印迹检测细胞增殖和细胞凋亡相关CDK2，cyclin D1，P21，Bcl-2，cleaved-caspase-3，caspase-3，p-Rb，Rb，p-Akt和Akt的蛋白表达水平。双荧光素酶报告基因分析法预测及验证其可能的靶基因。结果：MiRNA-205在各型食管癌细胞中高表达。沉默miRNA-205后，TE1细胞的增殖能力降低并表现为细胞周期阻滞，而细胞凋亡率显著升高(P<0.05)。同时细胞中cyclin D1，CDK2，bcl-2，p-Rb及p-Akt的蛋白表达水平均显著降低，p21及cleaved-caspase-3的蛋白表达水平显著升高。双荧光素酶报告基因分析显示PTEN是miRNA-205的可能作用靶点，TE1中共转染miRNA-205抑制剂和PTEN siRNA可部分逆转miRNA-205介导细胞增殖抑制及凋亡诱导作用。结论：沉默miRNA-205可靶向PTEN抑制食管癌细胞株TE1的增殖，并促进其凋亡，提示miRNA-205可作为食管癌诊疗的一个潜在作用靶点。
关键词：
miRNA-205
食管癌
增殖
凋亡
PTEN
Knockdown microRNA-205 inhibits proliferation and prompts apoptosis by targeting PTEN in TE1 cells
CorrespondingAuthor: SONG Shuang Email: songxingrongmed@126.com
DOI: 10.3978/j.issn.2095-6959.2017.04.016
Abstract
Objective: To investigate the regulatory effects and its underlying mechanisms of miRNA-205 on TE1 proliferation and apoptosis. Methods: The abundance of miRNA-205 in normal esophageal cell line Het-1A and different esophageal cancer cell lines (KYSE70, TE12, TE1) were measured by qRT-PCR and Western blot. After knockdown of miRNA-205 with inhibitor in TE1 cells, cell proliferation, cell cycle distribution and cell apoptosis were detected by CCK-8 assay, flow cytometry. Furthermore, the expression of cell cycle and cell apoptosis related proteins, such as cyclin D1, CDK2, P21, bcl-2, cleaved-caspase-3, caspase-3, p-Rb, Rb p-Akt and Akt were measured by Western blot assay. Luciferase reporter assay was used to forecast and verify target gene of miRNA-205. Results: MiRNA-205 was highly expressed in esophageal cancer cells. knockdown of miRNA-205 inhibited TE1 cell proliferation, caused cell cycle arrest and cell apoptosis. Furthermore, the protein expression of cyclin D1, CDK2, Bcl-2 , p-Rb and p-Akt significantly decreased after knockdown of miRNA-205, accompanied with the up-regulation of p21 and cleaved-caspase3 protein expression. Luciferase reporter assay demonstrated that PTEN was a potential target gene of miRNA-205. Down-regulated of PTEN in TE1 cells transfected with miRNA-205 inhibitor partially reversed the effect of miRNA-205 on cell proliferation and apoptosis. Conclusion: Knockdown of miRNA-205 restrained cell proliferation and promoted cell apoptosis in TE1 cells by targeting PTEN, suggesting that miRNA-205 may be a potential new therapeutic target of esophageal cancer.