摘要
目的探讨miR-143对肾癌GRC-1细胞增殖凋亡及其靶点缺氧诱导因子1α(HIF-1α)的影响。方法转染miR-143的模拟物于肾癌细胞株GRC-1细胞,采用实时定量PCR(q PCR)法检测转染的效果,根据实验将GRC-1细胞分为3组:未转染组、miR-143对照组和miR-143转染组,采用MTT法检测各组细胞的增殖情况,流式细胞术检测各组细胞凋亡情况;q PCR及Western blotting检测各组细胞的HIF-1αmRNA和蛋白水平;双荧光素酶报告基因实验验证miR-143与HIF-1α基因间的靶向关系。结果 miR-143转染组的miR-143水平高于未转染组和miR-143对照组,差异有统计学意义(P<0.05);与其余两组比较,miR-143转染组的增殖率降低而凋亡率升高,差异有统计学意义(P<0.05);瞬时过表达miR-143可降低GRC-1细胞的HIF-1αmRNA和蛋白水平(P<0.01),双荧光素酶报告基因实验证明HIF-1α是miR-143的直接作用靶点。结论miR-143可以调控肾癌GRC-1细胞的增殖凋亡,而HIF-1α是miR-143的直接靶点。
Objective To investigate the effects of miR-143 on proliferation and apoptosis of renal cell carcinoma GRC-1 cells and its target hypoxia inducible factor-1α (HIF-1α). Methods Renal cell carcinoma cell line GRC-1 cells were transfected with miR-143 mimics, and real time quantitative polymerase chain reaction (qPCR) was performed to evaluate the efficiency of transfection. According to the experiment, the GRC-1 cells were divided into 3 groups: non-transfection group, miR-143 control group and miR-143 transfection group. MTT method was used to detect the proliferation of cells in each group. Flow cytometry was used to detect the apop- tosis of each group. The qPCR and Western blotting were employed to measure the mRNA and protein level of HIF-1α. Dual luciferase reporter gene was applied to verify the relationship between miR-143 and HIF-1α. Results The level of miR-143 in miR-143 transfec- tion group was higher than those in non-transfection group and miR-143 control group, and the difference was statistically significant (P 〈0.05). Compared with other two groups, there were decreased proliferation rates but increased apoptotic rates in miR-143 transfection group (P〈0.05). Transient overexpression of miR-143 in GRC-1 cells decreased the expression of HIF-1α on both mRNA and protein levels. Moreover, dual-luciferase reporter assay confirmed that HIF-1α was a direct target of miR-143 in GRC-1 cells. Conclusion miR-143 may regulate the proliferation of GRC-I cells, and HIF-lct is a direct target of miR-143.
出处
《临床肿瘤学杂志》
CAS
2016年第11期967-971,共5页
Chinese Clinical Oncology
基金
溧阳科技局2014科技支撑(社会发展)资助项目(LC2014001)
关键词
肾癌
增殖
凋亡
双荧光素酶报告基因
MIR-143
缺氧诱导因子1Α
Renal cell carcinoma
Proliferation
Apoptosis
Dual luciferase reporter gene
MiR-143
Hypoxia in- ducible factor-1α