miR-143对肾癌GRC-1细胞增殖与凋亡及其靶点HIF-1α的影响
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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-1cells 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( q PCR) 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 apoptosis of each group. The q PCR 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 transfection 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-1 cells,and HIF-1α is a direct target of miR-143.
Objective To investigate the effects of miR-143 on proliferation and apoptosis of renal cell carcinoma GRC-1cells 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( q PCR) 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 apoptosis of each group. The q PCR 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 transfection 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-1 cells,and HIF-1α is a direct target of miR-143.
引文
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[14]彭启宇,温儒民,薛松.HIF-1α与CAⅨ在肾细胞癌中的表达及临床意义[J].徐州医学院学报,2008,28(4):229-231.
[2]Li M,Wang Y,Song Y,et al.MicroRNAs in renal cell carcinoma:a systematic review of clinical implications(Review)[J].Oncol Rep,2015,33(4):1571-1578.
[3]Yoshino H,Enokida H,Itesako T,et al.Tumor-suppressive microRNA-143/145 cluster targets hexokinase-2 in renal cell carcinoma[J].Cancer Sci,2013,104(12):1567-1574.
[4]田晓琳,杨臻,王建英,等.微小RNA与肿瘤的关系[J].癌症进展,2016,14(1):22-25.
[5]Zhang HB,Sun LC,Ling L,et al.miR-143 suppresses the proliferation of NSCLC cells by inhibiting the epidermal growth factor receptor[J].Exp Ther Med,2016,12(3):1795-1802.
[6]Wang L,He J,Xu H,et al.MiR-143 targets CTGF and exerts tumor-suppressing functions in epithelial ovarian cancer[J].Am J Transl Res,2016,8(6):2716-2726.
[7]He Z,Yi J,Liu X,et al.MiR-143-3p functions as a tumor suppressor by regulating cell proliferation,invasion and epithelialmesenchymal transition by targeting QKI-5 in esophageal squamous cell carcinoma[J].Mol Cancer,2016,15(1):51.
[8]Yang Z,Chen D,Nie J,et al.MicroRNA-143 targets CD44 to inhibit breast cancer progression and stem cell-like properties[J].Mol Med Rep,2016,13(6):5193-5199.
[9]Liu H,Wang H,Liu H,et al.Effect of miR-143 on the apoptosis of osteosarcoma cells[J].Int J Clin Exp Pathol,2015,8(11):14241-14246.
[10]马杰,茹国庆,赵仲生,等.缺氧诱导因子-1αmRNA表达与胃癌血管生成、侵袭转移及预后的关系[J].中华普通外科杂志,2006,21(10):752-753.
[11]余平,刘晓旺,钟丽菲,等.低氧微环境相关因子HIF-1α与癌症的研究[J].湖南生态科学学报,2015,2(2):52-56.
[12]王健,郝继辉.HIF-1α在肿瘤乏氧微环境中的作用及其靶向治疗的研究进展[J].中国肿瘤临床,2013,40(17):1072-1075.
[13]刘俊,崔龙.肾癌患者血清VHL、HIF-1α和HIF-2α的表达及临床意义[J].实用癌症杂志,2016,31(6):895-898.
[14]彭启宇,温儒民,薛松.HIF-1α与CAⅨ在肾细胞癌中的表达及临床意义[J].徐州医学院学报,2008,28(4):229-231.