舒尼替尼调控miR-143对人肾癌细胞增殖、侵袭的影响

Effect of regulation of miR-143 by sunitinib on the proliferation and invasion of human renal carcinoma cells

目的:探讨miR-143在肾癌细胞中的作用、舒尼替尼对miR-143表达的影响及与细胞增殖、侵袭的关系。方法:培养人肾癌786-O细胞株,分为对照组、miR-143组、舒尼替尼组、共同作用组。对照组细胞转染阴性对照序列,miR-143组细胞转染miR-143序列,舒尼替尼组细胞用舒尼替尼处理48 h,共同作用组细胞转染miR-143序列并用舒尼替尼处理48 h。MTT法检测细胞的增殖,Transwell检测其侵袭,qRT-PCR检测miR-143RNA表达水平,Western blotting检测DNA甲基转移酶3B(DNMT3B)、p-Akt-S473蛋白表达量。结果:成功构建稳定、高表达miR-143的人肾癌786-O细胞株,miR-143转染组细胞miR-143表达量增高(P<0.01)。舒尼替尼作用于786-O细胞48 h的IC50值为6μmol/L,选取该值作为后续实验用药浓度。786-O细胞经舒尼替尼作用48 h后miR-143表达量升高(P<0.01)。miR-143组和舒尼替尼组细胞存活率、细胞侵袭数、DNMT3B和p-AktS473蛋白表达量均低于对照组,且高于共同作用组,差异均有统计学意义(P<0.05~P<0.01)。结论:miR-143可以降低786-O细胞增殖、侵袭能力,这可能与miR-143降低DNMT3B、p-Akt的表达有关。舒尼替尼可能通过上调miR-143水平抑制786-O细胞的增殖与侵袭。

Objective:To investigate the role of miR-143 in renal cell carcinoma,the effect of sunitinib on the expression of miR-143 and its association with cell proliferation and invasion.Methods:The human renal carcinoma 786-O cells were divided into control group,miR-143 group,sunitinib group and co-action group.The negative control sequence was transfected into the cells of control group,the miR-143 sequence was transfected into the cells of miR-143 group,sunitinib group cells were treated with sunitinib for 48h,the co-acting group was transfected miR-143 sequence and treated with sunitinib for 48h.Proliferation and invasion of 786-O cells were detected by MTT and transwell experiment,miR-143 RNA expression was detected by qRT-PCR,DNA methyltrans-ferase3B(DNMT3B).The expressions of p-Akt-S473 protein were detected by Western blotting.Results:The human renal cancer 786-O cells with stable and high expression of miR-143 were successfully constructed.The expression of miR-143 was increased in the miR-143 transfected group(P<0.01).The IC50 value in 786-O cells with the treatment of sunitinib for 48 h was 6μmol/L,which was selected as the drug concentration in the subsequent experiment.The expression of miR-143 in 786-O cells was increased after the treatment of sunitinib for 48 h(P<0.01).Cell survival rate,cell invasion number,expressions of DNMT3B and p-Akt-S473 protein in miR-143 and sunitinib group were all lower than control group and higher than co-action group(P<0.05 to P<0.01).Conclusions:miR-143 reduced the proliferation and invasion capacity of 786-O cells,which may be related to the decreased expression of DNMT3B and p-Akt regulated by miR-143.Sunitinib may inhibit the proliferation and invasion of 786-O cells by upregulating miR-143.