Let-7a下调k-Ras和c-Myc癌基因的表达抑制肾癌细胞增殖

目的：MicroRNA 是近年发现的一类单链小分子RNA，对它的研究已成为一个新的热点。最近的研究发现，1et-7a 在细胞内影响着基因的表达调控，在疾病发生中起着及极重要的作用，尤其是在肿瘤的发展过程中，let-7a扮演着不可替代的角色。本文主要研究let-7a 在肾癌细胞株中的表达情况及其调控的靶基因、抑制细胞增殖的机制，对探索肾癌的致病基因，寻求肾癌新的治疗途径有重要意义。方法：应用化学合成的let-7a 模拟物（mimics）用脂质体Lipofectamine 2000 在体外瞬时转染786-O 和Caki-1肾癌细胞株，转染48 小时后采用荧光定量RT-PCR的方法检测let-7a 及c-Myc、k-Ras mRNA的表达情况，Western blot 检测这两株肾癌细胞转染了let-7a mimics 后c-Myc 及k-Ras 蛋白的表达变化；转染let-7a mimics 后分别在24、48、72 小时三个时间点用CCK-8 试剂盒检测对肾癌细胞株增殖的影响。结果：786-O 和Caki-1 肾癌细胞株中let-7a 的表达量明显低于正常肾小管上皮细胞株HK-2（P〈0.05）; 转染了let-7amimics的786-O 和Caki-1 肾癌细胞株，RT-PCR 及Western blot 结果显示c-Myc、k-Ras 在基因及蛋白的表达水平明显下调（P〈0.05）；CCK-8 检测结果显示转染了let-7a mimics的肾癌细胞株细胞增殖能力明显明显受到抑制，与阴性对照组比较差异有统计学意义（P〈0.05）。结论：Let-7a 在在肿瘤细胞与正常细胞中存在明显差异，let-7a 通过调控c-Myc、k-Ras的表达能抑制肾癌细胞增殖。Let-7a mimics 可以抑制肾癌细胞的增殖，因此上调Let-7a 的表达有可能成为肾癌基因治疗的一种有效治疗手段。

Let-7a Inhibits the Proliferation in Renal Cell Carcinoma Cell Lines by Regulating k-Ras and c-Myc Gene Expression

Objective： MicroRNA is a type of the single small RNA discovered in recent years, and the study of it has become a new hot spot. Let-7a plays a very important role and affects the expression of genes in cells. Especially let-7a plays an irreplaceable role in the process of tumor development.This article mainly researches the expression of let-7a in kidney cancer cell lines and regulation of target genes and the mechanism of inhibition of cell proliferation. It is a great significance to explore the pathogenic genes and new treat- ment approaches in kidney cancer. Methods： Application of chemical synthesis of the let-7a mimics with Lipofectamine 2000 transient transfects in 786-0 and Caki-1 kidney cancer cell lines was taken. The let-7a and c-Myc, k-Ras mRNA expressions were detected with method of RT-PCR after transfection. The expression changeswere tested with Western blot. Effects of let-7a in cell proliferation were eval- uated by CCK-8 assay. Results： Transfection of the let-7amimics in Cak-i and 786-Orenal cancer cell lines, that the c-Myc, k-Ras gene and protein expression level significantly reduced（P〈0.05）. The expression levels of let-7a in 786-0 and Caki-1 ells were lower than nor- mal renal tubular epithelial cell lines HK-2 （P〈0.05）. Over-expression of let-7a in 786-O and Caki- 1 cells suppressed c-Myc, k-Ras gene and protein level. The result showed by RT-PCR and Western blot （P〈0.05）. CCK-8 test results showed that transfection let-7a mimics inhibit their proliferation ability was significantly suppressed compared with negative control group（P〈0.05）. Conclusion： These findings suggest that Let-7a plays an important role in kidney cancer cell lines proliferation through suppressing c-Myc, k-Ras in vitro, which pro- vides a potential development of a new approach for the treatment of renal cancer.