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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。
Objective: MicroRNA is a kind of single-stranded small molecule RNA discovered in recent years. Its research has become a new hot spot. Recent studies have found that 1et-7a affects the gene expression and regulation in the cell and plays a very important role in the pathogenesis of the disease, especially let-7a plays an irreplaceable role in the development of the tumor. This article mainly studies the expression of let-7a in renal cell carcinoma cell lines, its target genes and the mechanism of inhibiting cell proliferation. It is of great significance to explore the causative genes of renal cell carcinoma and to seek new therapeutic approaches for renal cell carcinoma. METHODS: Chemically synthesized let-7a mimics were transiently transfected into 786-O and Caki-1 cells using Lipofectamine 2000. The transfected cells were transfected 48 hours later by fluorescence quantitative RT-PCR The expressions of let-7a and c-Myc, k-Ras mRNA were detected by Western blot. The expressions of c-Myc and k-Ras protein in let-7a mimics were detected by Western blot. After 7d mimics, CCK-8 kit was used to detect the effect on the proliferation of renal cancer cell lines at 24, 48 and 72 hours respectively. Results: The expression of let-7a in 786-O and Caki-1 cell lines was significantly lower than that in normal renal tubular epithelial cell line HK-2 (P <0.05). 786-O and Caki transfected let-7 amimics The results of RT-PCR and Western blot showed that the expression of c-Myc and k-Ras was down-regulated in gene and protein (P <0.05). The results of CCK-8 assay showed that transfected let-7a mimics Of renal cell carcinoma cell proliferation was significantly inhibited, compared with the negative control group, the difference was statistically significant (P <0.05). Conclusion: Let-7a in tumor cells and normal cells there is a significant difference, let-7a through the regulation of c-Myc, k-Ras expression can inhibit renal cell proliferation. Let-7a mimics can inhibit the proliferation of renal cell carcinoma, so up-regulation of Let-7a expression may become an effective treatment of renal cell carcinoma gene therapy.