miRNA-21-5p通过pdcd4靶向调控胃癌细胞对顺铂敏感性的机制研究

The mechanism research of miRNA-21-5p in the targeted regulation of the sensitivity of gastric cancer cells to cisplatin by pdcd4

目的:探究微小RNA-21-5p(miRNA-21-5p)通过下调pdcd4基因表达调控胃癌耐药细胞系SGC-7901/DDP对顺铂敏感性机制的研究。方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度的顺铂(0、1、2、4、8、16、32μmol/L)对胃癌细胞系SGC7901及胃癌耐药细胞系SGC7901/DDP增殖抑制的影响。通过实时荧光定量PCR检测SGC7901/DDP细胞中瞬时转染miRNA-21-5p inhibitors后miRNA-21-5p相对表达量的变化。采用Western blot及实时荧光定量PCR检测转染组、无关序列转染组(NC组)及空白转染组(CON组)分别转染miRNA-21-5p inhibitors及无关序列后对靶基因pdcd4蛋白及mRNA表达量的影响。结果:MTT实验结果显示耐药细胞SGC7901/DDP相对于亲本细胞SGC7901对顺铂不敏感(P<0.05)。实时荧光定量PCR结果表明,转染组miRNA-21-5p表达量均明显低于NC组和CON组(P<0.01),转染组pdcd4 mRNA表达量均明显高于NC组和CON组(P<0.01)。Western blot检测结果示,转染组pdcd4蛋白表达量均明显高于NC组和CON组(P<0.01)。结论:miRNA-21-5p表达水平的升高在一定程度上有利于胃癌细胞增殖,并通过下调pdcd4的表达,降低胃癌细胞对顺铂的敏感性。

Objective：To investigate the mechanism of microRNA-21-5p （miRNA-21-5p） in the targeted regulation of sensitivity of gastric cancer resistant cell line SGC7901/DDP to cisplatin by down-regulating the expression of pdcd4 gene. Methods：The inhibition effects of different concentrations of cisplatin（0,1,2,4,8,16,32 μmol/L） on the proliferation of gastric cancer cell line SGC7901 and gastric cancer drug-resistant cell line SGC7901/DDP were detected using the MTT. The relative expression of miRNA-21-5 in SGC7901/DDP cells at）er transient transtecting of miRNA-21-5p inhibitors was detected using real-time fluorescence quantitative poly-merase chain reaction（qRT-PCR）. The expressions of pdcd4 protein and mRNA in transtection group, ineelevant sequence transtection group（ NC group） and blank transtection group（ CON group） were detected after transtecting with miRNA-21-5p inhibitors and inrele-vant sequence, respectively. Results ：The results of MTT assay showed that the drug-resistant ceils SGC7901/DDP were insensitive to cisplatin compared with the parent ceils SGC7901 （P 〈 0.05）. The results of qRT-PCR showed that the expression level of miRNA-21- 5p in transtection group was significantly lower than that in NC group and CON group（ P 〈 0.01 ） , the expression level of pdcd4 mRNA in transtection group was significantly higher than that in NC group and CON group（P 〈 0.01 ）. The results of Western blot showed that the expression level of pdcd4 protein in transtection group was significantly higher than that in NC group and CON group（ P 〈 0.01 ）. Conclusions ：The increasing of miRNA-21-5p expression level is beneficial to the proliteration of gastric cancer ceils, and can reduce the sensitivity of gastric cancer ceils to cisplatin by down-regulating the expression of pdcd4.