miR-100通过Akt/mTOR信号通路对胃癌细胞增殖的负调控作用  

Negative regulation of miR-100 on gastric cancer cell proliferation through Akt/mTOR signaling pathway

作　　者：郭利华[1] 胡桂梅[1] 丁勇[1] 王静静 叶国良[1] GUO Lihua;HU Guimei;DING Yong;WANG Jingjing;YE Guoliang(Department of Gastroenterology,the Affiliated Hospital of Medical School,Ningbo University,Zhejiang,Ningbo 315020,China)

机构地区：[1]宁波大学医学院附属医院消化内科,浙江宁波315020

出　　处：《中国现代医生》2022年第28期41-45,53,共6页China Modern Doctor

基　　金：宁波市科技计划自然科学基金(2019A610328)。

摘　　要：目的探讨miR-100对胃癌细胞的生物学作用及对蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的调控作用。方法应用定量反转录聚合酶链反应(quantitative reverse transcription polymerase chain reaction,qRT-PCR)检测人胃癌细胞株BGC-823、SGC-7901、MKN-45和胃黏膜上皮细胞株GES-1中miR-100的表达差异;将人工合成的miR-100模拟剂(mimics)及阴性对照转染至人胃癌细胞株BGC-823中,使用CCK-8法检测细胞增殖情况,流式细胞术检测细胞周期和细胞凋亡;应用双荧光素酶报告基因验证miR-100靶基因,应用qRT-PCR和蛋白质印迹法分析Akt/mTOR信号通路相关基因及蛋白表达水平。结果胃癌细胞株miR-100的表达水平显著低于胃黏膜上皮细胞株(P<0.05);转染后48h、72h,miR-100 mimcs组吸光度值均显著低于对照组(P<0.05);流式细胞术显示miR-100 mimics组G_(0)/G_(1)期细胞比率显著高于对照组(P<0.05),S期、G_(2)/M期细胞比率均显著低于对照组(P<0.05);miR-100 mimics组细胞凋亡率与对照组比较差异无统计学意义(P>0.05);双荧光素酶报告显示miR-100 mimics组野生型mTOR荧光酶活性被抑制(P<0.05);miR-100 mimics组胃癌细胞Akt、mTOR、p70S6K的mRNA及蛋白表达水平均显著降低(P<0.05)。结论miR-100在胃癌细胞中表达降低,miR-100可靶向调控mTOR抑制胃癌细胞增殖,阻碍细胞周期进程。Objective To investigate the biological effects of miR-100 on gastric cancer cells and its regulation of protein kinase B/mammalian target of rapamycin(Akt/mTOR) signaling pathway. Methods Quantitative reverse transcription polymerase chain reaction(qRT-PCR) was used to detect the expression of miR-100 in human gastric cancer cell lines BGC-823, SGC-7901, MKN-45 and gastric mucosal epithelial cell line GES-1. The synthetic miR-100 mimics and negative control were transfected into human gastric cancer BGC-823 cells. The CCK-8 assay was used to detect cell proliferation activity, and the flow cytometry was used to detect cell cycle distribution and apoptosis. The miR-100 target gene was verified by double-luciferase reporter gene assay. The expression levels of Akt/mTOR signaling pathway related mRNA and proteins were further analyzed by qRT-PCR and Western blotting. Results The expression of miR-100 in gastric cancer cell line was significantly lower than that in normal gastric mucosal cell line(P<0.05);the absorbance value of miR-100 mimics group was significatly lower than that of control group after being transfected for 48 and 72 hours(P<0.05). Flow cytometry showed that the proportion of G_(0)/G_(1) cells in miR-100 mimics group was significantly higher than that in control group(P<0.05), the ratio of S phase and G_(2)/M phase cells was significantly lower than that in control group(P<0.05). There was no significant difference in apoptosis rate between miR-100 mimics group and control group(P<0.05). The dual-luciferase reporter gene assay showed that the luciferase activity of wild-type mT OR in miR-100 mimics group was inhibited(P<0.05). The expression of Akt, mTOR, p70S6K mRNA and protein decreased significantly in miR-100 mimics group(P<0.05). Conclusion The expression of miR-100 decreased in gastric cancer cells, and it can inhibit the proliferation and hinder cell cycle of gastric cancer cells by down regulating the expression of mTOR.

关 键 词：胃癌 miR-100 增殖 细胞周期 凋亡 Akt/mTOR信号通路 

分 类 号：R735.2[医药卫生—肿瘤]