长链非编码RNA MACC1-AS1调控AKT/m TOR通路介导胃癌顺铂耐药的研究  

Long non-coding RNA MACC1-AS1 mediates cisplatin resistance in gastric cancer through AKT/mTOR pathway

作　　者：努尔麦麦提·叶尔逊[1] 白洁[1] NUERMAIMAITI·Yeerxun;BAI Jie(Department of Gastroenterology,The Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830028,P.R.China)

机构地区：[1]新疆医科大学第二附属医院消化内科,乌鲁木齐830028

出　　处：《中国普外基础与临床杂志》2022年第4期458-464,共7页Chinese Journal of Bases and Clinics In General Surgery

摘　　要：目的探讨长链非编码RNA结肠癌相关转移基因1反义RNA(metastasis-associated in colon cancer 1-antisense RNA,MACC1-AS1)在顺铂耐药胃癌中的作用及其可能机制。方法选取人胃癌细胞株BGC823和顺铂耐药胃癌细胞BGC823/DDP为研究对象。对BGC823/DDP细胞进行转染,分为阴性对照组(si-NC组,转染si-NC空质粒)和MACC1-AS1基因沉默组(si-MACC1-AS1组,转染si-MACC1-AS1质粒);同时对BGC823细胞进行转染,分为阳性对照组(pcDNA-NC组,转染pcDNA-NC空质粒)和MACC1-AS1基因过表达组(pcDNA-MACC1-AS1组,转染pcDNA-MACC1-AS1质粒)。采用MTT法检测细胞增殖抑制情况和细胞半抑制浓度(50%inhibiting concentration,IC_(50))值;采用流式细胞术检测细胞凋亡情况;采用实时荧光定量PCR检测细胞中MACC1-AS1、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关的X基因(Bax)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、磷酸化mTOR(phosphorylated mTOR,p-mTOR)、蛋白激酶B(protein kinase B,AKT)和磷酸化AKT(phosphorylated AKT,p-AKT)的mRNA表达水平;采用Western blot法检测细胞Bax、Bcl-2、p-mTOR、mTOR、AKT和p-AKT蛋白表达水平。结果BGC823/DDP细胞中MACC1-AS1 mRNA的相对表达量高于BGC823胃癌细胞(P<0.01);si-MACC1-AS1组细胞中MACC1-AS1 mRNA相对表达量低于si-NC组(P<0.001);pc DNA-MACC1-AS1组细胞MACC1-AS1 mRNA相对表达量高于pc DNA-NC组(P<0.0001)。si-MACC1-AS1组的细胞生长抑制率和IC_(50)值均高于si-NC组(P<0.001);pcDNA-MACC1-AS1组的细胞生长抑制率和IC_(50)值均低于pcDNA-NC组(P<0.001)。pcDNA-MACC1-AS1组细胞的Bcl-2、p-AKT/AKT和p-mTOR/mTOR的蛋白和mRNA相对表达量均高于pcDNA-NC组(P<0.01),Bax蛋白和mRNA相对表达量低于pcDNA-NC组(P<0.01);pcDNA-MACC1-AS1组的细胞凋亡率低于pcDNA-NC组(P<0.01)。si-MACC1-AS1组细胞的Bcl-2、p-AKT/AKT和p-mTOR/mTOR的蛋白和mRNA相对表达量均低于si-NC组(P<0.01),Bax蛋白和mRNA相对表达量高于si-NC组(P<0.01);si-MACC1-AS1组的细胞凋亡率高于si-NCObjective To investigate the role of long non-coding RNA metastasis-associated in colon cancer 1-antisense RNA(MACC1-AS1)in cisplatin resistant gastric cancer and its possible mechanism.Methods Human gastric cancer cell line BGC823 and cisplatin resistant gastric cancer cell line(BGC823/DDP)were selected as the research objects.BGC823/DDP cells were transfected and divided into negative control group(si-NC group,transfected with si-NC empty plasmid)and MACC1-AS1 gene silencing group(si-MACC1-AS1 group,transfected with si-MACC1-AS1plasmid).The BGC823 cells were transfected and divided into positive control group(pcDNA-NC group,transfected with pcDNA-NC empty plasmid)and MACC1-AS1 gene overexpression group(pcDNA-MACC1-AS1 group,transfected with pcDNA-MACC1-AS1 plasmid).MTT was used to detect the inhibition and 50%inhibition concentration(IC_(50)).Flow cytometry was used to detect apoptosis.Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of MACC1-AS1,B-lymphoma-2 gene(Bcl-2),Bcl-2 related X gene(Bax),mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),protein kinase B(AKT),and phosphorylated AKT(p-AKT).Western blot was used to detect the protein expression levels of Bax,Bcl-2,p-mTOR,mTOR,AKT,and p-AKT.Results The relative expression level of MACC1-AS1 mRNA in BGC823/DDP cells was higher than that in BGC823 gastric cancer cells(P<0.01).The relative expression level of MACC1-AS1 mRNA in the si-MACC1-AS1 group cells was lower than that in the si-NC group cells(P<0.01).The relative expression level of MACC1-AS1 mRNA in the pcDNA-MACC1-AS1 group cells was higher than that in the pcDNA-NC group cells(P<0.01).The cell growth inhibition rate and IC_(50) of the si-MACC1-AS1 group were higher than those of the si-NC group(P<0.01).The cell growth inhibition rate and IC_(50) of the pcDNA-MACC1-AS1group were lower than those of the pcDNA-NC group(P<0.01).The mRNA and protein relative expression levels of Bcl-2,p-AKT/AKT and p-mTOR/mTOR in the pcDNA-MACC1-AS1 group were significan

关 键 词：长链非编码RNA 结肠癌相关转移基因1反义RNA 胃癌 耐药性 凋亡 

分 类 号：R735.2[医药卫生—肿瘤]