摘要
背景ARHI已经被证实表达缺失与肿瘤的发生与进展相关.但是,目前尚不清楚ARHI基因对胃癌(gastric cancer,GC)是否具有增殖抑制作用,此次实验以GC细胞株MKN28为例,构建高表达pcDNA3.1-ARHI质粒,通过细胞转染技术转染MKN28细胞株,进一步研究ARHI基因对GC细胞增殖、侵袭及凋亡的影响.目的以GC细胞MKN28为例,研究ARHI基因对GC细胞增殖、侵袭及凋亡的影响,并探讨其机制.构建pcDNA3.1-ARHI质粒,通过细胞转染技术转染MKN28细胞株,取处于对数期的空白对照组、阴性对照Mock组及ARHI高表达克隆株clone4细胞进行实验,以MTT比色法检测细胞增殖;细胞划痕检测细胞迁移能力;Transwell法检测细胞侵袭能力;流式细胞术检测细胞凋亡率;Western blot检测相关蛋白表达.结果与正常MKN28细胞48 h增殖率1.257%±0.006%相比,Mock组与ARHI高表达克隆株48h细胞增殖率分别为1.163%±0.003%(P>0.05),0.826%±0.005%(P<0.05);与正常MKN28细胞48 h迁移率(19.918%±0.233%)相比,Mock组与ARHI高表达克隆株48 h细胞迁移率分别为18.295%±0.534%(P>0.05),4.299%±1.572%(P<0.05);与正常MKN28细胞48 h侵袭率(234±3.61)相比,Mock组与ARHI高表达克隆株48 h细胞侵袭率分别为235±4.51(P>0.05),93.3±2.08(P<0.05);与正常MKN28细胞48 h总凋亡率(3.513%±0.015%)相比,Mock组与ARHI高表达克隆株48 h细胞总凋亡率分别为3.597%±0.25%(P>0.05),14.133%±0.032%(P<0.05);Western blot检测各组细胞内蛋白结果显示:与MKN28细胞相比,Mock组中ARHI蛋白为1.037±0.003(P>0.05),血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白为1.026±0.008(P>0.05),B淋巴细胞瘤-2(B-cel l lymphoma-2,Bcl-2)蛋白为1.014±0.010(P>0.05),蛋白激酶B(protein kinase B,AKT)蛋白为1.001±0.005(P>0.05),磷酸化AKT(p-AKT)蛋白为0.977±0.003(P>0.05);高表达克隆株clone4中ARHI蛋白为2.088±0.007(P<0.05),VEGF蛋白为0.456±0.004(P<0.05),Bcl-2蛋白为0.468±0.005(P<0.05),AKT蛋白为0.969±0.005(P>0.05),p-AKT蛋白为0.502±0.001(P<0.05).结论ARHI基因过表达可抑制GC细胞MKN28的增殖,促进细胞凋亡,这可能与ARHI基因高表达后导致磷脂酰肌醇3-激酶/AKT通路中的VEGF、p-AKT蛋白表达降低有关.
BACKGROUND ARHI has been proved to be associated with tumorigenesis and progression.However,it is not clear whether ARHI gene overexpression can inhibit the proliferation of gastric cancer(GC).In this study,we investigated the effect of ARHI gene overexpression on cell proliferation,invasion,and apoptosis in GC cell line MKN28.AIM To investigate the effect of ARHI overexpression on the proliferation,invasion,and apoptosis of gastric carcinoma MKN28 cells and to explore the possible mechanisms involved.METHODS The pcDNA 3.1-ARHI plasmid was constructed and used to transfect MKN28 cells.Meanwhile,a blank control group and a negative control mock group were set.Cell proliferation was detected by MTT assay.Cell scratch wound assay was used to detect the ability of cell migration.Transwell method was used to detect cell invasion ability.Flow cytometry was used to detect cell apoptosis.Western blot was used for detection of related protein expression.RESULTS Compared with blank control MKN28 cells(1.257%±0.006%),the proliferation rates at 48 h after transfection in the mock group was comparable(1.257%±0.006%vs 1.163%±0.003%,P>0.05),while that of the ARHI overexpression group was significantly decreased(1.257%±0.006%vs 0.826%±0.005%,P<0.05);the migration ability in the mock group was not significantly changed(19.918%±0.233%vs 18.295%±0.534%,P>0.05),while that of the ARHI overexpression group was significantly decreased(19.918%±0.233%vs 4.299%±1.572%,P<0.05);the invasion ability in the mock group was not significantly changed(234±3.61 vs 235±4.51,P>0.05),while that of the ARHI overexpression group was significantly decreased(234±3.61 vs 93.3±2.08,P<0.05);the total apoptosis rate in the mock group was not significantly changed(3.513%±0.015%vs 3.597%±0.25%,P>0.05),while that of the ARHI overexpression group was significantly increased(3.513%±0.015%vs 14.133%±0.032%,P<0.05).Western blot results showed that,compared with blank control MKN28 cells,the relative expression of ARHI protein(1.037±0.003),vascular endothelial growth factor(VEGF)(1.026±0.008),B-cell lymphoma-2(Bcl-2)(1.014±0.010),protein kinase B(AKT)(1.001±0.005),and p-AKT protein(0.977±0.003)was not significantly changed(P>0.05),while the expression of ARHI protein(2.088±0.007)was significantly up-regulated(P<0.05),that of VEGF protein(0.456±0.004),Bcl-2 protein(0.468±0.005),and p-AKT protein(0.502±0.001)was significantly down-regulated(P<0.05),and that of AKT protein was not significantly changed(P>0.05).CONCLUSION Excessive expression of ARHI gene can inhibit the proliferation of MKN28 cells and promote their apoptosis,which may be related to the reduction of VEGF and p-AKT protein expression.
作者
张晓威
Xiao-Wei Zhang(The First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000,Liaoning Province,China)
出处
《世界华人消化杂志》
CAS
2020年第2期50-57,共8页
World Chinese Journal of Digestology
基金
辽宁省教育厅2019年科学研究经费项目,No.32027~~
关键词
ARHI基因
胃癌
增殖
凋亡
ARHI gene
Gastric carcinoma
Proliferation
Apoptosis