Ⅳ型胶原蛋白基因α1对胃癌细胞增殖、转移及凋亡的调控作用与机制  

Regulation role and mechanism of type Ⅳ collagen geneα1 on the proliferation,metastasis and apoptosis of gastric cancer cells

作　　者：李怡君 杨振威[1] 陈晓露[1] 司望利[1] LI Yijun;YANG Zhenwei;CHEN Xiaolu;SI Wangli(Department of Gastroenterology,Xi'an Central Hospital,Xi'an 710003,Shaanxi Province,China)

机构地区：[1]西安市中心医院消化科,陕西西安710003

出　　处：《新乡医学院学报》2022年第4期311-317,共7页Journal of Xinxiang Medical University

摘　　要：目的探讨Ⅳ型胶原蛋白基因α1(COL4A1)对胃癌细胞增殖、转移及凋亡的影响以及其潜在的调控机制。方法采用实时荧光定量聚合酶链式反应(RT-qPCR)法检测胃癌组织和人正常胃黏膜上皮细胞GES-1及胃癌细胞(SGC-7901、MKN-28、BGC-803、MGC-823、MKN-45和AGS)中COL4A1基因的表达。根据6种胃癌细胞中COL4A1基因的表达情况,AGS和SGC-7901细胞被用于后续的细胞功能验证实验。将AGS、SGC-7901细胞接种于6孔板,并于融合度达到90%后,将2种细胞分为对照组、阴性对照(NC)小干扰RNA(siRNA)组、siCOL4A1-1组、siCOL4A1-2组和siCOL4A1-2+740 Y-P组。对照组细胞正常培养,不进行任何处理;NC siRNA组细胞转染NC siRNA,siCOL4A1-1组细胞转染siCOL4A1-1,siCOL4A1-2组细胞转染siCOL4A1-2,siCOL4A1-2+740 Y-P组细胞转染siCOL4A1-2,然后使用50 mg·L^(-1)的740 Y-P处理90 min。采用RT-qPCR检测5组细胞中COL4A1基因的表达情况,根据检测结果选择siCOL4A1-2组细胞进行后续实验。采用细胞计数试剂盒-8检测对照组、NC siRNA组、siCOL4A1-2组和siCOL4A1-2+740 Y-P组AGS和SGC-7901细胞培养1、2、3、4 d时的增殖能力,膜联蛋白-V/异硫氰酸荧光素实验检测4组AGS、SGC-7901细胞凋亡情况,Transwell实验检测4组AGS、SGC-7901细胞迁移和侵袭能力,Western blot检测4组AGS、SGC-7901细胞中磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)通路相关蛋白表达。结果siCOL4A1-2组AGS细胞的增殖能力在培养3 d和4 d时显著低于对照组和NC siRNA组(P<0.05),SGC-7901细胞的增殖能力在培养1、2、3、4 d时均显著低于对照组和NC siRNA组(P<0.05);siCOL4A1-2+740 Y-P组AGS细胞的增殖能力在培养3 d和4 d时显著高于siCOL4A1-2组(P<0.05),SGC-7901细胞的增殖能力在培养1、2、3、4 d时均显著高于siCOL4A1-2组(P<0.05);其余各组细胞增殖能力比较差异均无统计学意义(P>0.05)。siCOL4A1-2组AGS、SGC-7901细胞的凋亡率均显著高于对照组和NC siRNA组(P<0.05);siCOL4A1-2+740 Y-P�Objective To explore the effect of type Ⅳ collagen geneα1(COL4A1)on the proliferation,metastasis and apoptosis of gastric cancer cells and its potential regulatory mechanism.Methods The expression of COL4A1 gene in gastric cancer tissues,human normal gastric mucosal epithelial cells GES-1 and gastric cancer cells(SGC-7901,MKN-28,BGC-803,mgc-823,MKN-45 and AGS)was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).According to the expression of COL4A1 gene in six gastric cancer cells,AGS and SGC-7901 cells were used for subsequent cell function verification experiments.AGS and SGC-7901 cells were inoculated into 6-well plates.After the fusion reached 90%,the AGS and SGC-7901 cells were divided into control group,negative control(NC)small interfering RNA(siRNA)group,siCOL4A1-1 group,siCOL4A1-2 group and siCOL4A1-2+740 Y-P group.The cells in the control group were cultured normally without any treatment;the cells in NC siRNA group were transfected with NC siRNA,the cells in siCOL4A1-1 group were transfected with siCOL4A1-1,the cells in siCOL4A1-2 group were transfected with siCOL4A1-2,the cells in siCOL4A1-2+740 Y-P group were transfected with siCOL4A1-2,and then were treated with 740 Y-P(50 mg·L^(-1)) for 90 min.The expression of COL4A1 gene in AGS,SGC-7901 cells in the above five groups was detected by RT-qPCR,and the cells in the siCOL4A1-2 group was selected for follow-up experiments according to the detection results.The proliferation of AGS and SGC-7901 cells in control group,NC siRNA group,siCOL4A1-2 group and siCOL4A1-2+740 Y-P group was detected by cell counting kit-8 at 1,2,3,4 days of culture;the apoptosis of AGS and SGC-7901 cells in the four groups was detected by annexin-V/fluorescein isothiocyanate,the migration and invasion ability of AGS and SGC-7901 cells in the four groups were detected by Transwell experiment;the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway related protein in AGS and SGC-7901 cells was detected by Western blot.

关 键 词：Ⅳ型胶原蛋白基因α1 胃癌 磷脂酰肌醇3-激酶/蛋白激酶B通路 增殖 转移 

分 类 号：R735.2[医药卫生—肿瘤]