摘要
目的研究谷氨酰胺酶1(glutaminase1,GLS1)对胃癌细胞增殖、凋亡、迁移、侵袭和上皮间充质转化(EMT)的影响。方法体外培养人胃黏膜上皮细胞GES1和胃癌细胞N87、MKN28、BGC826和BGC803。设计合成靶向GLS1的siRNA片段(si-GLS1-1#和si-GLS1-2#),分别转染N87和BGC823细胞,以无意义siRNA为对照(si-con)。采用qRT-PCR检测细胞中GLS1 mRNA表达,CCK8和克隆形成实验检测细胞增殖能力,流式细胞术检测细胞凋亡情况,Transwell小室实验检测细胞迁移和侵袭能力,Western blot检测细胞中GLS1蛋白和上皮间充质转化(epithelial mesenchymal transition,EMT)相关蛋白表达。异种移植瘤实验评估干扰GLS1对胃癌细胞体内生长的影响。结果胃癌细胞中GLS1 mRNA和蛋白均呈高表达(P<0.05)。与si-con组比较,si-GLS1-1#和si-GLS1-2#组细胞中GLS1 mRNA和蛋白表达均明显降低(P<0.05)。细胞功能实验结果显示,与si-con组比较,si-GLS1-1#和si-GLS1-2#组细胞的增殖活力、克隆形成能力及迁移、侵袭能力均被抑制(P<0.05),而细胞凋亡率升高(P<0.05),细胞中N-cadherin和Vimentin蛋白表达明显降低(P<0.05),E-cadherin蛋白表达明显升高(P<0.05)。异种移植瘤实验结果显示,与si-con组比较,si-GLS1-1#和si-GLS1-2#组移植瘤体积和重量均明显减小(P<0.05),移植瘤中N-cadherin和Vimentin蛋白表达明显降低(P<0.05),E-cadherin蛋白表达明显升高(P<0.05)。结论干扰GLS1能够抑制胃癌细胞的增殖、迁移和侵袭能力及EMT,促进细胞凋亡,抑制胃癌细胞的体内生长。
Objective To investigate the regulatory effects of glutaminase 1(GLS1)on the proliferation,apoptosis,migration,invasion and epithelial-mesenchymal transition(EMT)of gastric cancer cells.Methods The human gastric epithelial cell line GES-1 and gastric cancer cell lines N87,MKN28,BGC826 and BGC803 were cultured in vitro.GLS-1 siRNAs(si-GLS1-1#and si-GLS1-2#)were designed and synthesized.They were transfected into N87 and BGC823 cells,with those transfected with nonsense siRNA as controls(si-con).The mRNA expression of GLS1 was detected by quantitative real-time PCR(qRT-PCR).Cell proliferation was detected by CCK-8 and clone formation assay.Cell apoptosis was detected by flow cytometry,and cell migration and invasion were detected by Transwell assay.The protein expressions of GLS1 and EMT-related proteins were detected by Western blot.A xenograft model was created in mice to evaluate the in vivo effect of silencing GLS1 on the growth of gastric cancer.Results Both the mRNA and protein expressions of GLS1 were significantly upregulated in gastric cancer cells.Compared with those of si-con group,the mRNA and protein expressions of GLS1 in gastric cancer cells transfected with si-GLS1-1#or si-GLS1-2#were significantly downregulated.Knockdown of GLS1 significantly inhibited proliferation,colony formation ability,migration and invasion,while the apoptotic rate was significantly enhanced in gastric cancer cells(P<0.05).In addition,knockdown of GLS1 downregulated N-cadherin and Vimentin,and upregulated E-cadherin in gastric cancer cells(P<0.05).In mice implanted with gastric cancer cells transfected with si-GLS1-1#or si-GLS1-2#,the volume and weight of xenograpfted tumor were significantly lower than those of controls(P<0.05).Meanwhile,significantly downregulated N-cadherin and Vimentin,and significantly upregulated E-cadherin were detected in xenografted tumor tissues collected from mice implanted with gastric cancer cells transfected with si-GLS1-1#or si-GLS1-2#than those of controls(P<0.05).Conclusion Knockdown of GLS1
作者
徐慧鲜
刘振锋
XU Huixian;LIU Zhenfeng(Department of Gastroenterology,Chongming Hospital Affiliated to Shanghai University of Medicine&Health Sciences(Shanghai Chongming District Central Hospital),Shanghai 202150,China)
出处
《河北医药》
CAS
2023年第13期1935-1940,共6页
Hebei Medical Journal
关键词
胃癌
谷氨酰胺酶1
增殖
凋亡
迁移
侵袭
上皮间充质转化
gastric cancer
glutaminase 1
proliferation
apoptosis
migration
invasion
epithelial-mesenchymal transition
