miR-376a-3p通过靶向调控GRP78抑制胃癌细胞生长与转移的机制研究  

The mechanism of miR-376a-3p inhibiting the growth and metastasis of gastric cancer cell by targeting GRP78

作　　者：张陈霏 沈晓雯[2] 何晓波 俞静 ZHANG Chenfei;SHEN Xiaowen;HE Xiaobo;YU Jing(Department of Medical Oncology,Nantong Tumor Hospital,Nantong 226000;Department of Laboratory Medicine,the Second People′s Hospital of Nantong;Basic Medical Research Center of Nantong University,China)

机构地区：[1]南通市肿瘤医院肿瘤内科,江苏南通226000 [2]南通市第二人民医院检验科 [3]南通大学基础医学研究中心

出　　处：《胃肠病学和肝病学杂志》2022年第8期908-914,共7页Chinese Journal of Gastroenterology and Hepatology

基　　金：南通市卫生和计划生育委员会科研课题(QA2019025,WKZL2018053)。

摘　　要：目的 探讨miR-376a-3p通过靶向调控GRP78抑制胃癌细胞生长与转移的机制。方法 qRT-PCR测定临床胃癌组织与细胞系中miR-376a-3p的表达,并分析miR-376a-3p与胃癌患者预后的关系;双荧光素酶报告基因系统与Western blotting验证miR-376a-3p与GRP78的靶向调控关系,同时检测临床胃癌组织中GRP78蛋白质的表达情况;分别采用Pre-miR-376a、pcDNA-3.1-NC与pcDNA-3.1-GRP78转染MKN45细胞系,CCK-8与EdU实验测定细胞增殖情况,Annexin V-FITC/PI双染实验检测细胞凋亡情况,划痕愈合实验与Transwell实验测定细胞迁移能力,Western blotting测定细胞上皮-间充质转化(epithelial-mesenchymal transition,EMT)标志蛋白的表达水平。结果 与正常胃黏膜细胞系GES-1相比,胃癌细胞系(MKN45、AGS、GC9811、HGC27)中miR-376a-3p表达水平显著降低(P<0.05),且miR-376a-3p低表达患者预后更差(P<0.05);miR-376a-3p靶向抑制GRP78蛋白质的表达(P<0.05);上调miR-376a-3p表达能抑制MKN45细胞增殖、迁移及EMT(P<0.05),促进细胞凋亡(P<0.05),而同时上调miR-376a-3p与GRP78的表达则能逆转此现象(P<0.05)。结论 miR-376a-3p通过靶向抑制GRP78蛋白表达抑制胃癌细胞生长与转移,提示miR-376a-3p/GRP78分子轴是胃癌可能的诊断和治疗靶标。Objective To investigate the potential underlying mechanisms by which miR-376a-3p targeted GRP78 to inhibit the development of growth and metastasis in gastric cancer cell.Methods qRT-PCR was conducted to determine the expression of miR-376a-3p in gastric cancer tissues and cell lines,and the correlation between miR-376a-3p levels and gastric cancer patients′ prognosis was analyzed.The regulating mechanisms of miR-376a-3p and GRP78 were verified by dual-luciferase reporter gene system assay and Western blotting analysis.The Pre-miR-376a,pcDNA-3.1-NC and pcDNA-3.1-GRP78 vectors were transfected into the MKN45 cells,and cell proliferation was examined by CCK-8assay and EdU assay,and Annexin V-FITC/PI double staining assay was performed to examine cell apoptosis.Cell invasion and migration were respectively determined by Transwell assay and wound scratch assay,and the epithelialmesenchymal transition(EMT) associated biomarkers were measured by Western blotting analysis.Results MiR-376a-3p was significantly downregulated in the gastric cancer cell lines(MKN45,AGS,GC9811 and HGC27) compared with normal GES-1 cell line(P<0.05),and gastric cancer patients with low-expressed miR-376a-3p tended to have a unfavorable prognosis(P<0.05).In addition,miR-376a-3p targeted inhibition GRP78 protein expression(P<0.05),and upregulation of miR-376a-3p suppressed cell proliferation,migration and EMT(P<0.05),and promoted cell apoptosis(P<0.05),at the same time,upregulated the miR-376a-3p and GRP78 expression could reverse this phenomenon.Conclusion MiR-376a-3p inhibits gastric cancer cells growth and metastasis by targeting inhibition GRP78 protein,indicating that the miR-376a-3p/GRP78 axis might be used as diagnostic and therapeutic biomarkers for gastric cancer in clinic.

关 键 词：胃癌 miR-376a-3p GRP78 增殖 凋亡 转移 

分 类 号：R735.2[医药卫生—肿瘤]