miR-582-5p靶向PDCD10对胃癌细胞迁移、侵袭及凋亡的影响  

Effects of miR-582-5p targeting PDCD10 on migration,invasion and apoptosis of gastric cancer cells

作　　者：翟宏芳[1] 刘丽丽[1] 刘爱娟[1] 李青科[1] 胡万宁 ZHAI Hongfang;LIU Lili;LIU Aijuan;LI Qingke;Hu Wanning(Tangshan People′s Hospital,Tangshan 063000,China;不详)

机构地区：[1]唐山市人民医院,河北唐山063000 [2]唐山市工人医院

出　　处：《山东医药》2020年第28期37-40,44,共5页Shandong Medical Journal

摘　　要：目的探讨微小RNA(miR)-582-5p靶向程序性细胞死亡因子10(PDCD10)对胃癌细胞迁移、侵袭、凋亡的影响。方法常规培养人正常胃黏膜上皮细胞株(GES-1)、胃癌细胞系(HGC-27、MKN74、NUGC-4),用qPCR法检测细胞中miR-582-5p表达,筛选miR-582-5p表达最低的HGC-27作为实验细胞。将对数生长期的HGC-27随机分为8组,各组采用脂质体转染法分别转染miR-582-5p模拟物(使miR-582-5p过表达)、miR-582-5p抑制剂(使miR-582-5p低表达)、miR-582-5p模拟物阴性对照miR-NC、miR-582-5p抑制剂阴性对照anti-miR-NC、PDCD10小干扰RNA si-PDCD10(使PDCD10低表达)、PDCD10小干扰RNA阴性对照si-NC、PDCD10过表达载体pcDNA-PDCD10(使PDCD10过表达)、过表达载体阴性对照pcDNA-NC。用qPCR法检测细胞内miR-582-5p、PDCD10 mRNA表达,证实转染成功。用Western blotting法检测细胞内PDCD10、活化半胱氨酸天冬氨酸蛋白水解酶3(Cleaved-caspase-3)、基质金属蛋白酶2(MMP2)、MMP9蛋白表达,用流式细胞术测算细胞凋亡率,用Transwell小室法检测细胞迁移和侵袭能力,用双荧光素酶报告基因检测法验证miR-582-5p与PDCD10的靶向调控关系。结果与miR-NC组比较,miR-582-5p组Cleaved-caspase-3蛋白相对表达量高,MMP2、MMP9蛋白相对表达量低,细胞凋亡率高,细胞迁移数、侵袭数少(P均<0.05)。与si-NC组比较,Cleaved-caspase-3蛋白相对表达量低,MMP2、MMP9蛋白相对表达量高,细胞凋亡率低,细胞迁移数、侵袭数多(P均<0.05)。miR-582-5p与PDCD10的3′UTR存在靶向结合位点。miR-582-5p与WT-PDCD10报告质粒共转染HGC-27后的荧光素酶活性低于miR-NC与WT-PDCD10报告质粒共转染(P<0.05);miR-582-5p或miR-NC与MUT-PDCD10报告质粒共转染HGC-27后的荧光素酶活性比较差异无统计学意义(P>0.05)。与miR-NC组比较,miR-582-5p组PDCD10蛋白相对表达量低(P<0.05);与anti-miR-NC组比较,anti-miR-582-5p组PDCD10蛋白相对表达量高(P<0.05)。与miR-582-5p+pcDNA-NC组比较,miR-582-5p+pcDNA-PDCObjective To investigate the effect of microRNA(miR)-582-5p targeting programmed cell death factor 10(PDCD10)on the migration,invasion and apoptosis of gastric cancer cells.Methods Normal human gastric mucosal epithelial cell lines(GES-1)and gastric cancer cell lines(HGC-27,MKN74,NUGC-4)were routinely cultured.The expression of miR-582-5p in the cells was detected by qPCR.HGC-27 with the lowest expression of miR-582-5p was screened out and used as the experimental cells.The HGC-27 in the logarithmic growth phase was randomly divided into 8 groups,which were transfected with miR-582-5p mimic(to overexpress miR-582-5p),miR-582-5p inhibitor(to make miR-582-5p low expression),miR-582-5p mimic negative control miR-NC,miR-582-5p inhibitor negative control anti-miR-NC,PDCD10 small interfering RNA si-PDCD10(to make PDCD10 low expression),PDCD10 small interfering RNA negative control si-NC,PDCD10 overexpression vector pcDNA-PDCD10(to overexpress PDCD10),and overexpression vector negative control pcDNA-NC by the liposome transfection method,respectively.The expression of miR-582-5p and PDCD10 mRNA in the cells was detected by qPCR,and PDCD10,Cleaved-Caspase-3(Cleaved-Caspase-3)and matrix metalloproteinase 2(MMP-2),and MMP-9 were detected by Western blotting.Flow cytometry was used to measure the apoptosis rate,the Transwell chamber method was used to detect cell migration and invasion abilities,and the dual luciferase reporter gene detection method was used to verify the target regulatory relationships of miR-582-5p and PDCD10.Results Compared with the miR-NC group,the relative expression of Cleaved-Caspase-3 protein was higher,the relative expression of MMP-2 and MMP-9 protein was lower,the apoptosis rate was higher,and the number of cell migration and invasion was less in the miR-582-5p group(all P<0.05).Compared with the si-NC group,the relative expression of Cleaved-Caspase-3 protein was lower,the relative expression of MMP-2 and MMP-9 protein was higher,the apoptosis rate was lower,and the number of cell migration and

关 键 词：胃癌 微小RNA-582-5p 程序性细胞死亡因子10 细胞迁移 细胞侵袭 细胞凋亡 

分 类 号：R735.2[医药卫生—肿瘤]