LHFPL3-AS1靶向miR-515-5p对胃癌顺铂耐药细胞增殖、迁移和侵袭的影响

Effect of LHFPL3-AS1 on the proliferation,migration and invasion of gastric cancer cisplatin-resistant cells by targeting miR-515-5p

目的探讨LHFPL3-AS1靶向miR-515-5p对胃癌顺铂耐药细胞增殖、迁移和侵袭的影响。方法实时定量聚合酶链反应分析胃黏膜上皮细胞GES-1、亲本细胞HGC-27、顺铂耐药细胞HGC-27/DDP中LHFPL3-AS1和miR-515-5p表达。将HGC-27/DDP细胞分为对照(Con)组、si-NC组、si-LHFPL3-AS1组、miR-NC组、miR-515-5p mimic组、si-LHFPL3-AS1+miR-515-5pinhibitor组。双荧光素酶报告实验确定LHFPL3-AS1和miR-515-5p的靶向关系。CCK-8法检测细胞存活率,平板克隆形成实验检测细胞克隆形成数,Transwell实验检测细胞迁移和侵袭数,蛋白质印迹法检测E-cadherin和N-cadherin蛋白表达。结果随着顺铂浓度的增加,HGC-27和HGC-27/DDP细胞的存活率逐渐降低,HGC-27/DDP细胞的IC50(236.20μmol/L)高于HGC-27细胞(5.83μmol/L)。与GES-1细胞比较,HGC-27和HGC-27/DDP细胞中LHFPL3-AS1相对水平显著升高,miR-515-5p相对水平显著降低(P<0.05)。LHFPL3-AS1和miR-515-5p直接特异性结合。与Con组、si-NC组比较,si-LHFPL3-AS1组HGC-27/DDP细胞miR-515-5p相对水平、E-cadherin蛋白表达显著升高,克隆形成数、迁移数、侵袭数、细胞存活率、N-cadherin蛋白表达显著降低(P<0.05);与miR-NC组比较,miR-515-5pmimic组HGC-27/DDP细胞E-cadherin蛋白表达显著升高,克隆形成数、迁移数、侵袭数、细胞存活率、N-cadherin蛋白表达显著降低(P<0.05);与si-LHFPL3-AS1组比较,si-LHFPL3-AS1+miR-515-5p inhibitor组HGC-27/DDP细胞E-cadherin蛋白表达显著降低,克隆形成数、迁移数、侵袭数、细胞存活率、N-cadherin蛋白表达显著升高(P<0.05)。结论干扰LHFPL3-AS1通过上调miR-515-5p可抑制胃癌细胞HGC-27/DDP增殖、迁移和侵袭。

Objective To investigate the effect of LHFPL3-AS1 targeting miR-515-5p on the proliferation,migration and invasion of gastric cancer cisplatin-resistant cells.Methods Real-time quantitative polymerase chain reaction analyzed the expression of LHFPL3-AS1 and miR-515-5p in gastric mucosal epithelial cells GES-1,parental cells HGC-27 and cisplatin-resistant cells HGC-27/DDP.HGC-27/DDP cells were divided into control(Con)group,si-NC group,si-LHFPL3-AS1group,miR-NC group,miR-515-5p mimic group,si-LHFPL3-AS1+miR-515-5p inhibitor group.Dual luciferase reporter experiment confirmed the targeting relationship between LHFPL3-AS1 and miR-515-5p.CCK-8 method detected cell survival rate,plate colony formation experiment detected cell colony formation number,Transwell experiment detected cell migration and invasion number,Western blotting calculated E-cadherin and N-cadherin protein expression.Results With the increase of cisplatin concentration,the survival rate of HGC-27 and HGC-27/DDP cells decreased gradually.The IC50of HGC-27/DDP cells(236.20μmol/L)was higher than that of HGC-27 cells(5.83μmol/L).Compared with GES-1 cells,the relative level of LHFPL3-AS1 in HGC-27 and HGC-27/DDP cells was significantly increased,while the relative level of miR-515-5p was significantly decreased(P<0.05).LHFPL3-AS1 directly bound to miR-515-5p.Compared with the Con group and the si-NC group,the relative levels of miR-515-5p,E-cadherin protein expression of HGC-27/DDP cells in si-LHFPL3-AS1 group were notably increased,clonal formation number,migration number,invasion number,cell survival rate and N-cadherin protein expression were notably reduced(P<0.05).Compared with miR-NC group,E-cadherin protein expression of HGC-27/DDP cells in miR-515-5p mimic group was notably increased,clonal formation number,migration number,invasion number,cell survival rate and N-cadherin protein expression were notably reduced(P<0.05).Compared with si-LHFPL3-AS1 group,E-cadherin protein expression of HGC-27/DDP cells in si-LHFPL3-AS1+miR-515-5p inhibitor group was notably reduced,clonal formation number,migration number,invasion number,cell survival rate and N-cadherin protein expression were notably increased(P<0.05).Conclusion Interference with LHFPL3-AS1 can inhibit the proliferation,migration and invasion of gastric cancer cells HGC-27/DDP by up-regulating miR-515-5p.