lncRNA-PCAT6通过调控miR-185/SIX1轴对肺癌A549细胞上皮间质转化及化疗敏感性的影响

Effects of lnc RNA-PCAT6 on epithelial-mesenchymal transition and chemotherapy sensitivity of lung cancer A549 cells by adjusting miR-185/SIX1 axis

目的:探讨长链非编码RNA-PCAT6(lncRNA-PCAT6)对肺癌A549细胞上皮间质转化(EMT)和化疗敏感性的影响,以及对微小RNA-185(miR-185)/SIX1轴的调控作用。方法:取A549细胞,分为空白组、lncRNA-PCAT6低表达腺病毒(PCAT6-Si)组、不含lncRNA-PCAT6-Si的空病毒载体(eGFP-Si-1)组、miR-185低表达腺病毒(miR-185-Si)组、不含miR-185-Si的空病毒载体(eGFP-Si-2)组、PCAT6-Si与miR-185-Si共转染(PCAT6-Si+miR-185-Si)组。RT-qPCR法检测lncRNA-PCAT6、miR-185表达;Transwell法测定细胞侵袭、迁移能力;免疫荧光检测EMT标志物N-cadherin阳性表达;Western blot法测定SIX1、EMT标志蛋白E-cadherin、Vimentin及N-cadherin表达。取A549细胞系及其顺铂耐药细胞株A549/DDP,RT-qPCR及Western blot法检测lncRNA-PCAT6、miR-185表达。取A549/DDP转染PCAT6-Si与miR-185-Si,并分为A549组、A549/DDP组、PCAT6-Si组、miR-185-Si组、PCAT6-Si+miR-185-Si、eGFP-Si-1组、eGFP-Si-2组;RT-qPCR检测lncRNA-PCAT6、miR-185表达;CCK-8法检测各组细胞半数抑制浓度(IC50)及耐药指数(RI);Western blot法检测SIX1、耐药相关蛋白P-gp表达;裸鼠荷瘤试验验证PCAT6对A549/DDP细胞化疗敏感性的作用。双荧光素酶试验验证PCAT6与miR-185之间的靶向关系。结果:与A549细胞相比,A549/DDP细胞IC50及RI、lncRNA-PCAT6、SIX1及P-gp表达升高(P<0.05),miR-185表达降低(P<0.05)。敲低A549细胞中lncRNA-PCAT6表达后,miR-185表达升高,SIX1蛋白表达降低,细胞侵袭、迁移能力及EMT进程降低,IC50及RI降低、肿瘤体积减小,且各指标变化差异显著(P<0.05)。敲低miR-185可逆转lncRNA-PCAT6的上述作用。双荧光素酶报告试验证实lncRNAPCAT6与miR-185之间有靶向负调控作用。结论:肺癌A549细胞中lncRNA-PCAT6低表达可靶向上调miR-185,下调SIX1,抑制EMT进程,增强对DDP化疗的敏感性。

Objective:To investigate the effects of long non-coding RNA-PCAT6(lncRNA-PCAT6)on the epithelial-mesenchymal transition(EMT)and chemotherapy sensitivity of lung cancer A549 cells,and its regulation effect on microRNA-185(miR-185)/SIX1 axis.Methods:A549 cells were divided into blank group,lncRNA-PCAT6 low-expressing adenovirus(PCAT6-Si)group,empty viral vector without lncRNA-PCAT6-Si(eGFP-Si-1)group,and miR-185 low-expressing adenovirus group(miR-185-Si)group,empty virus vector without miR-185-Si(eGFP-Si-2)group,PCAT6-Si and miR-185-Si co-transfection(PCAT6-Si+miR-185-Si)group.RT-qPCR method was used to detect the expressions of lncRNA-PCAT6 and miR-185;Transwell method was used to measure cell invasion and migration abilities;immunofluorescence was used to detect the positive expressions of EMT marker N-cadherin;Western blot method was used to determine the expressions of SIX1,EMT marker protein E-cadherin,Vimentin and N-cadherin.The A549 cell line and its cisplatin-resistant cell line A549/DDP were taken to detect the expressions of lncRNA-PCAT6 and miR-185 by RT-qPCR and Western blot methods.A549/DDP was taken to transfect PCAT6-Si and miR-185-Si,and they were divided into A549group,A549/DDP group,PCAT6-Si group,miR-185-Si group,PCAT6-Si+miR-185-Si group,eGFP-Si-1 group,eGFP-Si-2 group;RT-qPCR was used to detect lncRNA-PCAT6,miR-185;CCK-8 method was used to detect the half inhibitory concentration(IC50)and resistance index(RI)of each group of cells;Western blot was used to detect expressions of SIX1,resistance-related protein P-gp;a nude mouse tumor-bearing test was used to verify the effect of PCAT6 on the chemotherapy sensitivity of A549/DDP cells.The dual luciferase test was used to verify the targeting relationship between PCAT6 and miR-185.Results:Compared with A549 cells,the expressions of IC50and RI,lncRNA-PCAT6,SIX1 and P-gp of A549/DDP cells were increased(P<0.05),and the expression of miR-185 was decreased(P<0.05).After knocking down the expression of lncRNA-PCAT6 in A549 cells,the expression of miR-185was increased,the expression of SIX1 protein was decreased,the cell invasion,migration and EMT process were decreased,IC50and RI were decreased,tumor volume was decreased,and the changes of various indicators were significantly different(P<0.05).Knockdown of miR-185 could reverse the above effects of lncRNA-PCAT6.The dual luciferase report test confirmed that lncRNA-PCAT6 and miR-185 have a targeted negative regulation effect.Conclusion:The low expression of lncRNA-PCAT6 in lung cancer A549 cells can target to up-regulate miR-185,down-regulate SIX1,inhibit EMT process,and enhance the sensitivity to DDP chemotherapy.