lncRNA TDRG1、miR-194-3p在直肠癌组织中的表达变化及其对直肠癌细胞凋亡和放疗敏感性影响观察

Expression changes of lncRNA TDRG1 and miR-194-3p and their effects on apoptosis and radiosensitivity of rectal cancer cells

目的观察长链非编码RNA睾丸发育相关基因1(lncRNA TDRG1)、微小RNA-194-3p(miR-194-3p)在直肠癌组织中的表达变化,及其对直肠癌细胞凋亡、放疗敏感性的影响。方法收集直肠癌组织及癌旁组织,采用RT-qPCR法检测直肠癌组织及癌旁组织中lncRNA TDRG1、miR-194-3p。使用Lnc Base Predicted v.2在线预测网站分析lncRNA TDRG1的靶基因,采用双荧光素酶报告基因实验验证lncRNA TDRG1的靶基因。取人直肠癌细胞系SW1643分别转染si-TDRG1、si-NC、miR-194-3p mimics、miR-NC、si-TDRG1+anti-miR-194-3p、si-TDRG1+anti-miRNC,记为si-TDRG1组、si-NC组、miR-194-3p组、miR-NC组、si-TDRG1+anti-miR-194-3p组、si-TDRG1+anti-miR-NC组,采用流式细胞术测算各组细胞凋亡率,采用Western blotting法检测各组细胞中Bcl-2、Bax蛋白表达,采用克隆形成实验测算各组细胞放疗敏感性[以细胞存活分数、放射增敏比(SER)表示]。结果直肠癌组织中lncRNA TDRG1的相对表达量高于癌旁组织,miR-194-3p的相对表达量低于癌旁组织(P均<0.05)。转染WT-TDRG1、miR-194-3p mimics的SW1643细胞荧光素酶活性低于转染WT-TDRG1、miR-NC的SW1643细胞(P<0.05)。siTDRG1组细胞凋亡率高于si-NC组,miR-194-3p组细胞凋亡率高于miR-NC组,si-TDRG1+anti-miR-194-3p组细胞凋亡率低于si-TDRG1+anti-miR-NC组(P均<0.05)。si-TDRG1组Bcl-2蛋白相对表达量低于si-NC组,Bax蛋白相对表达量高于si-NC组(P<0.05);miR-194-3p组Bcl-2蛋白相对表达量低于miR-NC组,Bax蛋白相对表达量高于miR-NC组;si-TDRG1+anti-miR-194-3p组Bcl-2蛋白相对表达量高于si-TDRG1+anti-miR-NC组,Bax蛋白相对表达量低于si-TDRG1+anti-miR-NC组(P均<0.05)。si-TDRG1组、si-NC组细胞存活分数分别为0.330、0.657,SER为2.016;miR-194-3p组、miR-NC组细胞存活分数分别为0.370、0.669,SER为1.913;si-TDRG1+anti-miR-194-3p组、si-TDRG1+anti-miR-NC组细胞存活分数分别为0.547、0.320,SER为0.574。结论直肠癌组织中lncRNA TDRG1高表达、miR-194-3p低表达;干扰lncRNA TDRG1可能是通过靶向miR-194-3p促进直肠癌细胞凋亡、提高放疗敏感性。

Objective To observe the expression changes of long non-coding RNA testis development-related gene 1(lncRNA TDRG1)and microRNA-194-3 p(miR-194-3 p)in the rectal cancer tissues,and further to explore their effects on apoptosis and radiotherapy sensitivity of rectal cancer cells.Methods The expression of lncRNA TDRG1 and miR-194-3 p in the rectal cancer tissues and adjacent tissues were detected by RT-qPCR.Lnc Base Predicted v.2 website and dual luciferase reporter gene experiment were used to analyze and verify the target genes of lncRNA TDRG1.The si-TDRG1,si-NC,miR-194-3 p mimics,miR-NC,si-TDRG1+anti-miR-194-3 p,si-TDRG1+anti-miR-NC were transfected into human rectal cancer cell line SW1643,which were then taken as the si-TDRG1 group,si-NC group,miR-194-3 p group,miR-NC group,si-TDRG1+anti-miR-194-3 p group,and si-TDRG1+anti-miR-NC group.Flow cytometry was used to measure the apoptosis rate of each group.Western blotting was used to detect the expression levels of Bcl-2 and Bax proteins.The clone formation experiment was applied to measure the radiotherapy sensitivity[expressed as cell survival fraction,sensitive enhencement ratio(SER)].Results The relative expression level of lncRNA TDRG1 in the rectal cancer tissues was higher than that in the adjacent tissues,whereas the relative expression level of miR-194-3 p was lower than that in adjacent tissues(both P<0.05).The luciferase activity of SW1643 cells transfected with WT-TDRG1 and miR-194-3 p mimics was lower than that of SW1643 cells transfected with WT-TDRG1 and miR-NC(P<0.05).The apoptosis rate of the si-TDRG1 group was higher than that of the si-NC group,the apoptosis rate of the miR-194-3 p group was higher than that of the miR-NC group,and the apoptosis rate of the si-TDRG1+anti-miR-194-3 p group was lower than that of the siTDRG1+anti-miR-NC group(all P<0.05).The relative expression level of Bcl-2 protein in the si-TDRG1 group was lower than that in the si-NC group,whereas the relative expression level of Bax protein was higher than that of the si-NC group(both P<0.05);the relative expression level of Bcl-2 protein was lower in the miR-194-3 p group than in the miRNC group,whereas the relative expression of Bax protein was higher;the relative expression of Bcl-2 protein in the siTDRG1+anti-miR-194-3 p group was higher than that in the si-TDRG1+anti-miR-NC group,whereas the relative expression of Bax protein was lower(both P<0.05).The cell survival fractions of the si-TDRG1 group and the si-NC group were0.330 and 0.657,respectively,and the SER was 2.016;the cell survival scores of the miR-194-3 p group and miR-NC group were 0.370 and 0.669,respectively,and the SER was 1.913;the cell survival scores of the si-TDRG1+anti-miR-194-3 p group and si-TDRG1+anti-miR-NC group were 0.547 and 0.320,respectively,and SER was 0.574.Conclusion In the rectal cancer tissues,lncRNA TDRG1 is highly expressed in the rectal cancer tissues,whereas miR-194-3 p is low expressed;interfering with lncRNA TDRG1 may promote the apoptosis and increase the radiosensitivity of rectal cancer cells by targeting miR-194-3 p.