circEIF6调节miR-129-5p/TRAF6轴对胰腺癌细胞增殖、凋亡和吉西他滨敏感性的影响

Influences of circEIF6 on the proliferation,apoptosis and gemcitabine sensitivity of pancreatic cancer cells by regulating the miR-129-5p/TRAF6 axis

目的:探讨环状RNA真核起始因子6(circEIF6)调节miR-129-5p/肿瘤坏死因子受体相关因子6(TRAF6)轴对胰腺癌细胞增殖、凋亡和吉西他滨(GEM)敏感性的影响。方法:将胰腺癌细胞SW1990分为对照组(正常培养)、si-NC组(转染si-NC)、si-circEIF6组(转染si-circEIF6)、si-circEIF6+inhibitor-NC组(si-circEIF6和inhibitor-NC共转染)、si-circEIF6+miR-129-5p inhibitor组(si-circEIF6和miR-129-5p inhibitor共转染);RT-qPCR检测circEIF6、miR-129-5p的表达水平;MTT法检测细胞增殖能力;流式细胞术检测细胞凋亡;免疫印迹检测TRAF6、增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶3(caspase-3)的表达;MTT法检测不同浓度GEM对细胞增殖抑制率的影响;双荧光素酶报告基因实验分别验证circEIF6和miR-129-5p、miR-129-5p和TRAF6的关系。结果:与对照组、si-NC组比较,si-circEIF6组circEIF6表达、OD490值、TRAF6、PCNA蛋白表达降低,miR-129-5p表达、细胞凋亡率、Bax、caspase-3蛋白表达升高;与si-circEIF6组、si-circEIF6+inhibitor-NC组比较,si-circEIF6+miR-129-5p inhibitor组OD490值、TRAF6、PCNA蛋白表达升高,miR-129-5p表达、细胞凋亡率、Bax、caspase-3蛋白表达降低;细胞增殖抑制率在GEM处理下以剂量依赖性的方式显著增加;与对照组比较,GEM处理下细胞的增殖抑制率、凋亡率显著升高,敲低circEIF6可提高GEM处理下细胞的增殖抑制率、凋亡率,而miR-129-5p inhibitor可减弱敲低circEIF6发挥的上述作用;circEIF6靶向负调控miR-129-5p的表达,miR-129-5p靶向负调控TRAF6的表达;双荧光素酶报告实验证实miR-129-5p与circEIF6和TRAF6存在靶向关系。结论:敲低circEIF6可能通过靶向miR-129-5p下调TRAF6表达,进而抑制胰腺癌细胞增殖、促进细胞凋亡、提高胰腺癌细胞对GEM的敏感性。

Objective:To investigate the influences of circular RNA eukaryotic initiation factor 6(circEIF6)on the proliferation,apoptosis and gemcitabine(GEM)sensitivity of pancreatic cancer(PC)cells by regulating miR-129-5p/tumor necrosis factor receptor associated factor 6(TRAF6)axis.Methods:PC cells SW1990 were divided into a control group(normal culture),a si-NC group(transfected with si-NC),a si-circEIF6 group(transfected with si-circEIF6),a si-circEIF6+inhibitor NC group(co-transfected with si-circEIF6 and inhibitor NC),and a si-circEIF6+miR-129-5p inhibitor group(co-transfected with si-circEIF6 and miR-129-5p inhibitor).The expression levels of circEIF6 and miR-129-5p were detected by RT-qPCR.The proliferation ability of cells was detected by MTT assay.Cell apoptosis was detected by flow cytometry.Western blotting was applied to detect the expression of TRAF6,proliferating cell nuclear antigen(PCNA),Bax and caspase-3.MTT assay was used to detect the effect of different concentrations of GEM on the proliferation inhibition rate.Double luciferase reporter gene experiment was used to verify the relationship between circEIF6 and miR-129-5p,between miR-129-5p and TRAF6 respectively.Results:Compared with the control group and si-NC group,the expression of circEIF6,OD490 value,the protein expression of TRAF6 and PCNA in si-circEIF6 group were decreased,the expression of miR-129-5p,apoptosis rate,the protein expression of Bax and caspase-3 were increased.Compared with the si-circEIF6 group and si-circEIF6+inhibitor NC group,there was no obvious difference in the expression of circEIF6 in the si-circEIF6+miR-129-5p inhibitor group,OD490 value,and the protein expression of TRAF6 and PCNA were increased,the expression of miR-129-5p,apoptosis rate,the protein expression of Bax and caspase-3 were decreased.The proliferation inhibition rate was obviously increased in a dose-dependent manner under GEM treatment.Compared with the control group,the proliferation inhibition rate and apoptosis rate under GEM treatment were obviously higher,knocking-down circEIF6 could increase the proliferation inhibition rate and apoptosis rate under GEM treatment.However,miR-129-5p inhibitor could weaken the above effects of knockdown circEIF6.circEIF6 negatively regulated the expression of miR-129-5p,and miR-129-5p negatively regulated the expression of TRAF6.Conclusion:Knocking down circEIF6 may down-regulate the expression of TRAF6 by targeting miR-129-5p,thereby inhibiting the proliferation of PC cells,promoting apoptosis,and increasing the sensitivity of PC cells to GEM.