circ_0005230通过靶向miR-548c-3p影响胆管癌细胞增殖和凋亡的机制

Mechanism of affecting proliferation and apoptosis of cholangiocarcinoma cells from circ_0005230 by targeting miR-548c-3p

目的:探讨环状RNA(circRNA)circ_0005230对miR-548c-3p的靶向调控和对胆管癌细胞增殖和凋亡的作用与机制。方法:实时定量PCR(qPCR)检测胆管癌组织中circ_0005230和miR-548c-3p表达。将人胆管癌细胞RBE分为si-NC组、si-circ_0005230组、pcDNA-NC组、pcDNA-circ_0005230组、miR-NC组、miR-548c-3p组、si-circ_0005230+anti-miR-NC组、si-circ_0005230+anti-miR-548c-3p组,分别通过Lipofectamine 2000转染si-NC、si-circ_0005230、pcDNA-NC、pcDNA-circ_0005230、miR-NC、miR-548c-3p mimic、si-circ_0005230和anti-miR-NC、si-circ_0005230和miR-548c-3p inhibitor。以CCK-8法、克隆形成实验、免疫印迹实验(Western blot)和流式细胞术分别进行细胞的增殖、克隆形成、细胞周期蛋白D1(CyclinD1)、p21、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)、Bcl-2相关x蛋白(Bax)表达及细胞凋亡测定。双荧光素酶报告实验分析circ_0005230与miR-548c-3p靶向关系。结果:30例胆管癌组织中circ_0005230表达量高于癌旁组织,miR-548c-3p表达量低于癌旁组织,差异有统计学意义(P<0.05)。si-circ_0005230组或miR-548c-3p组RBE细胞的活性、克隆形成数、增殖蛋白CyclinD1和p21表达量减少,凋亡蛋白Caspase-3、Bax表达量和凋亡率增加,差异有统计学意义(P<0.05)。circ_0005230靶向调控miR-548c-3p的表达。pcDNA-circ_0005230组比pcDNA-NC组降低miR-548c-3p表达量,si-circ_0005230组较si-NC组提高miR-548c-3p表达量,差异均有统计学意义(P<0.05)。与si-circ_0005230+anti-miR-NC组比较,si-circ_0005230+anti-miR-548c-3p组RBE细胞的活性、克隆形成数、增殖蛋白CyclinD1和p21表达量升高,凋亡蛋白Caspase-3、Bax表达量和凋亡率降低,差异有统计学意义(P<0.05)。结论:circ_0005230在胆管癌组织中高表达,抑制circ_0005230表达通过靶向miR-548c-3p减弱胆管癌细胞的增殖和诱导其凋亡。

Objective:To investigate the targeted regulation of circRNA(circRNA)circ_0005230 on miR-548c-3p and its effect and mechanism on the proliferation and apoptosis of cholangiocarcinoma cells.Methods:Quantitative real-time polymerase chain reaction(qPCR)was used to detect the expression of circ_0005230 and miR-548c-3p in cholangiocarcinoma tissues.Human cholangiocarcinoma cell RBE was divided into si-NC group,si-circ_0005230 group,pcDNA-NC group,pcDNA-circ_0005230 group,miR-NC group,miR-548c-3p group,si-circ_0005230+anti-miR-NC group,si-circ_0005230+anti-miR-548c-3p group,and si-NC,si-circ_0005230,pcDNA-NC,pcDNA-circ_0005230,miR-NC,miR-548c-3p mimic,si-circ_0005230 and anti-miR-NC,si-circ_0005230 and miR-548c-3p inhibitor were transfected with Lipofectamine 2000,respectively.Cell proliferation,clone formation,proliferation proteins Cyclin D1(CyclinD1),p21,apoptosis protein aspartate specific caspase-3(Caspase-3),Bcl-2 related x protein(Bax)and cell apoptosis were performed by Cell Counting Kit-8(CCK-8)method,clone formation experiment,western blot and flow cytometry,respectively.The dual luciferase report experiment analyzed the targeting relationship between circ_0005230 and miR-548c-3p.Results:In 30 cases of cholangiocarcinoma,the expression of circ_0005230 was higher than that of adjacent tissues,and the expression of miR-548c-3p was lower than that of adjacent tissues.The difference was statistically significant(P<0.05).In si-circ_0005230 group or miR-548c-3p group,the activity,number of clone formation,expression of proliferating proteins CyclinD1 and p21 of RBE cells were decreased,while the expression of apoptotic proteins Caspase-3 and Bax and apoptosis rate were increased,and the difference was statistically significant(P<0.05).circ_0005230 targets and regulates the expression of miR-548c-3p.Compared with the pcDNA-NC group,the pcDNA-circ_0005230 group reduced the expression of miR-548c-3p;the si-circ_0005230 group increased the expression of miR-548c-3p than the si-NC group;the difference was statistically significant(P<0.05).Compared with si-circ_0005230+anti-miR-NC group,the activity,number of clone formation,expression of proliferating proteins CyclinD1 and p21 of RBE cells were increased,and the expression levels of apoptotic proteins Caspase-3 and Bax and the apoptotic rate were decreased in si-circ_0005230+anti-miR-548c-3p group,and the differences were statistically significant(P<0.05).Conclusion:circ_0005230 is highly expressed in cholangiocarcinoma tissues.Inhibition of circ_0005230 expression can reduce the proliferation and induce apoptosis of cholangiocarcinoma cells by targeting miR-548c-3p.