肝癌细胞系中miR-9和转录因子E2F1的表达及其与药物敏感性的关系

Expressions of miR-9 and transcription factor E2F1 in liver cancer cell lines and their relationships with drug-sensitivity

目的:研究肝癌细胞系中微小RNA(microRNA,miR)-9和转录因子E2F1的表达及其与药物敏感性的关系。方法:体外培养肝细胞系HL-7702,肝癌细胞系HepG2、Hep3B2.1-7,处理细胞分miRNA NC组、miR-9 inhibitor组、顺铂(CDDP)组、CDDP+miRNA NC组、CDDP+miR-9 inhibitor组。实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)检测miR-9表达水平;免疫印记(Western blot,WB)法检测E2F1蛋白水平;CCK-8法检测miR-9对细胞存活的影响;克隆计数检测miR-9对细胞数量的影响。结果:与正常肝细胞系HL-7702相比,肝癌细胞系HepG2、Hep3B2.1-7中miR-9、E2F1蛋白水平升高(P<0.05)。在HepG2、Hep3B2.1-7细胞系中,与miRNA NC组相比,miR-9 inhibitor组、CDDP+miR-9 inhibitor组miR-9、E2F1蛋白表达水平、细胞克隆数量降低(P<0.05)。与miR-9 inhibitor组相比,CDDP组、CDDP+miRNA NC组miR-9、E2F1蛋白表达水平、细胞克隆数量升高(P<0.05);CDDP+miR-9 inhibitor组细胞克隆数量、48 h细胞OD450降低(P<0.05);与CDDP组、CDDP+miRNA NC组相比,CDDP+miR-9 inhibitor组miR-9、E2F1蛋白表达水平、细胞克隆数量降低(P<0.05)。在HepG2细胞系中,与miRNA NC组、CDDP组、CDDP+miRNA NC组相比,miR-9 inhibitor组、CDDP+miR-9 inhibitor组(12、24、48 h)细胞OD450降低(P<0.05)。在Hep3B2.1-7细胞系中,与miRNA NC组相比,miR-9 inhibitor组(12、24、48 h)、CDDP+miR-9 inhibitor组(6、12、24、48 h)细胞OD450降低(P<0.05);与CDDP组相比,miR-9 inhibitor组(12、24、48 h)、CDDP+miR-9 inhibitor组(3、6、12、24、48 h)细胞OD450降低(P<0.05);与CDDP+miRNA NC组相比,miR-9 inhibitor组(24、48 h)、CDDP+miR-9 inhibitor组(12、24、48 h)细胞OD450降低(P<0.05)。结论:肝癌细胞系HepG2、Hep3B2.1-7中miR-9和E2F1表达上调;干扰miR-9可抑制E2F1表达,抑制细胞增殖、抑制细胞克隆形成,提高药物敏感性。

Objective:To study the expressions of microRNA(miR)-9 and transcription factor E2F1 in hepatoma cell lines and their relationships with drug-sensitivity.Methods:HL-7702,HepG2 and Hep3B2.1-7 were cultured in vitro,and the cells were divided into miRNA NC group,miR-9 inhibitor group,cisplatin(CDDP)group,CDDP+miRNA NC group and CDDP+miR-9 inhibitor group.The expression of miR-9 was detected by real-time fluorescent quantitative PCR(qRT-PCR).The protein level of E2F1 was detected by Western blot(WB).CCK-8 method was used to detect the effect of miR-9 on cell survival,and the effect of miR-9 on cell number was detected by clone count.Results:The levels of miR-9 and E2F1 proteins in HepG2 and Hep3B2.1-7 were higher than that in HL-7702(P<0.05).In HepG2 and Hep3B2.1-7,compared with miRNA NC group,miR-9 and E2F1 protein expression levels and cell clone number in miR-9 inhibitor group,CDDP+miR-9 inhibitor group were lower(P<0.05).Compared with miR-9 inhibitor group,the expression levels of miR-9 and E2F1 proteins and the number of cell clones in CDDP group and CDDP+miRNA NC group were higher(P<0.05).The number of clones and 48 h OD_(450) of cells in CDDP+miR-9 inhibitor group were lower(P<0.05).Compared with CDDP group and CDDP+miRNA NC group,the expression levels of miR-9 and E2F1 proteins and the number of cell clones in CDDP+miR-9 inhibitor group were lower(P<0.05).In HepG2,compared with miRNA NC group,CDDP group and CDDP+miRNA NC group,(12,24,48 h)OD_(450) of cells in miR-9 inhibitor group and CDDP+miR-9 inhibitor group were lower(P<0.05).In Hep3B2.1-7,compared with miRNA NC group,(12,24,48 h)OD_(450) of cells in miR-9 inhibitor group,(6,12,24,48 h)OD_(450) of cells in CDDP+miR-9 inhibitor group were lower(P<0.05).Compared with CDDP group,(12,24,48 h)OD_(450) of cells in miR-9 inhibitor group,(3,6,12,24,48 h)OD_(450) of cells in CDDP+miR-9 inhibitor group were lower(P<0.05).Compared with CDDP+miRNA NC group,(24,48 h)OD_(450) of cells in miR-9 inhibitor group,(12,24,48 h)OD_(450) of cells in CDDP+miR-9 inhibitor group were lower(P<0.05).Conclusion:The expressions of miR-9 and E2F1 in HepG2 and Hep3B2.1-7 cell lines are up-regulated.Interfering miR-9 can inhibit the expression of E2F1,inhibit proliferation,inhibit clone formation,and improve the drug-sensitivity.