生长抑制因子4对宫颈癌细胞CaSki增殖和凋亡的影响及作用机制研究

Effect of inhibitor of growth family member 4 on the proliferation and apoptosis of cervical cancer CaSki cells and its underlying mechanism

目的探讨生长抑制因子4(ING4)对宫颈癌细胞CaSki增殖和凋亡的影响及作用机制。方法取50例宫颈癌患者的宫颈癌组织和癌旁组织,宫颈癌细胞系Hela、SiHa、CaSki和正常宫颈细胞系Ect1/E6E7,定量逆转录聚合酶链反应(qRT-PCR)检测宫颈癌组织和细胞中ING4 mRNA的相对表达量,蛋白质印迹(Western blot)法检测组织和细胞中ING4蛋白的相对表达量。分别将转染ING4过表达慢病毒和稳定阴性对照慢病毒的宫颈癌细胞CaSki作为Lv-ING4组和Lv-NC组,将没有转染慢病毒的细胞作为对照组,CCK8法检测细胞增殖能力,流式细胞仪检测细胞凋亡能力,Western blot法检测caspase 3、β-catenin、c-myc蛋白的相对表达量。采用WNT/β-catenin信号激活剂LiCl处理ING4表达上调的宫颈癌CaSki细胞,检测细胞增殖能力、凋亡能力,以及caspase 3、β-catenin、c-myc蛋白的相对表达量。结果宫颈癌组织中ING4 mRNA的相对表达量明显低于癌旁组织(P﹤0.01),宫颈癌细胞CaSki中ING4 m RNA和ING4蛋白的相对表达量均明显低于宫颈癌细胞Hela、SiHa和正常宫颈细胞Ect1/E6E7(P﹤0.01)。慢病毒转染后,Lv-ING4组细胞中ING4 mRNA和ING4蛋白的相对表达量均明显高于对照组和Lv-NC组(P﹤0.01),光密度(OD)值明显低于对照组和Lv-NC组(P﹤0.01),细胞凋亡率明显高于对照组和Lv-NC组(P﹤0.01);Lv-ING4组宫颈癌细胞caspase 3蛋白的相对表达量明显高于对照组和Lv-NC组(P﹤0.01),β-catenin、c-myc蛋白的相对表达量均明显低于对照组和Lv-NC组(P﹤0.01)。WNT/β-catenin信号激活剂LiCl处理ING4表达上调的宫颈癌细胞CaSki后,Lv-ING4+LiCl组细胞OD值明显高于Lv-ING4组(P﹤0.01),凋亡率明显低于Lv-ING4组(P﹤0.01),Lv-ING4+LiCl组细胞caspase 3蛋白相对表达量明显低于Lv-ING4组(P﹤0.01),β-catenin、c-myc蛋白相对表达量均明显高于Lv-ING4组(P﹤0.01)。结论 ING4在宫颈癌中表达下调,其可能通过下调WNT/β-catenin信号激活通路抑制宫颈癌细胞增殖并诱导细胞凋亡。

Objective To explore the effect and mechanism of inhibitor of growth family member 4(ING4)on the proliferation and apoptosis of cervical cancer CaSki cells.Method A total of 50 cases of cervical cancer patients’cervical cancer tissues and adjacent tissues,and cervical cancer cell lines Hela,SiHa,CaSki,and normal cervical cell lines Ect1/E6 E7,were taken as the subjects.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect the relative expression of ING4 m RNA in cervical cancer tissues and cells,and Western blot was used to detect the relative expression of ING4 protein in tissues and cells.Cervical cancer cells CaSki transfected with ING4 overexpressing lentivirus and stable negative control lentivirus were used as Lv-ING4 group and Lv-NC group,and the cells not infected with lentivirus were used as the control group.CCK8 method was used to detect cell proliferation ability,flow cytometry was used to detect cell apoptosis ability,and Western blot method was used to detect the relative expression of caspase 3,β-catenin and c-myc protein.LiCl,a WNT/β-catenin signal activator,was used to treat up-regulated ING4 cervical cancer CaSki cells.The cell proliferation,apoptosis,and the expression of caspase 3,β-catenin,and c-myc were also detected.Result The relative expression of ING4 mRNA in cervical cancer tissues was significantly lower than that in adjacent normal tissues(P<0.01),and the relative expressions of ING4 mRNA and ING4 protein in cervical cancer cells CaSki were significantly lower than those of cervical cancer cells Hela,SiHa and normal cervical cells Ect1/E6 E7(P<0.01).After lentivirus transfection,the relative expression levels of ING4 mRNA and ING4 protein in the cells of the LvING4 group were significantly higher than those of the control group and the Lv-NC group(P<0.01),the optical density(OD)value was significantly lower than those of the control group and Lv-NC group(P<0.01),and the cell apoptosis rate was higher than the control group and Lv-NC group(P<0.01).After lentivirus transfection,the relative expression of caspase 3 protein in cervical cancer cells in the Lv-ING4 group was significantly higher than that of the control group and Lv-NC group(P<0.01),and the relative expression levels ofβ-catenin and c-myc protein were significantly lower than those of the control group and Lv-NC group(P<0.01).After the WNT/β-catenin signal activator LiCl treated cervical cancer cell CaSki whose ING4 expression was up-regulated,the OD value of cells in the Lv-ING4+LiCl group was significantly higher than that in the Lv-ING4 group(P<0.01),and the apoptosis rate was significantly lower than that of the LvING4 group(P<0.01),the relative expression of caspase 3 protein in the Lv-ING4+LiCl group was significantly lower than that in the Lv-ING4 group(P<0.01),and the relative expression ofβ-catenin and c-myc protein was significantly higher than that in the Lv-ING4 group(P<0.01).Conclusion ING4 is down-regulated in cervical cancer,and it may inhibit the proliferation of cervical cancer cells and induce cell apoptosis by down-regulating the activation of WNT/β-catenin signaling.