miR-10b通过抑制胶质瘤相关癌基因2蛋白表达对卵巢癌细胞克隆、凋亡的影响研究

The effect of miR-10b on the cloning and apoptosis of ovarian cancer cells by inhibiting the expression of GLI2 protein

目的探究miR-10b通过抑制胶质瘤相关癌基因2(GLI2)蛋白表达对卵巢癌细胞克隆、凋亡影响的作用机制。方法选取2019年3月至2021年4月于河北大学附属医院接受卵巢癌手术的60例患者的卵巢癌组织及距离病灶5cm处的癌旁组织作为研究对象,采用实时荧光定量聚合酶链反应(qRT-PCR)检测组织中miR-10b、GLI2 mRNA的表达;培养卵巢癌细胞,将其分为miR-10b组、GLI2蛋白组、联合组及对照组,采用克隆实验检测细胞的克隆数量,采用流式细胞仪检测细胞的凋亡,采用双荧光素酶报告基因检测miR-10b与GLI2的靶向性;选取20只裸鼠,右腋皮下接种卵巢癌细胞以建立裸鼠卵巢癌模型,建模成功后,随机分为抑制剂组和空白组,每组10只,取肿瘤组织以计算肿瘤体积。结果卵巢癌组织中miR-10b mRNA表达明显低于癌旁组织,GLI2 mRNA表达明显高于癌旁组织(P<0.05)。联合组细胞克隆及侵袭数量低于miR-10b组,细胞凋亡率高于miR-10b组(P<0.05);与对照组比较,miR-10b组、GLI2蛋白组和联合组细胞克隆及侵袭数量明显减少,细胞凋亡率明显增加,且联合组细胞克隆及侵袭数量少于miR-10b组及GLI2蛋白组,细胞凋亡率高于miR-10b组及GLI2蛋白组(P<0.05);双荧光素酶报告显示,转染miR-10b后可显著降低GLI2蛋白活性(P<0.05),但对突变基因无显著影响(P>0.05),且GLI2蛋白与miR-10b呈现负相关。抑制剂组瘤体体积显著小于空白组(P<0.05)。结论miR-10b可降低卵巢癌细胞的克隆、侵袭数量,并促进其凋亡,作用机制与靶向抑制GLI2蛋白有关。

Objective To explore the mechanism of miR-10 b inhibiting glioma-associated oncogene 2(GLI2)protein on the cloning and apoptosis of ovarian cancer cells.Methods From March 2019 to April 202160 patients with ovarian cancer who underwent surgery in Affiliated Hospital of Hebei University were selected as the research objects.The expression of miR-10 b and GLI2 mRNA in ovarian cancer tissues and adjacent tissues 5 cm away from the lesion were detected by quantitative real-time polymerase chain reaction(qRT-PCR).Ovarian cancer cells were cultured and divided into miR-10 b group,GLI2 protein group,combination group and the control group.The number of clones was detected by cloning assay,cell apoptosis was detected by flow cytometry,and the targeting of miR-10 b and GLI2 was detected by double luciferase reporter gene.20 nude mice were selected and subcutaneously inoculated with ovarian cancer cells in the right axilla to establish ovarian cancer model.After successful modeling,they were randomly divided into inhibitor group and blank group with 10 mice in each group.Tumor tissue was collected to calculate tumor volume.Results The expression of miR-10 b mRNA in ovarian cancer tissue was significantly lower than that of adjacent tissues,and the expression of GLI2 mRNA was significantly higher than that of adjacent tissues(P<0.05).The number of clones and invasion of ovarian cancer cells in the combination group was lower than that of the miR-10 b group,and the apoptosis rate was higher than that of the miR-10 b group(P<0.05).Compared with the control group,the number of clones and invasion of ovarian cancer cells in the miR-10 b group,the GLI2 protein group and the combination group was significantly reduced,the rate of cell apoptosis increased significantly,and the number of ovarian cancer cell clones and invasion in the combination group was lower than that of the miR-10 b group and the GLI2 protein group,the apoptosis rate was higher than that of miR-10 b group and GLI2 protein group(P<0.05).The dual luciferase report showed that transfection of miR-10 b can significantly reduce the GLI2 activity(P<0.05),but has no significant effect on the mutant gene(P>0.05),and the GLI2 protein is negatively correlated with miR-10 b.The tumor volume in the inhibitor group was significantly lower than that in the blank group(P<0.05).Conclusions MiR-10 b can reduce the number of cloning and invasion of ovarian cancer cells,and promote their apoptosis.Its mechanism of action is related to the targeted inhibition of GLI2 protein.