miR-449b-5p通过靶向Cyclin E2 抑制卵巢癌细胞生长和细胞周期进展

目的： 探讨miR-449b-5p对卵巢癌细胞生长的作用和机制。方法： 选取2018年6月至2020年6月于四川省人民医院妇产科接受手术的20例卵巢癌患者的癌组织和癌旁组织，以及正常卵巢上皮细胞系HOSEpiC和6种人宫颈癌细胞系SKOV3、ES-2、OVCAR-3、HO8910、CaOV-3和A2780，用qPCR检测卵巢癌组织和细胞中miR-449b-5p与CCNE2 mRNA的表达。将miR-NC、miR-499b-5p mimic、miR-499b-5p inhibitor、pc-CCNE2质粒分别或联合转染进入SKOV3细胞，通过CCK-8法测定细胞生长，流式细胞术检测细胞周期，WB法检测CCNE2蛋白的表达。双荧光素酶报告实验验证miR-449b-5p与CCNE2的靶向关系。裸鼠皮下注射转染miR-499b-5p mimic的SKOV3细胞悬液构建移植瘤模型，每周检测移植瘤体积，第 42 天处死，电子天平称皮下肿瘤重量，免疫组化法检测移植瘤组织中CCNE2和Ki67的表达。结果： 与正常卵巢组织和上皮细胞系HOSEpiC相比，miR-499b在人宫颈癌组织和细胞系SKOV3、ES-2、OVCAR-3、HO8910、CaOV-3和A2780中表达明显下调（P<0.01）。与Control组相比，miR-499b mimic组SKOV3细胞增殖明显受到抑制（P<0.01），G0/G1期细胞比例明显升高（P<0.01）；miR-499b inhibitor组SKOV3细胞增殖水平升高（P<0.01），G0/G1期细胞比例明显降低（P<0.01）。miR-499b-5p高表达明显抑制了含有野生型CCNE2质粒的荧光素酶活性（P<0.01），但对突变型CCNE2质粒的荧光素酶活性无影响。与miR-499b mimic组相比，miR-499b mimic+ pc-CCNE2组SKOV3细胞生长明显增加（P<0.01），G0/G1期细胞比例明显降低（P<0.01）。与miR-NC组相比，miR-499b mimic组裸鼠移植瘤体积明显变小（P<0.01），瘤质量明显减轻（P<0.01），CCNE2和Ki67阳性细胞明显减少（P<0.01）。结论： miR-449b-5p通过靶向Cyclin E2 抑制卵巢癌细胞生长和细胞周期进展。

miR-449b-5p inhibits ovarian cancer cell growth and cell cycle progression by targeting Cyclin E2

Objective: To investigate the effect and mechanism of miR-449b-5p on the proliferation of ovarian cancer cells.Methods: Cancer tissue and corresponding para-cancerous tissue specimens from 20 patients who underwent surgery in the Department of Obstetrics and Gynecology of Sichuan Provincial People''s Hospital from June 2018 to June 2020 were collected for this study; in addition, normal ovarian epithelial cell line (HOSEpiC) and six human cervical cancer cell lines (SKOV3, ES-2, OVCAR-3, HO8910, CaOV-3 and A2780) were also selected. mRNA expressions of miR-449b-5p and CCNE2 in ovarian cancer tissues and cells were detected by qPCR. The plasmids miR-NC, miR-499b-5p mimic, miR-499b-5p inhibitor and pc-CCNE2 were transfected into SKOV3 cells separately or in combination. Cell growth and cell cycle were measured by the CCK-8 method and Flow cytometry, the expression of CCNE2 protein was detected by WB assay, respectively. The targeting relationship between miR-449b-5p and CCNE2 was verified by Dual luciferase reporter assay. miR-499b-5p transfected SKOV3 cells were injected subcutaneously in nude mice to construct xenograft model, and the tumor volume was measured weekly. Nude mice were sacrificed at day 42. The weight of the subcutaneous tumors was weighed by an electronic balance, and the expressions of CCNE2 and Ki67 were detected by immunohistochemistry.Results: Compared with normal ovarian tissues and epithelial cell line HOSEpiC, miR-499b expression was significantly down-regulated in human cervical cancer tissues and cell lines SKOV3, ES-2, OVCAR-3, HO8910, CaOV-3 and A2780 (P<0.01). Compared with the Control group, the proliferation of SKOV3 cells in the miR-499b mimic group was significantly reduced (P<0.01) and the cell proportion in G0/G1 phase was significantly increased ( P<0.01); while the proliferation of SKOV3 cells in the miR-499b inhibitor group was significantly increased (P<0.01) and the cell proportion in G0/G1 phase was significantly reduced (P<0.01). Over-expression of miR-499b-5p significantly inhibited the luciferase activity of wild type CCNE2 plasmid (P<0.01) but had no effect on the luciferase activity of the mutant CCNE2 plasmid. Compared with the miR-499b mimic group, the growth of SKOV3 cells in the miR-499b mimic+pc-CCNE2 group was significantly increased (P<0.01) and the cell proportion in G0/G1 phase was significantly reduced (P<0.01). Compared with the miR-NC group, the tumor volume and weight of nude mice in the miR-499b mimic group were significantly reduced (all P<0.01), and the proportion of CCNE2 and Ki67 positive cells was significantly decreased (P<0.01).Conclusion: miR-449b-5p inhibits the growth and cell cycle progression of ovarian cancer cells by targeting Cyclin E2.