miR-98-5p靶向PYGO2基因对宫颈癌细胞增殖、迁移和侵袭的影响及其作用机制研究

目的探讨miR-98-5p对宫颈癌细胞增殖、迁移和侵袭的影响以及其作用机制。 方法选取2016年1月至2019年1月郴州市第一人民医院收治的宫颈癌患者的癌组织和癌旁组织；予以miR-98-5p、si-PYGO2及anti-miR-98-5p单独或共培养Siha细胞，记为：miR-NC组、miR-98-5p组、si-NC组、si-PYGO2组、anti-miR-NC组、anti-miR-98-5p组、miR-98-5p+pcDNA组、miR-98-5p+pcDNA-PYGO2组。运用qRT-PCR检测宫颈癌组织和细胞中miR-98-5p和PYGO2 mRNA的表达水平；Western blot检测蛋白表达；MTT法检测细胞增殖活性；Transwell检测细胞迁移和侵袭；将WT-PYGO2、MUT-PYGO2分别与miR-NC、miR-98-5p共转染至Siha细胞中，双荧光素酶报告基因检测实验检测荧光活性。采用方差分析和t检验进行统计学分析。 结果与癌旁组织相比，宫颈癌组织中miR-98-5p表达水平降低（0.98±0.08比0.47±0.05），PYGO2 mRNA （1.00±0.07比2.43±0.24）和蛋白表达水平（0.27±0.03比0.62±0.05）均升高（P均< 0.001）。与正常宫颈细胞Ect1/E6E7相比，宫颈癌细胞Siha、Hela、Caski中PYGO2 mRNA （0.98±0.09比2.76±0.23、2.46±0.24、2.55±0.21）和蛋白表达水平（0.21±0.03比0.62±0.06、0.51±0.05、0.57±0.06）升高；miR-98-5p的表达水平降低（1.00±0.08比0.34±0.04、0.56±0.05、0.46±0.04） （P均< 0.05）。与miR-NC组相比，miR-98-5p组宫颈癌Siha细胞活性（48 h：0.61±0.05比0.42±0.04，72 h：1.02±0.09比0.59±0.06）、迁移数量[ （112.46±10.27）个比（48.35±4.96）个]及侵袭数量[ （92.47±9.56）个比（39.46±3.52）个]均降低（P均< 0.05）。与si-NC组相比，si-PYGO2组宫颈癌Siha细胞活性（48 h：0.64±0.06比0.46±0.05，72 h：1.05±0.08比0.67±0.06）、Siha迁移数量[ （106.48±9.75）个比（42.16±4.25）个]和侵袭数量[ （87.63±8.11）个比（35.42±6.20）个]均降低（P均< 0.05）；Cyclin D1、MMP-2、MMP-9、MMP-14表达水平降低，p21、p27表达水平升高，差异有统计学意义（P均< 0.05）。与miR-NC组比较，miR-98-5p组转染WT-PYGO2的Siha细胞荧光素酶活性（0.38±0.04比0.99±0.08）降低（P < 0.05），转染MUT-PYGO2的Siha细胞荧光素酶活性（1.03±0.08比1.01±0.09）差异无统计学意义（P > 0.05）。PYGO2过表达逆转了miR-98-5p过表达对宫颈癌Siha细胞的增殖、迁移和侵袭的抑制作用。 结论miR-98-5p可抑制宫颈癌细胞增殖、迁移和侵袭，其机制可能与其靶向调控PYGO2的表达有关，将可为宫颈癌的预防和治疗提供新靶点。

Effect and mechanisms of miR-98-5p targeting PYGO2 gene on proliferation,migration and invasion of cervical cancer

ObjectiveTo investigate the effects and mechanisms of miR-98-5p on the proliferation, migration and invasion of cervical cancer cells. MethodsCancer tissues and adjacent tissues of cervical cancer patients admitted to the first people's Hospital of Chenzhou City from January 2016 to January 2019 were collected; miR-98-5p, si-PYGO2 and anti-miR-98-5p were individually or co-cultured in Siha cells, and named miR-NC group, miR-98-5p group, and si-NC Group, si-PYGO2 group, anti-miR-NC group, anti-miR-98-5p group, miR-98-5p+pcDNA group, miR-98-5p+pcDNA-PYGO2 group. qRT-PCR was used to detect the expressions of miR-98-5p and PYGO2 mRNA in cervical cancer tissues and cells; Western blot was used to detect protein expression; MTT method to detect cell proliferation activity; Transwell to detect cell migration and invasion. WT-PYGO2 and MUT-PYGO2 were co-transfected with miR-NC and miR-98-5p into Siha cells, and the fluorescent activity was detected by dual luciferase reporter gene detection experiment. Statistical analysis was performed using method analysis and t-test. ResultsCompared with adjacent tissues, the expression of miR-98-5p in cervical cancer tissue was significantly reduced (0.98±0.08 vs 0.47±0.05) , PYGO2 mRNA (1.00±0.07 vs 2.43±0.24) and protein expression (0.27±0.03 vs 0.62±0.05) was increased (all P < 0.001) . Compared with normal cervical cells Ect1/E6E7, PYGO2 mRNA (0.98±0.09 vs 2.76±0.23, 2.46±0.24, 2.55±0.21) and protein levels (0.21±0.03 vs 0.62±0.06, 0.51±0.05, 0.57±0.06) in cervical cancer cells Siha, Hela, and Caski was increased; the expression of miR-98-5p (1.00±0.08 vs 0.34±0.04, 0.56±0.05, 0.46±0.04) was decreased (all P < 0.05) . Compared with the miR-NC group, the activity of cervical cancer Siha cells in the miR-98-5p group (48 h: 0.42±0.04 vs 0.61±0.05, 72 h: 0.59±0.06 vs 1.02±0.09) , the number of migration (48.35±4.96 vs 112.46±10.27) and invasions (39.46±3.52 vs 92.47±9.56) were decreased (all P < 0.05) . Compared with the si-NC group, the activity of cervical cancer Siha cells in the si-PYGO2 group (48 h: 0.46±0.05 vs 0.64±0.06, 72 h: 0.67±0.06 vs 1.05±0.08) , the number of migration (42.16±4.25 vs 106.48±9.75) and invasions cells (35.42±6.20 vs 87.63±8.11) were decreased (all P < 0.05) ; the expressions of Cyclin D1, MMP-2, MMP-9, MMP-14 proteins were decreased, the expressions of p21 and p27 proteins were increased, and the difference was statistically significant (all P < 0.05) . Compared with the miR-NC group, the luciferase activity (0.38±0.04 vs 0.99±0.08) was decreased of Siha cells transfected with WT-PYGO2 in miR-98-5p group (P < 0.05) , and the fluorescence of Siha cells transfected with MUT-PYGO2 (1.03±0.08 vs 1.01±0.09) was not significantly different (P > 0.05) . Overexpression of PYGO2 reversed the inhibitory effect of miR-98-5p overexpression on the proliferation, migration and invasion of cervical cancer Siha cells. ConclusionmiR-98-5p could inhibit the proliferation, migration and invasion of cervical cancer cells, which may be related to its targeted regulation of PYGO2 expression, which will provide a new target for the prevention and treatment of cervical cancer.