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中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (03) : 138 -145. doi: 10.3877/cma.j.issn.2095-1221.2020.03.002

所属专题: 文献

论著

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
首泉1,(), 王茜1, 谭志远2   
  1. 1. 423000 郴州,湖南省郴州市第一人民医院妇科
    2. 423000 郴州,湖南省郴州市第四人民医院麻醉科
  • 收稿日期:2019-07-23 出版日期:2020-06-01
  • 通信作者: 首泉

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

Quan Shou1,(), Qian Wang1, Zhiyuan Tan2   

  1. 1. Department of Gynecology, the First People's Hospital of Chenzhou City, Chenzhou 423000, China
    2. Department of Anesthesiology, Chenzhou Fourth People's Hospital, Chenzhou 423000, China
  • Received:2019-07-23 Published:2020-06-01
  • Corresponding author: Quan Shou
  • About author:
    Corresponding author: Shou Quan, Email:
引用本文:

首泉, 王茜, 谭志远. 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[J/OL]. 中华细胞与干细胞杂志(电子版), 2020, 10(03): 138-145.

Quan Shou, Qian Wang, Zhiyuan Tan. Effect and mechanisms of miR-98-5p targeting PYGO2 gene on proliferation, migration and invasion of cervical cancer[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(03): 138-145.

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。

Objective

To investigate the effects and mechanisms of miR-98-5p on the proliferation, migration and invasion of cervical cancer cells.

Methods

Cancer 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.

Results

Compared 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.

Conclusion

miR-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.

表1 PYGO2及miR-98-5p干扰序列
表2 引物序列信息
图1 PYGO2蛋白在宫颈癌组织中的表达
表3 不同临床特征宫颈癌患者的宫颈癌组织中miR-98-5p、PYGO2基因相对表达量(n = 20)
表4 miR-98-5p和PYGO2在宫颈癌组织中的表达(±s
图2 PYGO2蛋白在宫颈癌细胞和正常宫颈细胞中的表达
表5 miR-98-5p和PYGO2在宫颈癌细胞和正常宫颈细胞中的表达(±sn = 3)
图3 miR-98-5p过表达对宫颈癌Siha细胞中增殖、迁移侵袭相关蛋白的表达的影响
图4 荧光倒置显微镜下观察miR-98-5p过表达抑制宫颈癌Siha细胞迁移侵袭(结晶紫染色,×200)
表6 miR-98-5p过表达抑制宫颈癌Siha细胞中增殖、迁移侵袭能力比较(±sn = 3)
图5 PYGO2和增殖、迁移侵袭相关蛋白表达
表7 抑制PYGO2表达抑制宫颈癌Siha细胞增殖、迁移和侵袭能力比较(±sn = 3)
图6 PYGO2的3'UTR中含有与miR-98-5p互补的核苷酸序列
图7 miR-98-5p对PYGO2蛋白表达的影响
表8 miR-NC组和miR-98-5p组荧光素酶相对活性(±sn = 3)
图8 PYGO2过表达逆转了miR-98-5p过表达对宫颈癌Siha细胞中PYGO2和增殖、迁移侵袭相关蛋白表达的影响
表9 宫颈癌Siha细胞增殖和迁移侵袭的比较(±sn = 3)
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