天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (10): 1031-1037.doi: 10.11958/20210777

• 细胞与分子生物学 • 上一篇    下一篇

miR-422a靶向激肽释放酶-4抑制宫颈癌细胞的增殖、迁移与侵袭

宋鹏霞，李群锋，曹焰晖，姚水洪   

  1. 衢州职业技术学院医学院生殖健康研究所（邮编324000）
  • 收稿日期:2021-04-01 修回日期:2021-06-28 出版日期:2021-10-15 发布日期:2021-10-15
  • 通讯作者: 宋鹏霞 E-mail:songpengxiasdu@163.com
  • 基金资助:
    衢州市科技计划指导性项目;校级重点科研项目

miR-422a targeting Kallikrein 4 inhibits proliferation, migration and invasion of cervical cancer cells

SONG Peng-xia, LI Qun-feng, CAO Yan-hui, YAO Shui-hong   

  1. Institute for Reproductive Health, School of Medicine, Quzhou College of Technology, Quzhou 324000, China
  • Received:2021-04-01 Revised:2021-06-28 Published:2021-10-15 Online:2021-10-15
  • Contact: SONG PengXia E-mail:songpengxiasdu@163.com

PDF (PC)

2878

摘要： 目的 探讨miR-422a靶向激肽释放酶-4（KLK4）对宫颈癌细胞增殖、迁移和侵袭的影响。方法 实时荧 光定量PCR法（qPCR）检测人宫颈癌细胞HeLa、SiHa和人正常宫颈上皮细胞H8中miR-422a的表达；将宫颈癌HeLa 细胞分为 blank 组、miR-NC 组、miR-422a 组、mut miR-422a 组、miR-422a+pcDNA 组、miR-422a+pcDNA-KLK4 组。 CCK-8实验和Transwell实验检测各组宫颈癌HeLa细胞增殖、迁移和侵袭的能力；TargetScan软件预测miR-422a可 能调控结合的靶基因，双荧光素酶活性实验对靶向关系进行验证。Western blot检测各组细胞KLK4蛋白表达水平。 结果 与人正常宫颈上皮细胞H8相比，宫颈癌HeLa、SiHa细胞中miR-422a呈现低表达（P＜0.01），选择宫颈癌HeLa 细胞进行后续实验；与miR-NC组和blank组相比，miR-422a组培养4、6 d细胞增殖能力降低，培养6、12 h细胞迁移 和侵袭数量减少（P＜0.05）；与miR-422a+pcDNA组相比，miR-422a+pcDNA-KLK4组培养4、6 d细胞增殖能力明显 升高，培养6、12 h细胞迁移和侵袭数量增加（P＜0.01）。TargetScan软件预测发现KLK4基因与miR-422a存在结合位 点，并经双荧光素酶报告基因实验验证。与 miR-NC 组相比，miR-422a 组中 KLK4 蛋白的表达水平降低，与 miR- 422a 组相比，mut miR-422a 组中 KLK4 蛋白的表达水平升高（P＜0.01），与 miR-422a+pcDNA 组相比，miR-422a+ pcDNA-KLK4组中KLK4蛋白的表达水平升高（P＜0.01）。结论 miR-422a可通过靶向KLK4蛋白的表达抑制宫颈 癌细胞增殖、迁移和侵袭。

关键词: 宫颈肿瘤, 激肽释放酶类, 微RNAs, HeLa细胞, 细胞增殖, 细胞运动, 细胞侵袭, miR-422a

Abstract: Objective To investigate the effects of miR-422a targeting kallikinase 4 (KLK4) on proliferation, migration and invasion of cervical cancer cells. Methods The expression levels of miR-422a in human cervical cancer cells (HeLa, SiHa) and human normal cervical epithelial cells (H8) were quantified by quantitative real-time PCR. Cervical cancer HeLa cells were divided into the blank group, the miR-NC group, the miR-422a group, the mut miR-422a group, the miR-422a+ pcDNA group and the miR-422a+pcDNA-KLK4 group. CCK-8 assay and transwell assay were used to detect the proliferation, migration and invasion of HeLa cells in each group. TargetScan software was used to predict the binding target genes. Double luciferase activity test was used to verify the targeting relationship. Western blot assay was used to detect the KLK4 protein expression in each group. Results Compared with normal human cervical epithelial cells H8, the expression levels of miR-422a were low in both cervical cancer HeLa cells and SiHa cell lines (P＜0.01), and cervical cancer HeLa cells were selected for the subsequent experiments. Compared with that in the miR-NC group and blank group, the OD450 of the miR-422a group decreased after cells were cultured for 4, 6 d, the numbers of invasion and migration cells decreased in the miR-422a group after cells were cultured for 6 and 12 h (P＜0.05). Compared with miR-422a+pcDNA group, OD450 of the miR-422a+pcDNA-KLK4 group was significantly increased at 4 and 6 d after culturing, and the number of cell migration and invasion was increased at 6 and 12 h after culturing (P＜0.01). TargetScan software predicted that there was a binding site between miR-422a and KLK4, which was verified by double luciferase reporter gene experiment. Compared with the miR-NC group, the expression level of KLK4 protein was decreased in the miR-422a group, and the expression level of KLK4 protein was increased in the mut miR-422a group (P＜0.01). Compared with the miR-422a+pcDNA group, the expression level of KLK4 protein increased in the miR-422a+pc DNA-KLK4 group (P＜0.01). Conclusion miR-422a can inhibit the HeLa cell proliferation, migration and invasion by targeting the KLK4 protein expression.

Key words: uterine cervical neoplasms, kallikreins, microRNAs, HeLa cells, cell proliferation, cell movement, cell invasion, miR-422a