天津医药 ›› 2022, Vol. 50 ›› Issue (1): 46-52.doi: 10.11958/20211424

• 细胞与分子生物学 • 上一篇    下一篇

LINC01123通过miR-449a调控HGF/c-MET通路参与宫颈癌发生发展的分子机制

杜晨旭1,公建庄2,张艺1,张适1,燕巍1,谢林森1△   

  1. 1郑州大学附属郑州中心医院检验科(邮编450007),2胰腺炎科
  • 收稿日期:2021-06-18 修回日期:2021-09-12 出版日期:2022-01-15 发布日期:2022-01-19
  • 通讯作者: 杜晨旭 E-mail:cxsunny80@163.com
  • 作者简介:基金相付,较新;统计见二修附件
  • 基金资助:
    2021年度河南省高等学校重点科研项目

The molecular mechanism of LINC01123 regulates HGF/c-MET pathway through miR-449a and participates in the occurrence and development of cervical carcinoma

DU Chenxu1, GONG Jianzhuang2, ZHANG Yi1, ZHANG Shi1, YAN Wei1, XIE Linsen1△   

  1. 1 Department of Laboratory, 2 Department of Pancreatitis, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, China
  • Received:2021-06-18 Revised:2021-09-12 Published:2022-01-15 Online:2022-01-19

摘要:

摘要:目的 探讨长链非编码RNA(lncRNA)LINC01123对宫颈癌(CC)细胞增殖、迁移及侵袭的影响及可能的作用机制。方法 在GEPIA数据库分析CC组织中差异表达的lncRNA;体外培养人CC细胞系SiHa、HeLa、CaSki和人正常子宫颈上皮细胞HcerEpic,实时荧光定量PCR(qPCR)检测细胞中LINC01123、microRNA-449a(miR-449a)的表达水平。取对数生长期的CaSki细胞,分成对照组(NC)组、pcDNA3.1-NC组、LINC01123过表达组、si-NC组、LINC01123沉默组、LINC01123沉默+inhibitor-NC组、LINC01123沉默+miR-449a抑制剂组,转染48 h后,收集各组细胞用qPCR法检测转染效果;CCK-8法检测细胞增殖活性;流式细胞术分析细胞凋亡;划痕实验和Transwell小室实验检测细胞迁移、侵袭能力;Western blot检测细胞中肝细胞生长因子(HGF)/细胞间质上皮转化因子(c-MET)通路相关蛋白的表达;双荧光素酶报告基因验证LINC01123与miR-449a的靶向关系。结果 LINC01123在CC组织和细胞中的表达升高,而miR-449a在人CC细胞中呈低表达(P<0.05)。与NC组比较,LINC01123沉默组LINC01123表达、细胞增殖活性、迁移和侵袭能力、HGF表达和磷酸化c-MET(p-c-MET)/c-MET比值降低,miR-449a表达、细胞凋亡率升高(P<0.05);下调miR-449a的表达可明显减弱LINC01123沉默对CaSki细胞增殖、迁移和侵袭的抑制作用(P<0.05)。双荧光素酶检测结果显示,miR-449a是LINC01123的靶基因。结论 沉默LINC01123可通过上调miR-449a表达,抑制HGF/c-MET信号通路的激活,从而抑制CC细胞的生长和转移。

关键词: 宫颈肿瘤;肝细胞生长因子;RNA, 长链非编码;间质上皮转化因子;LINC01123;microRNA-449a 

Abstract:

Abstract: Objective To investigate the effects of long non-coding RNA (lncRNA) LINC01123 on the proliferation, migration and invasion of cervical carcinoma (CC) cells and its possible mechanism. Methods The differentially expressed lncRNA in CC tissues was analyzed in the GEPIA database. The human CC cell lines SiHa, HeLa, CaSki and human normal cervical epithelial cells HcerEpic were cultured in vitro. The real-time fluorescent quantitative PCR (qPCR) was used to detect the expression levels of LINC01123 and microRNA-449a (miR-449a) in cells. The CaSki cells in logarithmic growth phase were divided into the control group (NC group), the pcDNA3.1-NC group, the LINC01123 overexpression group, the si-NC group, the LINC01123 silence group, the LINC01123 silence+inhibitor-NC group and the LINC01123 silence+ miR-449a inhibitor group. After 48 hours of transfection, the cells of each group were collected and real-time fluorescent quantitative PCR (qPCR) was used to detect the transfection effect. CCK-8 method was used to detect cell viability. Flow cytometry was used to analyze the cell apoptosis. Scratch healing test and Transwell chamber experiment were used to detect cell migration and invasion ability. Western blot assay was used to detect the expression of hepatocyte growth factor (HGF)/cellular-mesenchymal epithelial transition factor (c-MET) pathway related proteins in cells. The dual luciferase reporter gene was used to verify the targeting relationship between LINC01123 and miR-449a. Results The expression levels of LINC01123 in CC tissues and cells were significantly increased, while the expression of miR-449a was low in human CC cells (P<0.05). Compared with the NC group, the LINC01123 expression, cell proliferation activity, migration and invasion ability, HGF expression and p-c-MET/c-MET ratio were significantly decreased in the LINC01123 silence group, the miR-449a expression and apoptosis rate were significantly increased (P<0.05). The down-regulated expression of miR-449a could significantly reduce the inhibitory effect of LINC01123 silencing on the proliferation, migration and invasion of CaSki cells (P<0.05). The result of dual luciferase test showed that miR-449a was the target gene of LINC01123. Conclusion Silencing LINC01123 can inhibit the activation of HGF/c-MET signaling pathway and inhibit the growth and metastasis of CC cells by up-regulating the expression of miR-449a.

Key words: uterine cervical neoplasms, hepatocyte growth factor, RNA, long noncoding, mesenchymal epithelial transformation factor, LINC01123, microRNA-449a