摘要
目的探究miR-21对宫颈癌Hela细胞增殖、凋亡、侵袭以及放射敏感性的影响和潜在作用机制。方法利用RT-qPCR方法检测宫颈癌组织和相邻非肿瘤组织、正常宫颈上皮细胞(H8)以及宫颈癌细胞系(HeLa、SiHa、ME180)中miR-21表达水平。通过CCK-8检测、Caspase3/7活细胞凋亡检测、伤口愈合试验、Transwell侵袭、克隆形成试验、蛋白印迹和间接免疫荧光方法分别检测miR-21降低或RECK低表达对HeLa细胞活力、凋亡、迁移、侵袭、放射敏感性以及相关蛋白影响;双荧光素酶报告基因试验验证miR-21是否直接靶向RECK发挥调控作用。结果miR-21在宫颈癌组织中表达明显高于相邻非肿瘤组织(P<0.05)。与H8细胞相比,HeLa、SiHa、ME180细胞中miR-21表达水平显著增加(均P<0.05)。下调miR-21表达可显著性抑制HeLa细胞活力、促进细胞凋亡、降低其放射耐受性,并且抑制Cyclin D1、Bcl-2、MMP-2和MMP-9蛋白的表达、促进p21和Bax蛋白表达(均P<0.05)。miR-21可直接靶向结合RECK mRNA的3’-UTR,从而负向调控RECK表达;沉默RECK逆转了miR-21降低对HeLa细胞凋亡、迁移、侵袭、放射敏感性的影响。结论抑制miR-21表达能明显抑制HeLa细胞活力、诱导细胞凋亡、降低细胞迁移和侵袭能力、增强细胞放射敏感性,其作用机制与靶向促进RECK基因的表达密切相关。
Objective To explore the effect of miR-21 on cell proliferation,apoptosis,invasion and radiosensitivity of cervical cancer HeLa cells and unravel the underlying mechanism.Methods RT-qPCR assay was used to detect the expression levels of miR-21 in cervical cancer tissues and adjacent non-tumor tissues,normal cervical epithelial cells(H8)and cervical cancer cell lines(HeLa,SiHa,ME180).HeLa cell line with inhibition of miR-21 or knockdown of RECK were constructed.CCK-8,Caspase3/7 live cell apoptosis detection,wound healing test,Transwell invasion,clone formation assay,Western blot and immunofluorescence were performed to detect cell viability,apoptosis,migration,invasion,radiosensitivity and related proteins.The dual luciferase assay verified whether miR-21 targeted RECK.Results MiR-21 level in the cervical cancer tissues was significantly higher than that in its corresponding adjacent non-tumor tissues(P<0.05).The expression levels of miR-21 in cervical cancer cell lines HeLa,SiHa and ME180 were significantly up-regulated compared with those in normal cervical epithelial cells H8(all P<0.05).MiR-21 knockdown significantly inhibited HeLa cell viability,promoted cell apoptosis,reduced radiation tolerance,down-regulated the expression of Cyclin D1,Bcl-2,MMP-2 and MMP-9,and up-regulated the expression P21 and Bax proteins(all P<0.05).miR-21 targeted the 3’-UTR of RECK mRNA and negatively regulated the expression of RECK.Silencing RECK reversed the effects of miR-21 knockdown on HeLa cell apoptosis,migration,invasion and radiosensitivity.Conclusions Inhibiting the expression of miR-21significantly decreases cell viability,induces cell apoptosis,weakens cell migration and invasion capabilities,and enhances the radiosensitivity of HeLa cells.The potential mechanism is closely related to the targeted up-regulation of RECK.
作者
苏玥辉
张梦真
Su Yuehui;Zhang Mengzhen(Department of Obstetrics andGynecology,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)
出处
《中华放射肿瘤学杂志》
CSCD
北大核心
2022年第3期277-283,共7页
Chinese Journal of Radiation Oncology
基金
河南省高等学校重点科研项目(19A320007)。
