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。
Objective To investigate the effects of miR-422 a targeting kallikinase 4(KLK4)on proliferation,migration and invasion of cervical cancer cells.Methods The expression levels of miR-422 a 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-422 a group,the mut miR-422 a group,the miR-422 a+pcDNA group and the miR-422 a+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-422 a 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-422 a group decreased after cells were cultured for 4,6 d,the numbers of invasion and migration cells decreased in the miR-422 a group after cells were cultured for 6 and 12 h(P<0.05).Compared with miR-422 a+pcDNA group,OD450 of the miR-422 a+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-422 a 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-422 a group,and the expression level of KLK4 protein was increased in the mut miR-422 a group(P<0.01).Compared with the miR-422 a+pcDNA group,the expression level of KLK4 protein increased in the miR-422 a+pc DNA-KLK4
作者
宋鹏霞
李群锋
曹焰晖
姚水洪
SONG Peng-xia;LI Qun-feng;CAO Yan-hui;YAO Shui-hong(Institute for Reproductive Health,School of Medicine,Quzhou College of Technology,Quzhou 324000,China)
出处
《天津医药》
CAS
北大核心
2021年第10期1031-1037,共7页
Tianjin Medical Journal
基金
衢州市科技计划指导性项目(2019122)
衢州市重点创新团队项目。
