miR-200a对宫颈癌细胞系HeLa增殖、迁移、侵袭调控作用观察及其靶向mRNA和lncRNA预测分析

Regulatory effects of miR-200a on proliferation,migration,and invasion of cervical cancer cell line HeLa and prediction and analysis of their targeted mRNA and lncRNA

目的观察微小RNA-200a(miR-200a)对宫颈癌细胞系HeLa增殖、迁移和侵袭的调控作用,对miR-200a的靶向mRNA及靶向长链非编码RNA(lncRNA)进行预测分析。方法取人宫颈癌细胞系HeLa分为1、2、3及4组,分别转染miRNA模拟物miR-200a mimics、miRNA抑制物miR-200a inhibitor、对照物mimics NC及inhibitor NC。培养48 h时采用qRT-PCR法检测各组细胞miR-200a表达;采用CCK8法检测各组细胞增殖能力,采用Tran⁃swell侵袭实验检测细胞侵袭能力,采用Transwell迁移实验和细胞划痕实验检测各组细胞迁移能力。使用miRNA数据库在线预测miR-200a靶向mRNA和靶向lncRNA,采用基因本体论功能注释和京都基因和基因组百科全书富集分析法分析宫颈组织中miR-200a的靶向mRNA和靶向lncRNA的生物学功能。结果与3组比较,1组细胞miR-200a相对表达量高;与4组比较,2组细胞miR-200a相对表达量低(P均<0.05)。与3组比较,培养72、96 h时1组细胞增殖能力低,培养48 h时侵袭细胞数及细胞穿膜数少,细胞迁移率低(P均<0.05);与4组比较,培养72、96 h时2组细胞增殖能力高,培养48 h时侵袭细胞数及细胞穿膜数多,细胞迁移率高(P均<0.05)。miR-200a靶向mRNA及lncRNA分别有83、6个,miR-200a靶向mRNA功能主要有与转录调控、自噬体组装、神经元迁移和轴突导向等生物学过程,信号通路主要有Wnt信号通路、Hippo信号通路及内质网蛋白加工信号通路等。结论上调miR-200a表达可抑制HeLa细胞的增殖、迁移和侵袭,抑制miR-200a表达可促进HeLa细胞的增殖、迁移和侵袭。miR-200a可能通过转录调控、核酸结合等生物学过程,Wnt信号通路、Hippo信号通路及内质网蛋白加工信号通路,参与调控宫颈癌的增殖、迁移和侵袭。

Objective To investigate the regulatory effects of microRNA-200a(miR-200a)on the proliferation,mi⁃gration,and invasion of cervical cancer cell line HeLa,and to predict and analyze the targeted mRNA and lncRNA of miR-200a.Methods Human cervical cancer HeLa cells were divided into four groups,which were transfected with miR-200a mimics,miR-200a inhibitor,mimics NC,and inhibitor NC,respectively.The expression level of miR-200a in the transfected cells was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Cell prolifera⁃tion and invasion were detected by CCK8 assay and Transwell invasion assay,respectively.Migration rate was measured by Transwell migration assay and wound healing assay.The miRNA database was used to predict the targeted mRNA and lncRNA of miR-200a on line.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway anal⁃yses were used to analyze the function of miR-200a target genes and signal transduction pathways in cervical tissues.Results Compared with group 3,the relative expression of miR-200a in the group 1 increased;compared with group 4,the relative expression of miR-200a in the group 2 decreased(both P<0.05).Compared with group 3,the cell proliferation ability of the group 1 was lower at 72 and 96 h of culture,the number of invasion cells and the number of transmembrane cells were smaller at 48 h of culture,and the cell migration rate was lower(all P<0.05).Compared with group 4,the cell proliferation ability of the group 2 was higher at 72 and 96 h culture,the number of invading cells and the number of trans⁃membrane cells were higher at 48 h culture,and the cell migration rate was higher(all P<0.05).A total of 83 miR-200a target mRNAs and 6 target lncRNAs were predicted.MiR-200a target mRNA functions were concentrated in transcription⁃al regulation,autophagosome assembly,neuronal migration and axonal guidance,and the signaling pathways mainly in⁃cluded Wnt signaling pathway,Hippo signaling pathway and endoplasmic reticulum protein processing signaling pathway.Conclusion Up-regulation of miR-200a expression can inhibit the proliferation,migration,and invasion of HeLa cells,while inhibition of miR-200a expression can promote them;miR-200a may participate in the regulation of proliferation,mi⁃gration and invasion of cervical cancer through biological processes such as transcriptional regulation,nucleic acid bind⁃ing,Wnt signaling pathway,Hippo signaling pathway and endoplasmic reticulum protein processing signaling pathway.