IKCa1通道对HeLa细胞迁移、侵袭及miRNA表达谱的影响

摘要

目的 探讨钙激活钾离子(IKCa1)通道对宫颈癌HeLa细胞迁移、侵袭及miRNA表达谱的影响.方法 取生长期HeLa细胞,随机分为观察1~5组及DMSO组,分别给予0、10.0、20.0、30.0、40.0μmol/L TRAM-34及等体积DMSO(TRAM-34溶剂)处理24、48和72 h,用划痕实验检测HeLa细胞迁移特性;用Transwell侵袭实验检测48 h细胞侵袭特性.用高通量测序法检测IKCa1通道阻断前后miRNA表达谱的差异,并用qRT-PCR法对部分差异表达miRNAs进行验证.运用miRanda软件预测差异miRNA可能调控的靶基因,并对这些靶基因进行通路富集分析.结果 划痕实验,TRAM-34干预后HeLa细胞迁移受抑制,随着TRAM-34浓度增加及作用时间延长,抑制迁移作用越明显(P<0.001).Transwell侵袭实验显示,HeLa细胞的侵袭率随TRAM-34浓度增加呈递减趋势(F=384.050,P<0.001).通过高通量测序筛选出15个差异有统计学意义而表达上调的miRNA,5个表达下调的miRNA.qRT-PCR结果显示差异性miRNA的表达趋势与测序结果一致.生物信息学分析发现,hsa-miR-143-5p的靶基因在癌症相关通路、p53基因信号通路及MAPK信号通路上出现聚集;而hsa-miR-421的靶基因在肿瘤蛋白多糖、Rap1信号通路和Wnt信号通路出现聚集.结论 阻断IKCa1通道可抑制HeLa细胞迁移和侵袭,影响miRNAs表达谱.%Objective To investigate the effects of IKCa1 channels on the migration,invasion,and miRNA expres-sion profile of cervical cancer HeLa cells. Methods HeLa cells in the logarithmic phase were randomly divided into the observation groups 1-5 and the DMSO group,which were then treated with 0,10. 0,20. 0,30. 0,and 40. 0 umol/ L TRAM-34 (a characteristic inhibitor of IKCa1 channel),and equal volume of DMSO (TRAM-34 solvent),respectively, for 24,48 and 72 h. The migration characteristics of HeLa cells were detected by Scratch test. Transwell invasion assay was used to detect invasive properties. The miRNA expression profile of IKCa1 channel was detected by high-throughput se-quencing,and some differentially expressed miRNAs were verified by qRT-PCR. The miRanda software was used to predict the target genes that could differentiate miRNAs and underwent signal pathway enrichment analysis. Results Scratch test suggested that TRAM-34 inhibited the migration of HeLa cells,with a concentration- and time-dependent manner (P <0. 001). Transwell invasion showed that the invasion rate of HeLa cells decreased with the increase of TRAM-34 concentra-tions (F = 384. 050,P < 0. 001). High-throughput sequencing revealed 15 differentially expressed and up-regulated miR-NAs and 5 down-regulated miRNAs. The results of qRT-PCR showed that the differential expression of miRNAs was con-sistent with the sequencing results. Bioinformatics analysis showed that the target gene of hsa-miR-143-5p aggregated in the cancer-related pathways,p53 gene pathway,and MAPK signal pathway,while the target gene of hsa-miR-421 aggregated in the tumor proteoglycans,Rap1 signal pathway,and Wnt signaling pathway. Conclusion Blocking IKCa1 channel can in-hibit the migration and invasion of HeLa cells and influence the miRNAs expression profile.