Smoothened沉默胚胎成纤维细胞NIH/3T3的mRNA表达谱分析

Expression Profile Analysis of mRNAs in the Embryo Fibroblast Cell Line NIH/3T3 with Smoothened Knocked Down

目的探讨通过沉默Smoothened(Smo)基因抑制Hedgehog通路后,小鼠胚胎成纤维细胞NIH/3T3中mRNA表达谱的变化。方法提取Smo沉默和对照NIH/3T3细胞的总RNA,并进行测序。与对照组比较,分析差异表达的mRNA,通过BLAST2GO和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析和预测这些差异表达mRNA的相关功能。选取差异表达mRNA进行实时定量PCR验证。结果通过测序在Smo沉默的NIH/3T3细胞中发现有2289个mRNA差异表达,其中1646个mRNA在Smo沉默后表达下调,而643个mRNA表达上调。荧光实时定量PCR结果与测序结果一致。生物信息学分析显示,差异表达mRNA参与细胞过程、单有机体过程、生物学调节。差异表达mRNA富集在轴突导向(ko04360)、癌症通路(ko05200)、癌症中的microRNA(ko05206)等通路。利用GEPIA数据库分析CXCL12、IL-33、TGF-β2和FGF10及其受体在食管癌和正常食管组织中的表达情况,和预后的预测分析。结论在NIH/3T3细胞中沉默Smo抑制Hedgehog通路可能会通过细胞因子CXCL12、IL-33、TGF-β2和FGF10影响肿瘤细胞的增殖。

Objective To investigate the changes of mRNAs expression profile in mouse embryonic fibroblasts cell line NIH/3T3 after silencing the Hedgehog pathway by knocking down the Smoothened(Smo).Methods Total RNA from control and Smo knocked down NIH/3T3 cells was sequenced.The differentially expressed mRNA was compared with controls,and BLAST2GO and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to analyze and predict the functions of these differentially expressed mRNAs.The accuracy of sequencing was verified by real-time quantitative PCR using selected differentially expressed mRNAs.Results A total of 2289 mRNAs were found differentially expressed by sequencing in Smo knocked down NIH/3T3 cells,of which 1646 mRNAs were down-regulated and 643 mRNAs up-regulated.The results of real-time quantitative PCR were consistent with those of sequencing.Bioinformatics analysis revealed that differentially expressed mRNAs may be involved in cellular process,single-organism process and biological regulation.Differentially expressed mRNAs were enriched in axon guidance(ko04360),pathways in cancer(ko05200),and microRNAs in cancer(ko05206).The expression and prognostic prediction of receptors of CXCL12,IL-33,TGF-β2 and FGF10 in esophageal and normal esophageal tissues were analyzed using the GEPIA database.Conclusion Inhibition of Hedgehog pathway by Smo knocked down in NIH/3T3 cells may be inhibit the growth of tumor cells through CXCL12,IL-33,TGF-β2 and FGF10.