miR-181b对甲状腺癌发生发展调控机制转录组学方法分析

Regulatory mechanism of miR-181b in thyroid cancer of genesis and development analyzed by transcriptomics

目的随着甲状腺癌发病率的提高,甲状腺癌的筛查与治疗受到了社会的关注。微小RNA在甲状腺肿瘤的发生和发展中发挥着重要作用。本研究旨在运用高通量测序技术和生物信息学技术来寻找与miR-181b相关的差异表达基因及信号通路并探讨其临床应用价值。方法选取甲状腺癌细胞BCPAP为研究对象,将miR-181b-5p的模拟物、空白模拟物和miR-181b-5p的抑制物及空白抑制物分别转染进BCPAP细胞,荧光定量PCR检测细胞miR-181b-5p的表达,高通量测序检测差异基因;用Cluster Profiles 3.0.5软件进行GO、KEGG及reactome富集分析。结果经验证转染成功后,通过测序检测,与转染空白模拟物的细胞相比,在转染miR-181b-5p模拟物的细胞中,有149个基因的表达量升高,fold change>2,P<0.05;同时有416个基因的表达量下降了,fold change>2,P<0.05。GO、KEGG及Reactome富集分析显示,下调的416个基因主要被富集在了白三烯生物合成、代谢等过程,并且差异有统计学意义,P<0.05。通过蛋白印迹技术验证,miR-181b-5p模拟物中SYK蛋白表达较对照组明显减少,差异有统计学意义,P=0.002(n=3)。结论在甲状腺癌中,miR-181b高表达可能通过抑制抑癌基因SYK表达,促进甲状腺癌的发生和发展,为甲状腺癌潜在的诊断及治疗的靶点。

OBJECTIVE Screening and treatment of thyroid cancer has been concern in society with the increase in the incidence of thyroid cancer.MicroRNA plays an important role in the occurrence and development of thyroid tumors.The purpose of this study was to find different expressed genes and signaling pathways related to miR-181 band explore their clinical application value in thyroid cancer by transcriptome sequencing and Bioinformatics technology.METHODS Thyroid cancer BCPAP were selected for the study,miR-181b-5p mimic,mimic control,miR-181b-5p inhibitor,and inhibitor control were transfected into thyroid cancer BCPAP cells.The expression of miR-181b-5p in thyroid cancer cells was detected by fluorescence quantitative RT-PCR,Transcriptome sequencing was carried out to find different expressed genes,and Cluster Profiles 3.0.5 software was used to do GO,KEGG and reactome pathway enrichment analysis.RESULTS After successful transfection,the expression of 149 genes in the cells transfected with miR-181b-5p mimics was increased compared with cells transfected with blank mimics by sequencing detection(fold change>2 and P <0.05),while the expression level of 416 genes decreased(fold change>2 and P<0.05).GO,KEGG and Reactome pathway enrichment analysis showed that 416 genes that were down-regulated in cells transfected with miR-181b-5p mimics were mainly enriched in leukotriene biosynthetic and metabolic process compared to cells transfected with blank mimics,which was statistically different(P<0.05).The expression of SYK protein in the miR-181b-5p mimics was significantly reduced by Western blotting,which was statistically different(P=0.002,n=3).CONCLUSION The high expression of miR-181 bin thyroid cancer can inhibit the expression of the tumor suppressor gene SYK,which further affects the metabolism of leukotrienes,thereby promoting the occurrence and development of thyroid cancer,which is potential diagnostic and therapeutic target for thyroid cancer.