高通量测序分析胆囊结石患者microRNA表达谱差异

Analysis of differential microRNA expression in patient with gallbladder stones through high-throughput sequencing technologies

目的探讨胆囊结石和非结石患者胆囊黏膜中mi RNA表达谱的差异。方法选取30例胆结石患者(结石组)和30例胆囊息肉患者(非结石组)的胆囊黏膜组织,采用Illumina Hi Seq 2500高通量测序技术进行深度测序,比较2组的micro RNA表达差异。对差异表达的mi RNAs进行筛选与靶基因预测,并进行GO和KEGG功能显著性富集分析。荧光定量RT-PCR(q RT-PCR)对差异表达mi RNAs进行验证。结果结石组和非结石组分别获得2 215 832和1 424 770条序列,平均长度22 nt。2组间显著差异表达的mi RNA共计17个,其中9个表达上调,8个表达下调。GO分析结果示靶基因富集于离子的结合和转运、载脂蛋白结合、钙离子通道激活、蛋白激酶活性等分子功能以及大分子的合成和代谢、类固醇激素生物合成和代谢等生物进程。KEGG通路富集分析示靶基因多集中于癌症相关通路,包括WNT、Hippo等信号通路。q RT-PCR结果示差异性mi RNA的表达趋势与测序结果相一致。结论差异表达的mi RNA可能在胆囊结石的形成中具有重要作用。

Objective To detect the differential expression profile of microRNAs between patients with or without gall？bladder stone. Methods Samples from 30 patients with gallbladder stones （GS） and 30 without gallbladder stones （GP） were collected, in which microRNAs expression profiles were examined using high-throughput sequencing instrument Illumi？na HiSeq 2500. MicroRNA sequences were obtained and compared to Genebank and Rfam database for classification. Differ？entially expressed microRNAs were screened, and their target genes were predicted. Significant enrichment analysis of GO and KEGG were performed. Real-time quantitative PCR was performed on selected miRNAs in order to validate their expres？sion. Results Clean tags were obtained from both GS group （n=2 215 832） and GP group （n=1 424 770）. A total of 17 mi？croRNAs were differentially expressed between GS and GP groups with statistical significance, among which 9 were up-regu？lated and 8 were down-regulated in GS group compared to those in GP group. GO （Gene ocology） analysis showed that target genes were enriched in ion binding and transport, apolipoprotein binding, calcium channel activity, protein kinase activity, steroid hormone biosynthesis and metabolism. KEGG（Kyoto Encyclopedia of Genes and Genomes）analysis is shown for the target genes enriched in cancer related pathways, including WNT, HIPPO pathways. qRT-PCR validation of some differen？tially expressed miRNAs confirmed the result of high-throughput data analysis. Conclusion The differential expression levels of microRNAs may play an important role in occurrence and development of gallbladder stones.