摘要
目的:应用Solexa技术分析和筛选出肾脏特异性表达microRNA(miRNA),同时利用生物信息学技术预测特异表达miRNA的靶基因并对其功能进行初步分析,探讨特异表达miRNA在肾脏中的功能。方法:选取8周龄C57/B6雄性小鼠,取其全身11个组织器官,提取总RNA并从中分离小分子量RNA进行Solexa测序,筛选出相较其他组织器官,在肾脏中表达最为特异的miRNA;然后,利用实时荧光定量逆转录聚合酶链反应(qRTPCR)技术对筛选出的肾脏特异性表达miRNA进一步验证;最后,通过生物信息学技术对其进行靶基因预测,并对交集的基因集合分别进行GO分析和KEGG通路分析。结果:Solexa测序结果显示在正常小鼠中miR-196a和miR-196b在肾脏最特异性高表达。qRT-PCR验证了miR-196a和miR-196b在小鼠11个组织器官中的表达分布情况,定量结果与测序结果相一致。TargetScan6.2和PicTar两个软件预测到交集靶基因74个。GO分析显示miR-196a/b的预测靶基因集合显著富集在血小板源生长因子结合活性、转录调控活性、细胞外基质的结构组成等分子功能上并参与调控代谢、发育等生物学过程(P<0.01)。信号转导通路显著富集于促性腺激素释放激素信号通路、细胞外基质受体相互作用等多个信号通路和脑胶质瘤、前列腺癌等疾病通路中(P<0.05)。结论:miR-196a/b在肾脏中特异性高表达,靶基因功能广泛,与细胞代谢、生长发育等密切相关。
Objective：Solexa Sequencing was used to screen for kidney-specific microRNAs. The target genes of the identified miRNAs were analysed by bioinformatics to predict the biological functions of the miRNAs. Methodology： To screen for the miRNAs specifically expressed in kidney, leven tissues or organs from 8 weeks C57/B6 male mice were collected. Total RNA was extracted, followed by small RNA isolation. Solexa sequencing was performed to profile the small RNA samples. Real-time quantitative reverse transcription polymerase chain reaction （qRT-PCR） was used to validate the results from the sequencing. Bioinformatic analysis was performed to predict the target genes of the kidney specific miRNAs. The predicted target genes were subject to gene ontology （GO） and KEGG pathway analyses for function prediction. Results： Solexa sequencing results showed that miR-196a and miR-196b were the kidney specific miRNAs in normal mice. The distribution of miR-196a and miR-196b in 11 tissues or organs of mice were validated by q-PCR, and the results were consistent with those from sequencing. The number of overlapping predicted target genes by TargetScan6. 2 and PicTar was 74. GO analysis showed that the genes involved in the regulation of platelet-derived growth factor binding, transcriptional activity and extracellular matrix structural constituent were enriched in the list of the 74 genes. The genes associated with several biological processes, including metabolic process and developmental process, were also enriched among the 74 genes （P〈0. 01 ）. The pathway analysis showed that the genes involved in GnRH signaling, ECM-receptor interaction, and glioma and prostate cancer, were significantly enriched in the 74 genes （P〈0. 05）. Conclusion：miR-196a/b specifically expressed in the kidney, and their functions are predicted to be closely related with cell metabolism, growth and development. Thus, our study may help understand the mechanism underlying kidney disease. Further studies are required to understand the role of miR-196a/b in the physiology and pathology of kidney.
出处
《肾脏病与透析肾移植杂志》
CAS
CSCD
北大核心
2015年第2期107-112,共6页
Chinese Journal of Nephrology,Dialysis & Transplantation
基金
国家重点基础研究发展计划(973计划)(2012CB517603)
