miRNA在绝经后骨质疏松症肾阴虚证中的表达谱特征及生物信息学分析

Analysis of gene expression profiles and bioinformatics in postmenopausal osteoporosis patients with Kidney Yin deficiency

目的探讨绝经后骨质疏松症(postmenopausal osteoporosis,PMOP)肾阴虚证miRNA的表达谱特征,及对差异表达的miRNA进行生物信息学分析。方法随机选择绝经后骨质疏松症受试者,中医辨证,分为3组:PMOP肾阴虚组3例,PMOP肾阳虚组3例,健康绝经后妇女3例作为对照组。采用人miRNA芯片检测外周血单个核细胞miRNA的表达水平。肾阴虚证组分别与其他两组比较,筛选共同的差异表达基因,实时荧光定量PCR验证芯片结果,并对差异表达的miRNA进行pathway分析及靶基因预测。结果肾阴虚证组与对照组、肾阳虚证组两组比较,筛选出20条共同差异表达miRNA;实时定量PCR验证hsa-miR-411-5p、hsa-miR-143-3p、hsa-miR-95-3p表达的结果与芯片结果相符;pathway分析结果显示,这些差异miRNA主要参与代谢通路、癌症通路、Rap1信号通路、粘着斑信号通路、PI3K-Akt、MAPK信号通路、钙离子信号通路、内质网蛋白加工、c GMP-PKG信号通路、Ras信号通路、Wnt信号通路的调控;结合多个数据库对差异表达的miRNA进行靶基因预测,最终筛选出9个靶基因(RC3H1、SOX11、FUT4、GABRA4、NUFIP2、ONECUT2、PHF20、PURB、ZNF148)。结论建立了PMOP肾阴虚证的miRNA基因表达谱,且这20个差异表达的miRNA可能通过调控PI3K-Akt、MAPK、Wnt等与骨代谢相关信号通路参与PMOP肾阴虚证的发生发展过程。

Objective To explore the characteristics of gene expression profiles and bioinformatics in postmenopausal osteoporosis patients with Kidney Yin deficiency.Methods Patients with postmenopausal osteoporosis were randomly chosen and according to TCM syndrome differentiation assigned into kidney yin deficiency （n=3） and kidney yang deficiency groups （n=3）, and three healthy postmenopausal women were selected as control group.MicroRNA gene expression in single nuclear cells of peripheral blood was examined using microarray method.Patients in the kidney yin deficiency group were compared with the other two groups.The common differential expression genes were selected and the dysregulated miRNAs were detected by qRT-PCR for further confirmation of microarray data.The differential expression of miRNA was analysed by bioinformatics method including pathway analysis and target gene prediction.Results There were 20 common differential expression genes.The relative expression level of hsa-miR-411-5p, hsa-miR-143-3p and hsa-miR-95-3p was consistent with the microarray data.The pathway analysis showed that these differential expression genes were involved in metabolic pathways, in cancer, Rap1 signaling pathway, focal adhesion, pi3k-akt signaling pathway, MAPK signaling pathway, calcium signaling pathway, protein processing in endoplasmic reticulum, CGMP-PKG signaling pathway, Ras signaling pathway and WNT signaling pathway.At last we screened 9 target genes （RC3H1, SOX11, FUT4, GABRA4, NUFIP2, ONECUT2, PHF20, PURB and ZNF148）.Conclusion MiRNA expression profiling of postmenopausal osteoporosis patients with Kidney Yin deficiency was established successfully.The 20 differential expressions of miRNAs involved in postmenopausal osteoporosis with Kidney Yin deficiency by regulation of PI3K-Akt, MAPK and Wnt signaling pathway.