糖尿病视网膜病变伴糖尿病肾病患者转录组学差异分析

The analysis of transcriptome differences in patients with diabetic nephropathy and diabetic retinopathy

目的应用转录组学技术分析伴与不伴糖尿病视网膜病变(DR)和糖尿病肾病(DN)的T2DM患者的差异表达基因,寻找DR伴DN相关基因.方法入组T_(2)DM患者14例,其中8例伴DR和DN纳入DNDR组、6例不伴DN和DR纳入DM组.采集患者外周血,分离白细胞,抽提总RNA,反转录为cDNA,构建转录组文库,Illumina HiSeqTM2000系统测序,取得每份样本转录组数据.以两组间差异基因表达量均数和中位数差异倍数上下调均≥2.0倍为标准,筛选差异表达基因.并行基因本体(GO)显著性富集分析、生化代谢与信号转导通路(Pathway)显著性富集分析、基因关系网络分析.实时荧光定量PCR(RT-qPCR)检测外周血白细胞蛋白激酶C delta结合蛋白(PRKCDBP)基因、CD177抗原(CD177)基因的mRNA水平,验证转录组测序结果.结果与DM组比较,DNDR组筛选出差异表达基因98个,其中上调42个,下调56个.GO富集分析显示,差异基因在分子功能、细胞组分、生物学过程中富集最多的条目分别为结合功能、细胞和细胞成分、单一生物过程.Pathway显著性富集分析显示差异基因参与85个Pathway,抗原处理和递呈为富集最显著的Pathway之一,下调最显著的杀伤细胞免疫球蛋白样受体2DS1(KIR2DS1)参与其中.基因相互作用网络分析示差异表达基因中淋巴因子激活的杀伤T细胞来源蛋白激酶(PBK)与PRKCDBP、Ras关联域家族成员6(RASSF6)之间存在相互作用.杀伤细胞免疫球蛋白样受体2DL1(KIR2DL1)与KIR2DS1有相互作用关系.RT-qPCR示DNDR组PRKCDBPmRNA表达量低于DM组、CD177mRNA表达量高于DM组.结论两组外周血白细胞中有多种差异表达基因,下调基因中的KIR2DL1、KIR2DS1、SPINK4、PRKCDBP,上调基因中的ABHD11-AS1、WNT3、CD177是否与DNDR的发病和进展有关,有待于进一步验证.

Objecties To study the iflerentially expressed genes in diabetic patients with or without diabetic retinopathy(DR)and diabetic nephropathyd(DN),and to search for the related genes of DR and DN by applying the transcriptome techniques.Methods According to the inclusion criteria,14 patients with T_(2)DM were selected.Among them,8 T_(2)DM patients both with DR and DN were included in the DNDR group and 6 T_(2)DM patients without DN and DR were included in the DM group.The peripheral blood of each patient was collected,white blood cells were isolated,total RNA was extracted and retranscribed to cDNA.The transcriptome library was constructed and sequenced by Illumina HiSeqTM2000 system.The differential expression genes were screened according to the standard that the average and median difference multiple of differential gene expression between the two groups were more than or equal to 2.0 times(up/down regulated).Further,The difference expressed genes ontology(GO)enrichnient analysis,biochemical metabolism and signal transduction pathway(Pathway)enrichment analysis,and gene relationship network analysis were carried out.The Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect and validate the protein kinase C delta binding protein(PRKCDBP)and CD177 antigen(CD177)mKNA levels in peripheral leukocytes of the enrolled patients.Results Compared with the DM group,98 differentially expressed genes were screened out in the DNDR group,including 42 up-regulated genes and 56 down regulated genes.GO enrichment analysis showed that the most abundant items of differential genes in biological process,cell components and molecular functions were single biological process,cell and cell components,and binding function.Pathway analysis showed that the differentially expressed genes participated in 85 Pathways.One of the most significant enrichment pathways was antigen treatment and presentation.The most significant down regulation of killer cell immunoglobulin-like receptor 2DS1(KIR2DS1)gene was involved.Gene interaction network analysis showed that there was interaction between lyniphokine activated killer T cell-derived protein kinase(PBK)and PRKCDBP,RAS related domain family member 6(rassf6).Killer cell immunoglobulin like receptor 2dl1(KIR2DL1)interacts with KIK2DS1.That PRKCDBP mRNA expression was lower and CD177 mRNA expression was higher in DNDR group than those in DM group revealed by RT qPCR.Conclusion There are a variety of differentially expressed genes in peripheral blood leukocytes between DNDR group and DM group.Among them,KIR2DL1,KIR2DS1,SPINK4 and PRKCDBP in down-regulated genes and ABHD11-AS1,WNT3,CD177 in up-regulated genes,whether these genes are related to the pathogenesis and progression of DNDR remains to be further verified.