全基因组关联研究(GWAS)已经确定了>100个位点独立地导致2型糖尿病(T2D)风险。然而,翻译对精准医学和新治疗方法的发展的影响令人失望,由于对这些基因座如何影响T2D病理生理学知之甚少。这里,我们旨在测量位于T2D相关信号附近的基因的表达,并评估其对胰腺β细胞胰岛素分泌的影响.
在人类多组织面板中测量了104个候选T2D易感基因的表达,通过无PCR表达测定。接下来研究了β细胞富集基因的敲低对人EndoC-βH1β细胞系的胰岛素分泌的影响。最后,我们进行了RNA测序(RNA-seq),以评估影响EndoC-βH1胰岛素分泌的新基因敲除所影响的通路,并分析了新基因在胰腺β细胞功能改变的小鼠模型中的表达.
我们发现候选T2D易感基因的表达在通过激光捕获显微切割获得的胰腺β细胞或通过流式细胞术分选的胰腺β细胞和EndoC-βH1细胞中显著富集,但不在胰岛素敏感组织中。此外,七个T2D易感基因(CDKN2A,GCK,HNF4A,KCNK16,SLC30A8,TBC1D4和TCF19)在β细胞中具有已知的表达和/或功能,改变了胰岛素分泌,支持我们的功能方法。我们显示了四个候选T2D易感基因(PRC1,SRR,ZFAND3和ZFAND6),先前没有β细胞中存在和功能的知识。EndoC-βH1细胞中PRC1、SRR、ZFAND6或ZFAND3鉴定了与T2D病理生理学相关的特定基因网络。最后,Ins2的表达与Prc1、Srr的表达呈正相关,在β细胞功能改变的小鼠胰岛中发现了Zfand6和Zfand3。
这项研究表明,GWAS后功能研究能够鉴定参与人胰腺β细胞功能和T2D病理生理学的新基因和通路。
Genome-wide association studies (GWAS) have identified >100 loci independently contributing to type 2 diabetes (T2D) risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells.
The expression of 104 candidate T2D susceptibility genes was measured in a human multi-tissue panel, through PCR-free expression assay. The effects of the knockdown of beta-cell enriched genes were next investigated on insulin secretion from the human EndoC-βH1 beta-cell line. Finally, we performed RNA-sequencing (RNA-seq) so as to assess the pathways affected by the knockdown of the new genes impacting insulin secretion from EndoC-βH1, and we analyzed the expression of the new genes in mouse models with altered pancreatic beta-cell function.
We found that the candidate T2D susceptibility genes\' expression is significantly enriched in pancreatic beta cells obtained by laser capture microdissection or sorted by flow cytometry and in EndoC-βH1 cells, but not in insulin sensitive tissues. Furthermore, the knockdown of seven T2D-susceptibility genes (CDKN2A, GCK, HNF4A, KCNK16, SLC30A8, TBC1D4, and TCF19) with already known expression and/or function in beta cells changed insulin secretion, supporting our functional approach. We showed first evidence for a role in insulin secretion of four candidate T2D-susceptibility genes (PRC1, SRR, ZFAND3, and ZFAND6) with no previous knowledge of presence and function in beta cells. RNA-seq in EndoC-βH1 cells with decreased expression of PRC1, SRR, ZFAND6, or ZFAND3 identified specific gene networks related to T2D pathophysiology. Finally, a positive correlation between the expression of Ins2 and the expression of Prc1, Srr, Zfand6, and Zfand3 was found in mouse pancreatic islets with altered beta-cell function.
This study showed the ability of post-GWAS functional studies to identify new genes and pathways involved in human pancreatic beta-cell function and in T2D pathophysiology.