To investigated the functional pathways enriched and differential expressed genes in islet of tacrolimus induced post-transplantation hyperglycemia.

Differential expressed genes between tacrolimus and PBS treatment were filtered from the dataset GSE140230 from National Center for Biotechnology Information GEO database. Functional pathway annotations were conducted by gene set enrichment analysis and weighted gene co-expression network analysis. Master differential expressed genes screened from highly correlated modules of co-expression network analysis were then validated in dataset GSE156903 and tacrolimus induced diabetic mice islets. The results of quantiative real-time PCR (qRT-PCR) were compared with uncoupled t test in GraphPad Prism 6. A P<0.05 was considered statistically significant.

Altogether 268 differential expressed genes were filtered . The protein-protein interaction network constructed by these differential expressed genes had 126 nodes and 176 edges. INS, AGT, GAST and ADRA1D were the seed genes with highest degree of the protein-protein interaction network. MCODE plugin identified five close link modules in the protein-protein interaction network. Differential expressed genes mainly enriched in cell surface receptor signaling pathway, phosphorus metabolic process, phosphate-containing compound metabolic process, positive regulation of multicellular organismal process and G protein-coupled receptor signaling pathway. Altogether 38 differential expressed genes were contained in these pathways, and upregulated differential expressed genes were all enriched in G protein-coupled receptor signaling pathway, while downregulated differential expressed genes were enriched in other 4 pathways. Weighted gene correlation network analysis (WGCNA) screened 12 co-expression modules. Yellow module was the most positive correlated module (r=0.64, P=0.07), genes in yellow module were mainly enriched in insulin resistance and sulfur metabolism. Black module was the most negative correlated module (r=-0.60, P=0.09). Genes in black module were mainly enriched in adenosine 5′-monophosphate-activated protein kinase signaling pathway, Huntington disease, legionellosis and longevity regulatin pathway-multiple species. There were 61 and 56 seed genes in yellow module and black modules, respectively. The results of qRT-PCR showed that there was statistic difference for IER3 expression of islets between tacrolimus and DMSO treated mice (t=3.288, P<0.05), while KISS1R, LDLRAD3 and JMY were not (t=0.486, 0.742 and 1.731, all P>0.05). IER3 was also the differential expressed genes of tacrolimus treated islets in GSE156903. Immunofluorescence analysis showed that IER3 protein expression was decreased in tacrolimus-treated mice. The co-locoliazation analysis showed that almost 99% of insulin-positive staining area showed the postive-IER3 staining, while only 65% of the insulin-positive staining area was IER3 positive in tacrolimus-treated group. STRING database showed that IER3 was interacted with protein phosphatase 2 related genes, mitogen-activated protein kinase related genes, TNF and SLC37A2.

IER3 may play an important role in tacrolimus induced diabetes. Our findings may provide potential targets for prevention and treatment of tacrolimus induced post-transplantation diabetes mellitus.