| Silent information regulator 1(SIRT1)is an NAD~+dependent class III histone deacetylase.As a multifunctional transcriptional regulator,SIRT1 can regulate the activities of key transcription factors and cofactors in almost all tissues,and plays an important role in multiple pathways related to diabetes such as the aging and apoptosis of pancreatic isletĪ²cells,insulin secretion and resistance,glucose and lipid metabolism.Animal studies have shown that SIRT1 gene regulates insulin secretion and is involved in autoimmune diseases,and activation of SIRT1 gene can protect against type 1 diabetes(T1D)to a certain extent.Moreover,a study published in Cell Metabolism revealed the role of a rare variant of SIRT1 gene in human autoimmune diabetes,however,whether it has genetic variation in Chinese Han T1D population and its specific mechanism remains unclear.Aims:1)To explore the association between genetic variants of SIRT1 and classical T1D in Chinese Han population,and identified common and rare variants of SIRT1 gene,with a view to providing some molecular genetic evidence for exploring T1D susceptibility genes;2)To explore the effects of genetic variants on SIRT1 gene function and its possible mechanism of T1D,so as to provide a new explanation for the pathogenesis of T1D.Methods:1)We collected genetic samples of 979 Chinese Han patients with classic T1D and 529 healthy controls diagnosed at the Department of endocrinology of Second Xiangya Hospital of Central South University.All exonic regions of SIRT1 gene was captured and sequenced by Fast target technology to identify common and rare variants.SPSS 26.0 statistical software was used to compare the clinical data,genotype and allele frequencies between T1D patients and the control group,and to analyze the relationship between SIRT1 gene polymorphism and T1D susceptibility under different genetic models;2)Combined with bioinformatics methods,rare variants that may affect the function of SIRT1 were selected from the SIRT1 gene variant sites sequenced in the first part;3)The overexpressed plasmids of wild type and mutated types at different sites of SIRT1 gene were constructed,and established 293T cell models that stably overexpressed wild type and mutated types respectively;4)The functional study of screened SIRT1 gene variants:(1)Immunofluorescence was used to detect the localization of SIRT1 protein;(2)Western blot(WB)was used to detect the expressions of wild type and mutated SIRT1 protein and the protein stability;(3)CCK-8 assay was used to detected the proliferation activity of 293T cells stably expressing wild type and mutated SIRT1 gene;(4)ELISA was used to detected the insulin secretion of wild type and different SIRT1 genetic variants in MIN6 cell model.Results:1)A total of 21 rare variants of SIRT1 gene and 4 common variants of SIRT1 gene were found in all samples,of which 10 variants were reported for the first time;2)Among the common variants of SIRT1gene,there was no significant difference between T1D patients and the control group(P>0.05),and no statistically significant correlation between SIRT1 rs74790878 and T1D susceptibility under different genetic models;3)Among the rare variants of SIRT1 gene,the frequency of rs978078516(P=0.014)was significantly different between T1D patients and the control group;4)For the first time,9 novel SIRT1 genetic variants(10:69647232,10:69651251,10:69666703,10:69672390,10:69672498,10:69672641,10:69676324,10:69644845,10:69648852)were revealed that may affect the risk of T1D in the Chinese Han population;5)6 SIRT1 gene variant sites were screened by bioinformatics method,and wild type and mutated plasmid vectors were successfully constructed;6)Study on the effects of SIRT1 genetic variants on gene function:(1)Immunofluorescence results showed that SIRT1 gene variation did not alter the localization of its protein;(2)WB results showed that in 293T cells stably expressing wild type and mutated SIRT1,A488G,A1768C and G2134A mutated protein had stronger structural stability than wild type SIRT1;(3)CCK-8 results showed that 293T cells transfected with wild type or mutated SIRT1 were more proliferative than normal 293T cells without treatment(P<0.05),and 293T cells stably expressing wild type SIRT1 showed the strongest proliferation,while293T cells with the G2134A variation showed decreased proliferation,compared with 293T cells with wild type SIRT1(P<0.05);(4)ELISA results show that there was no significant difference in basal insulin secretion between wild type and mutated(C110T,A308G,A488G)MIN6cells(P>0.05),but there was a statistically significant difference in insulin secretion between wild type and mutated(G2134A)MIN6 cells(P=0.009).There was a statistically significant difference in insulin secretion stimulated by high glucose between wild type and mutated(A308G,G2134A)MIN6 cells(P<0.001).Conclusions:1)Common variants in the SIRT1 gene affect the risk of T1D in Chinese Han population;2)Rare variants of SIRT1 gene may affect the risk of T1D in Chinese Han population;3)SIRT1 genetic variants may not change the subcellular localization and their protein expression levels but it could slow down the protein degradation rate caused by cycloheximide,thereby improving the structural stability of their protein;4)The SIRT1 genetic variant may affect the proliferation of293T cell proliferation and insulin secretion levels in MIN6 cells. |
