Screening Of Serum Protein Biomarker Based On ITRAQ And Mechanism Research In Diabetes Mellitus

Objective: Using iTRAQ technology of proteomics to explore the types and abnormal level of serum low abundance protein expression in diabetic patients and healthy people,then to screen the biological markers relate to diabetes,for further research of the verification protein from proteomic in population studies.At last,explore the mechanisms of differentially expressed proteins on insulin signal transduction in basic research.In order to provide new data for the study of population proteome database and serum protein distribution and function in patients with diabetes mellitus,and explore the new research direction of protein associated with the diabetes pathogenesis.Methods: Using iTRAQ technology to observe the serum low abundance proteins in patients with newly diagnosed T2 DM compared with healthy people in order to select the expression level of abnormal protein,application of biomedical software to classify the identified protein and biological processes involved in the analysis of components and cells.From the first stage of the screened proteins selected two kinds of abnormal decrease protein TGFBIp and IGFLAS,then through ELISA method to observe these two protein expression levels in different populations,in patients with diabetes and non diabetes patients with chronic diseases and healthy people to verify the protein group study results.The correlation between the expression of TGFBIp and IGFLAS and the clinical data of patients with diabetes mellitus was further observed.The receiver operating characteristic curve was drawn to determine the effect of differential expressed protein on the diagnosis of diabetes.Insulin resistance(IR)cell model was induced by high concentration insulin and glucose in human hepatocellular carcinoma cells(Hep G2),and the expression of IGFALS was silenced by IGFALS-si RNA.ELISA method was used to detect the expression of IGFALS in each group of cells.Western-blot method was used to detect the expression of Akt,Phospho-Akt,NF-kappa B,Phospho-NF-kappa B in cells before and after IGFALS-siRNA transfection.Results: The number of serum proteins identified in T2 DM patients was 222,and detected 72 difference level protein.Among them,the expression of the 38 proteins was up-regulated and the expression of the 34 proteins was down regulated.Among the 72 differentially expressed proteins,17% of them were extracellular proteins,the other one was cytoplasmic protein,the other was membrane protein,and the other was the inner membrane protein,the percent was 16%,10% and 4%.There were 33% involved in the regulation of biological process,and 19% was involved in lipid metabolism.11% was involved in the metabolic process,8% was involved in the immune response,7% was involved in the immune response and 6% was involved in the inflammatory response.The TGFBIp increased significantly in the chronic disease group(CD group),diabetic group(DM group)showed a decreasing trend than in the control group(CON group),but the difference was not statistically significant(220.09 ng/ml±46.19 ng/ml vs 233.07 ng/ml±45.83 ng/ml,P=0.118).There are significant differences in the three groups of IGFALS,it was the lowest in DM group(12.21 ng/ml±4.21 ng/ml),followed by group CD(16.93 ng/ml±4.21 ng/ml),in CON group was the highest(18.85 ng/ml±5.63 ng/ml),the difference was statistically significant(F=48.90,P<0.01).There was no statistical correlation between TGFBIp and clinical indexes,IGFALS was negatively correlated with FBG and Hb A1 C,and the R value was-0.23,-0.32(P<0.05),which was positively correlated with DBP,and the R value was 0.35(P<0.01).The Cutoff value of IGFALS was 15.91 ng/ml,with a sensitivity of 65.2%,specificity of and AUC of 0.802,which was statistical(P<0.01).Hep G2 were not significantly affected by high insulin and high glucose cultured 48 h and 24 h respectively.When use the high concentration of insulin interfered with 24 h and the concentration of glucose at 44 mmo/L,55 mmol/L and 66 mmol/L interfered with 12 h,which had a greater effect on insulin sensitivity.Decreased glucose consuming in cells after silencing the expression of IGFALS(P<0.01).IGFALS secretion was significantly lower in insulin induced IR cells than that in the control group(136.83 ng/ml±7.45 ng/ml,142.27 ng/ml±10.11 ng/ml,147.65 ng/ml±8.14 ng/ml vs 187.99 ng/ml±6.13 ng/ml,P<0.01).The secretion of IGFALS in IR cells was significantly lower than that of control group(131.30 ng/ml±4.33 ng/ml,144.09 ng/ml±7.00 ng/ml vs 187.99 ng/ml±6.13 ng/ml,P<0.01)in the presence of the two concentrations of high glucose.The secretion of IGFALS in IGFALS-home-890 group was significantly lower than that in control group(114.55 ng/ml±7.50 ng/ml vs 187.99 ng/m±6.13 ng/ml,P<0.01).WB test results show that the cellular p-Akt levels in three groups were significantly lower than that in the control group(1.23±0.12,1.11±0.14,0.99±0.05 vs 1.48±0.11,F=10.832,P<0.01)The expression of Akt was difference only in insulin induced group(1.78±0.19 vs 2.42±0.29,P<0.05).The expression of p-NF-?B and NF-?B in three groups were significantly higher than that in control group(p-NF-?B:0.41±0.05,0.48±0.11,0.39 ±0.04 vs 0.23±0.03,F=7.706,P=0.01;NF-?B:0.86±0.08,0.90±0.07,0.96±0.07 vs 0.65±0.04,F=12.760,P=0.002).Conclusions: Differential proteomic analysis through iTRAQ technology to detect low abundance proteins can be used to screen differentially expressed proteins in diabetic patients,providing more optional proteins for the detection of serum biomarkers in patients with diabetes mellitus.The level of IGFALS in serum of patients with diabetes decreased significantly compared with the control group,and was negatively correlated with the levels of Hb A1 C,FBG.As a biological marker for diagnosing diabetes mellitus,IGFALS has better specificity and less sensitivity.The expression of IGFALS was significantly decreased in IR cells induced by high glucose and high insulin.After silencing the expression of IGFALS,the level of p-Akt in insulin signal transduction pathway decreased,at the same time the level of NF-?B and p-NF-?B in signal transduction pathway of inflammation increased.IGFALS may influence IR through insulin and inflammation signal transduction pathway.