Plasma Level And Biofunction Of MiR-9-3p And MiR-146a-5p In Patients Of Type 2 Diabetes

Diabetes,a common endocrine and metabolic disease,has been one of the major diseases in general population. With the rapid development of social economy and continuous improvement of living standards,the prevalence of diabetes continues to increase. Diabetes types include type 1 diabetes,type 2 diabetes,gestational diabetes,and other special type of diabetes. T2DM patients accounts for 90%-95% of the total number of diabetic patients, hyperglycemia and hypercholesterolemia are the main characteristics of them.Current research suggests that type 2 diabetes is a chronic low-grade inflammation, study after study has shown the T2DM patients often accompanied by high levels of c-reactive protein and lipopolysaccharide, tumor necrosis factor and interleukin-6 inflammatory factors. Nuclear factor-κB,protein kinase C,mitogen-activated protein kinase,and inhibitor of nuclear factor kappa-B kinase and other inflammatory reaction system were induced by inflammation factors and the effects of the cytotoxic through the oxidative stress reaction.Inflammatory reaction not only damage the mitochondria of islet cells to accelerate islet beta cells apoptosis and lead to insufficient insulin secretion,but also cause abnormal structure and function of endothelial cells which leading to insulin transport barriers in the tissue cells so as to promote the occurrence of insulin resistance.microRNAs (miRNA) are short,small RNAs,which play important roles in cell differentiation,proliferation and apoptosis via the mechanism of post-transcriptional inhibition to its target mRNA translation. Several evidences have confirmed that these miRNA are involved in the path of physiological changes of various diseases such as T2DM,Cancer and CHD. In 2008,the scientists reported miRNA existed stably in plasma or serum and their abnormal changes in circulation system are closely related with the patnoiogicai progress of several diseases,the miRNA can be transmittea by blood to receptor cells,and the change of miRNA in plasma is earlier than protein markers.These traits of miRNA represent potential ability of biomarkers for the diagnosis and prognosis of human diseases. Recently, miRNA are paid more attention in the field of diabetes. However,miRNA in diabetes diseases remain less unkown.The present study showed that the function of miR-9-3p and miR-146 a-5p is associated with chronic inflammation and insulin secretion regulation, the circulating level of miR-9-3p and miR-146a-5p is closely related with the disease such as type 2 diabetes,coronary heart disease,systemic lupus erythematosus.miR-9-3p gene,firstly reported to be originated from brain, located on choromosome 15.Its mature sequences contain two sequances, miR-9-5p and miR-9-3p. At present,some studies focused on the function of miR-9-3p in type 2 diabetes based on its roles in the inhibition of insulin secretion and reduce islet cells Sirtl protein levels.miR-146 is one of the members of miRNA,including miR-146a and miR-146b,their genes are located on chromosome 5 and chromosome 10.The mature sequences of miR-146a-5p contain two sequances,miR-146a-5p and miR-146a-3p.miR-146 was found to have immune regulation function and miR-146a-5p involved in the pathological process of chronic inflammation,the present study confirmed that the expression level of miR-146a-5p was changed significantly in diabetes cell model and cell inflammation models. Now only one paper reported that the plasma level of miR-9-3p and miR-146a-5p were changed in newly diagnosed type 2 diabetes mellitus patients,the plasma level of miR-9-3p and miR-146a-5p as a new diagnostic markers for type 2 diabetes is still lack of sufficient evidence.At present,the function of miR-9-3p and miR-146a-5p on HepG2 cell research has not yet been reported,a study shows:over expression of miR-9-3p in insulin-secreting cells causes a reduction in exocytosis elicited by glucose or potassium.One study showd that miR-9-3p acts by diminishing the expression of the transcription factor Onecut-2 and,in turn,by increasing the level of Granuphilin/Slp4,a Rab GTPase effector associated with beta-cell secretory granules that exerts a negative control on insulin release. One article has reported that miR-9-3p and Sirtl protein levels are actively regulated in vivo in β-islets during glucose dependent insulin secretion,the data also demonstrates that miR-9-3p targets and regulates Sirtl expression in insulin-secreting cells,where also showed a reduction in Sirtl protein levels when miR-9-3p expression is high during glucose-dependent insulin secretion.miR-146a-5p can changes the expression level of inflammation factor such as TNF alpha,IL-6,IL-1 beta,IRAK-1 and TRAF-6,prompt miR-146a-5p is the key regulatory factor of inflammation and play an important role in the repair of inflammation and inhibit the inflammatory response. At present,some studies focused on the function of miR-146a-5p in inflammatory reaction based on its roles in the inhibition of inflammatory response.One article has reported that miR-146a-5p was found to be a NF-kappa B-dependent gene. Importantly,miR-146a/b were predicted to base-pair with sequences in the 3’UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes,and we found that these UTRs inhibit expression of a linked reporter gene.These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors.Another article has reported that the expression levels of miR-146a-5p significantly increased in the Porphyromonas gingivalis LPS-stimulated HGFs compared to the non-stimulated HGFs.The inhibition of miR-146a-5p resulted in increased IL-1 β,IL-6 and TNF-a secretion.The mRNA and protein levels of IRAK1,but not TRAF6,also increased. They further found that miR-146a-5p directly bound to the IRAK1 3’-UTR.In this study,we first investigate Plasma level of miR-9-3p and miR-146a-5p in type 2 diabetes patients.We also explore the target inhibition of miR-9-3p to sirtuin-1 in vitro using the techniques of cell culture, transiently transfection technique,TaqMAN real-time quantitative polymerase chain reaction (PCR) and western blotting.Lastly,we use the techniques of cell culture,transiently transfection technique,western blotting to studied whether miR-146a-5p can impacts IRAK-1,ABCG1 and LXRa protein expression in P.gLPS stimulated HepG2 cell.PARTI Expression levels of miR-9-3p and miR-146a-5p in plasma from T2DM patientsObjective:This study is to investigate clinical significance of expression miR-9-3p and miR-146a-5p in T2DM patients using qRT-PCR.Methods:The volunteers including 47 T2DM patients and 28 healthy subjects enrolled from Department of Endocrinology and medical center in our Hospital between January 2012 and June 2013.Peripheral blood was handled by two-step centrifugation to remove blood cells,platelets,and petris. Total RNA from plasma were isolated using miRVana microRNA kit according to manufacture instruction.Using qRT-PCR method is to detect expression levels of miR-9-3p and miR-146a-5p.Receiver-operating curve (ROC) analysis were used to evaluate its diagnostic value for T2DM patients. Data expressed as mean ± SD or median (interquartile range).Relative levels of miRNA were reported as 2-△Ct method. Independent students’t test or non-parameters t test were used as appropriated.P<0.05 was considered as statistical significance,SPSS16.0 and GraphPAD Prism 5.0 software were used to analyzed data and drawing.Results:1 Clinical characteristics of these volunteers in this study.selected healthy people 28 cases including 14 males and 14 females,type 2 diabetic patients 47 cases including of 27 cases male and 20 cases female,the average age was 55±7,54±10 years old respectively (Tablel). Clinical data including age,sex,BMI,smoking,HP,total cholesterol levels,TG,HDL-C,and LDL-C were no significant among control group and T2DM group (P>0.05),except from FPG levels,2hPG levels and HbAlC levels of T2DM group was significantly increased (P<0.0001),compared with control group (Tablel).2 Compared with control group,expression levels of miR-9-3p in T2 DM group were significantly elevated (2-△Ct,0.004(0.002,0.006)vs0.033(0.0 04,0.209),P=0.0005)(Fig.1).3 Compared with control group,expression levels of miR-146a-5p in T2DM group were significantly depressed(2-△Ct,0.823±0.478 vs 0.016(0.0 03,0.153),P<0.0001)(Fig.2).4 AUC in 0.5 to 0.7 for the disease has a lower accuracy, AUC in 0.7-0.9 of the disease have a certain accuracy, AUC in 0.9 above for the disease had a higher accuracy,The ROC curves of miR-9-3p with an area under curve (AUC) of 0.742 (95%CI:0.628-0.857,P=0.0005) refected seperation between T2DM and control group(Fig.3).5 AUC in 0.5 to 0.7 for the disease has a lower accuracy, AUC in 0.7-0.9 of the disease have a certain accuracy, AUC in 0.9 above for the disease had a higher accuracy,The ROC curves of miR-146a-5p with an area under curve (AUC) of 0.947 (95%CI:0.901-0.993,P<0.0001)refected strong seperation between T2DM and control group (Fig.4).Conclusion:1 Expression levels of miR-9-3p in plasma from T2DM patients was elevated compared to controls.2 Expression levels of miR-146a-5p in plasma from T2DM patients was depressed compared to controls.3 Plasma miR-9-3p and miR-146a-5p in type 2 diabetes mellitus with high diagnostic value,clinical significance remains to be further research.PART2 miR-9-3p inhibits sirtuin-1 protein expression in HepG2 cell lineObjective:This study is to explore the inhibitory effect of miR-9-3p in the expression of Sirtl in vitro.Methods:In vitro experiments were performed using the techniques of cell culture,transiently transfection,qRT-PCR and western blotting. qRT-PCR data were analyed use 2-△Ct method.Independent students’t test or non-parameters t test were used as appropriated.P<0.05 was considered as statistical significance.SPSS16.0 and GraphPAD Prism 5.0 software were used to analyzed data and drawing.Results:1 qRT-PCR result indicated that there is no difference of Sirtl mRNA levels between miR-9-3p tranfected group and control group(P=0.30;Fig.5)2 Western blotting showed that protein levels of Sirtl gene after transfection are significantly lower than control group(P=0.017;Fig.6,Fig.7).Conclusion:1 In HepG2 cells,miR-9-3p had no effect of Sirtl expression at the transcriptional level.2 miR-9-3p inhibit Sirtl expression at translation level,rather than at transcriptiaonal level,which indicates that miR-9-3p regulate the expression of Sirtl via the mechanism of post-transcriptional gene silencing.PART 3 miR-146a-5p suppresses IRAK-1 and ABCG1 protein expression in P.gLPS stimulated HepG2 cell lineObjective:To investigate the potential role of miR-146a-5p in HepG2 cell line and P.gLPS stimulated HepG2 cell line.Methods:In vitro experiments were performed using the techniques of cell culture,transiently transfection and western blotting.To investigate the effect of miR-146a-5p in the expression of IRAK-1,ABCGl,LXRa in HepG2 cell line and Whether miR-146a-5p can affect the IRAK-1,ABCG1,LXRa protein expression levels in P.gLPS stimulated HepG2 cell line. Independent students’t test or non-parameters t test were used as appropriated.P<0.05 was considered as statistical significance.SPSS16.0 software and GraphPAD Prism 5.0 software were used to analysis the data and drawing,Results:1 Western blotting showed that protein levels of IRAK-1 gene after stimulated by.P.gLPS are significantly higher than control group(P=0.026; Fig.8,Fig.12,Fig.15).2 Western blotting showed that protein levels of ABCG1 gene after stimulated by.P.gLPS are significantly higher than control group(P=0.020; Fig.9,Fig.13,Fig.16).3 Western blotting showed that protein levels of LXRa gene after stimulated by.P.gLPS has no significantly change compared with control group(P>0.05;Fig.10, Fig.14).4 Our data revealed that protein levels of IRAK-1 gene after miR-146a-5p transfection are significantly lower than control group(P=0.028;Fig.11,Fig.12,Fig.15).5 Our data revealed that protein levels of ABCG1 gene after miR-146a-5p transfection has no significantly change compared with control group(P>0.05;Fig.13,Fig.16).6 Our data revealed that protein levels of LXRa gene after miR-146a-5p transfection has no significantly change compared with control group(P> 0.05;Fig.14).7 Our data revealed that miR-146a-5p+P.gLPS group can significantly downregulation the IRAK-1 protein level compared with P.gLPS stimulated HepG2 cell line(P=0.002;Fig.12,Fig.15).8 Our data revealed that miR-146a-5p+P.gLPS group can significantly downregulation the ABCG1 protein level compared with P.gLPS stimulated HepG2 cell line(P=0.008;Fig.13,Fig.16).9 Our data revealed that the LXRa protein expression level of miR-146a-5p+P.gLPS group compared with P.gLPS stimulated HepG2 cell line has no significantly change(P>0.05;Fig.14).Conclusion:1 P.gLPS stimulated HepG2 cell resulted in significant upregulation of IRAK-1 protein expression.2 P.gLPS stimulated HepG2 cell resulted in significant upregulation of ABCG1 protein expression.3 miR-146a-5p mimics transfected HepG2 cell resulted in significant downregulation of IRAK-1 protein levels.4 Manipulation with miR-146a-5p mimics significantly suppressed the expression of IRAK-1 in P.gLPS stimulated HepG2 cells.5 Manipulation with miR-146a-5p mimics significantly suppressed the expression of ABCG1 in P.gLPS stimulated HepG2 cells.