| Purpose and significance:Gestational diabetes mellitus(GDM)is defined as abnormal glucose metabolism that is first detected durimg pregnancy.In recent years,with the improvement of living standards and changes in dietary structure,the incidence of GDM has increased year by year.At present,the traditional viewpoint is that insulin resistance(IR)plays an important factor in the disease.Abnormalities in any aspect of insulin signaling may interfere with the normal function of insulin.It is worth noting that FABP4,as a low molecular weight cytoplasmic protein,can participate in insulin regulation,fatty acid transport,and mediation of lipase activity.It is highly expressed in GDM.The increased FABP4 can promote the accumulation of short chain fatty acids in cells,reduce the activity of PI3K-AKT.Therefore,FABP4 is closely related to insulin resistance in GDM.Recent reports suggest that tumor-suppressor phosphatase and tension homolog deleted on chromosome ten(PTEN)is a negative regulator of the PI3K-AKT pathway and an important modulator of insulin signaling.PTEN-FABP4 complex formation has been found,which provides an evidence of interaction between PTEN and FABP4 in regulation of glycolipid metabolism.MiR-291b-3p belongs to the miR-290 family,which regulates various cellular signaling pathways.Previous studies have confirmed that miR-291b-3p is involved in the regulation of AKT/GSK signaling pathway activation and PTEN protein expression.FABP4 and PTEN may participate in insulin resistance as the form of complex,and there are some correlations between FABP4,PTEN and AKT/GSK signaling pathway.MiR-291b-3p may participate in the AKT/GSK signaling pathway of liver insulin resistance by positively regulating the expression of PTEN.Based on the related research progress at home and abroad,this study boldly speculates that miR-291b-3p can regulate the activation of AKT/GSK signaling pathway through PTEN and the expression of FABP4 in insulin resistance of GDM.Therefore,this study investigated the mechanism of insulin resistance in GDM at the four nodes of FABP4,AKT/GSK pathway,PTEN and miR-291b-3p,in order to explore the value of miR-291b-3p in the diagnosis of GDM.The aim is to explore the value of miR-291b-3p in the diagnosis and treatment of GDM,and to provide new ideas for the search of biomarkers and drug targets of GDM.Method:First,Relationship between serum FABP4 and gene PTEN and insulin resistance in patients with gestational diabetesThe experiments in this chapter were divided into two groups:the GDM group(n=46)and the non-GDM group(n=55).The specific experimental methods were as follows:1.High-performance liquid phase,ELISA test,biochemical assay were used for detecting blood glucose,C-reactive protein,IL-6,adiponectin,HbA1c,aspartate aminotransferase,alanine aminotransferase,TC and TG in serum of patients with GDM and non-GDM.2.The content of FABP4 and TNF-a in serum were detected by ELISA.The relationship between FABP4 and GDM prediction and HOMA-IR was statistically analyzed by statistical software.3.ELISA and Real time PCR were used to detect the expression levels of protein PTEN and genes PTEN and miR-291b-3p in the serum of the experimenters.Second,Role of miR-291b-3p in PBMCs cellular insulin resistance mechanism1.PBMCs cells were isolated and purified by glycan-diammonia glucan density gradient centrifugation and immunomagnetic beads method,and the growth curve was determined by CCK8 method.2.The CCK8 method determines the safe use concentration of TNF-α stimulation on PBMCs cells.The multi-functional automatic biochemical analyzer glucose-hexose kinase method was used to detect the glucose content,and the optimal concentration and time of TNF-α stimulation on insulin production of PBMCs were established.3.Immunoblotting assay was used to detect the expression changes of proteins FABP4,GSK,p-GSK,AKT,p-AKT and PTEN in insulin resistance cell model.Real time PCR assay was used to detect the expression changes of insulin resistance cell model genes PTEN and miR-291b-3p.4.PBMCs insulin resistance cell model was transfected with miR-291b-3p mimic and miR-291b-3p inhibitor,and the expression of protein FABP4,GSK,p-GSK,AKT,p-AKT and PTEN were detected by immunoblotting.Real time PCR assay was used to detect changes in the expression of the cell model genes PTEN and miR-291b-3p.Result:First,Relationship between serum FABP4 and gene PTEN and insulin resistance in patients with gestational diabetes1.There were not difference in age,gestational weeks,and pre-pregnancy BMI between women with GDM and non-GDM controls(P>0.05).Serum C-reactive protein and IL-6 levels in women with GDM were slightly higher than those in the control group(P<0.05).There were not significant differences between SDM,DBP,TC,TG,AST,ALT and HbAlc between GDM and control groups(P>0.01).However,compared with non-GDM,fasting blood glucose,1-hour blood glucose,2-hour blood glucose,fasting insulin,and HOMA-IR were significantly elevated in GDM pregnant women(P<0.01).In addition,women with GDM gained more weight than women with normal glucose tolerance,but the difference was small and therefore did not reach statistical significance.The need for insulin therapy increased with the increase in BMI in the GDM group.Among women with normal weight(n =29),10.4%required insulin,while obese women had a probability of requiring insulin up to 29.4%(n = 1 7)(P<0.01).Neonatal results showed that the frequency of LGA(greater than gestational age)in the GDM group was 8.33%,and that in the control group was 4.44%.In the GDM group,infants whose 5-minute Apgar score were below 7 were more than the control group(5.56%vs 2.22%).2.In the GDM group,serum FABP4(4.79±2.11 vs 1.13±0.62 ng/mL,P<0.01)and TNF-α(90.17±73.07 vs 18.59±9.31 pg/mL,P<0.01)were significantly higher than control group.The area under the ROC curve used to predict GDM was 0.94(95%CI 0.90-0.98),P<0.01.The cutoff value of FABP4 used to distinguish GDM patients was 1.96 ng/mL,sensitivity%(95%CI):86.96(73.7-95.06%),specificity%(95%CI):89.09(77.75-95.89%).FABP4 concentrations were significantly higher in over-weight GDM patients(n = 17)than in normal-weight GDM patients(n = 29).In addition,a significant positive correlation was found between BMI and FABP4 levels(r = 0.88,P<0.01).FABP4 concentrations were positively correlated with insulin resistance(r = 0.95,P<0.01)and TNF-a(r = 0.90,P<0.01)in GDM patients.But serum TNF-a level was not associated with BMI(r =-0.33,P>0.05).3.The ELISA results showed that the serum PTEN concentration in the GDM group was significantly up-regulated comparing with the non-GDM group(0.43±0.14 vs 0.09±0.03 ng/mL,P<0.05).Real time PCR results showed that the serum levels of PTEN(0.63±0.15 vs 0.18±0.03,P<0.05)and miR-291b-3p(1.65±0.39 vs 1.00±0.02,P<0.05)were significantly up-regulated in the GDM group comparing with the non-GDM group.Second,Role of miR-291b-3p in PBMCs cellular insulin resistance mechanism1.PBMCs were successfully isolated and purified with a purity of 80%and a yield of 11%.2.CCK8 results showed that 0-12ng/mL TNF-α had no significant effect on the inhibition rate of PBMCs;Acting for 0-36h with TNF-α,there were not significant difference in the inhibition rate of PBMCs.The results of glucose-hexose kinase assay for glucose content showed that the concentration of glucose in the serum of 4,8 ng/mL group were significantly lower than control group,and the glucose concentration in the 12 ng/mL group was not significantly different from that of the control group.12 ng/mL TNF-α treatment PBMCs 0-48h had no significant effect on cell proliferation.However,the serum glucose concentration in the 12 and 24h groups were significantly lower than that in the control group.There were not significant difference in the glucose concentration between the 36 and 48h groups compared with the control group.The insulin resistance model of PBMCs was successfully established.12 ng/mL and 36 h were the optimal concentration and duration for TNF-α to stimulate PBMCs to produce insulin resistance.3.Compared with the control group,the expression of AKT and GSK in the insulin resistance group did not show significant change,the expression of p-AKT and p-GSK increased significantly,and the expression of FABP4 increased significantly.Compared with the control group,the expression of PTEN increased significantly in the insulin resistance group.Real time PCR results showed that the expression of the genes miR-291b-3p and PTEN in the insulin resistance group were significantly higher than that in the control group,which was statistically significant.4.Western blot results showed that there were not significant changes in expression of the proteins FABP4,GSK,AKT,p-GSK,p-AKT,p-AKT/AKT,p-GSK/GSK and PTEN in the miR-291b-3p inhibitor group comparing with the control group.The expression of FABP4 and PTEN were significantly increased in the TNF-α treated group,the miR-291b-3p negative group and the miR-291b-3p mimic group,and the miR-291b-3p mimic group was the most significantly elevated.The expression of p-GSK,p-AKT,p-AKT/AKT,p-GSK/GSK levels were significantly reduced in the TNF-α treated group,the miR-291b-3p negative group and the miR-291b-3p mimic group,and the miR-291b-3p mimic group was most significantly reduced.Compared with the TNF-α treated group,the expressions of the proteins FABP4,GSK,AKT,p-GSK,p-AKT,p-AKT/AKT,p-GSK/GSK and PTEN were not significantly different in the miR-291b-3p negative expression group.The expression of FABP4 and PTEN were significantly increased in the miR-291b-3p mimic group,and the levels of p-GSK,p-AKT,p-AKT/AKT,and p-GSK/GSK were significantly decreased.The expression of FABP4 and PTEN was significantly decreased in the miR-291b-3p inhibitor group,and the levels of p-GSK,p-AKT,p-AKT/AKT,and p-GSK/GSK were significantly increased.Real time PCR results showed that the expression of miR-291b-3p was significantly decreased in the miR-291b-3p inhibitor group,while the expression of PTEN was not significantly changed comparing with the control group;The genes miR-291b-3p and PTEN were significantly increased in the TNF-αtreatment group,miR-291b-3p negative expression group and the miR-291b-3p mimic group,and the high expression levels of the two groups of genes in the miR-291b-3p mimic group were most obvious.Conclusion:1.Compared with non-GDM,fasting blood glucose,1-hour blood glucose,2-hour blood glucose,fasting insulin,and HOMA-IR were significantly elevated in GDM pregnant women;In the GDM group,the frequency of LGA and infants whose 5-minute Apgar score were below 7 were more than the control group.2.FABP4 concentrations were positively correlated with insulin resistance and TNF-α in GDM patients.3.The serum protein PTEN concentration and the serum levels of gene PTEN and miR-291b-3p were significantly up-regulated in GDM patients.4.The protein FABP4 and insulin signaling pathway proteins of PBMCs from GDM patients returned to normal level after they were separated from the pregnant women.After TNF-α stimulation,the expression levels of protein FABP4,PTEN and gene PTEN,miR-291b-3p were up-regulated,and the expression levels of p-GSK and p-AKT were down-regulated.5.The results of transfection of miR-291b-3p mimic and inhibitor showed that after transfection of miR-291b-3p mimic,the abnormal elevation of protein FABP4,gene and protein PTEN induced by TNF-α in PBMCs cells were aggravated,and the abnormal inhibition of insulin signaling pathway(ATK/GSK pathway)was aggravated.After transfection of the inhibitor of miR-291b-3p,the abnormal elevation of protein FABP4,gene and protein PTEN induced by TNF-a in PBMCs were counteracted,and the abnormal inhibition of insulin signaling pathway(ATK/GSK pathway)was also counteracted.These results suggest that the expression of FABP4 and AKT/GSK pathway may be regulated by miR-291b-3p,which may affect insulin resistance in gestational diabetes mellitus,and may also affect the expression of PTEN at gene and protein levels,thus play a role in the mechanism of insulin sensitivity. |
PDF Full Text Request