CircPIK3R1 Targets MiR-29a-3p To Regulate The Molecular Mechanism Of Insulin Resistance In Diabetes And The Effect Of Isoquercetin

Objective: In vivo and in vitro experiments were performed to explore the molecular mechanism of circPIK3R1 targeting miR-29a-3p in regulating insulin resistance in diabetes and the effect of isoquercetin.Methods: 1.Cell experiments Bioinformatics analysis was first used to predict the binding sites and targeting relationship of circPIK3R1 with miR-29a-3p.Then the circPIK3R1 wild-type(PGL3-circPIK3R1 Wt)and mutant plasmids(PGL3-circPIK3R1 Mut)were constructed and co transfected with miR-29a-3p mimics HEK-293 T versus Hep G2 cells,respectively,The targeted binding sites of circPIK3R1 and miR-29a-3p were verified by double luciferase assay.Next we constructed a circPIK3R1 overexpression plasmid(hsa-circ-0072746)to detecting the expression of circPIK3R1 versus linear PIK3R1 in Hep G2 cells after Rnase R treatment using QRT PCR,and the same assay was used to measure the expression of circPIK3R1 versus miR-29a-3p in insulin resistant Hep G2 cells.Intervention with 20μM isoquercetin in insulin resistant Hep G2 cells transfected with circPIK3R1 or co transfected with circPIK3R1,miR-29a-3p,cell culture supernatants were harvested 48 h after transfection,and glucose uptake was measured by glucose oxidase assay;Total RNA and protein were extracted from cells,and m RNA and protein expression of genes involved in the PI3 K / Akt signaling pathway were determined by QRT PCR and Western blot.2.Animal experiments To construct circPIK3R1 overexpression adenovirus(mmu-circ-0004400),18 SPF grade male d B / db mice(C57 background),5 weeks old,were divided into diabetes group,empty plasmid adenovirus group as well as adenovirus group,6 mice per group,6 were fed under the same conditions as same week old C57 mice,and this group was considered as normal group.Mice in the adenovirus group with empty plasmid adenovirus as well as mice in the adenovirus group with circPIK3R1 overexpression adenovirus were injected via tail vein.Injection concentrations were all 1E + 10 PFU/ m L in an injection volume of200μL / mouse.Liver tissues from mice in each group were harvested 14 days later,and QRT PCR was used to determine circPIK3R1 expression.An additional 36 SPF grade male db / db mice(C57 background),7weeks old,were divided into diabetes,adenovirus with empty plasmid,and circPIK3R1 overexpression adenovirus group,as well as isoquercetin low,medium,and high(20μM、40μM、80μM)Dose groups,6 per group,were fed under the same conditions as 6 same week old C57 mice of the same sex,and this group was considered as the normal group.Tail vein injection of empty plasmid adenovirus in empty plasmid adenovirus group and circPIK3R1 overexpression adenovirus in circPIK3R1 overexpression adenovirus group and isoquercetin group.After 14 days,isoquercetin groups were given low,medium and high doses of isoquercetin by gavage,while the non administration group was given normal saline under the same conditions.Blood samples were taken after continuous gavage for 14 days.Roche blood glucose meter and supporting test paper were used to detect fasting blood glucose.Serum fasting insulin was detected by ELISA.The tissue morphology of pancreas was observed by HE staining and immunohistochemistry.QRTPCR was used to detect the expression of circPIK3R1 and miR-29a-3p.QRT-PCR and Western blot were used to detect the m RNA and protein expression of PI3 K / Akt signal pathway related factors in the downstream pathway of miR-29a-3p.Result:1.In HEK-293 T cells or Hep G2 cells,the fluorescence activity of circPIK3R1 wild-type and miR-29a-3p co transfected cells decreased significantly(P < 0.05),while the fluorescence activity of circPIK3R1 mutant and miR-29a-3p co transfected cells did not change significantly(P > 0.05).2.Compared with the control group,the expression of circPIK3R1 decreased significantly and the expression of miR-29a-3p increased significantly in insulin resistant Hep G2 cells(P <0.05).3.Isoquercetin intervened in the transfection of circPIK3R1 and co transfection of circPIK3R1 and miR-29a-3p into insulin resistant Hep G2 cells.Compared with the model group,isoquercetin significantly increased the glucose consumption of insulin resistant Hep G2 cells(P <0.05 or P < 0.01),reduced the expression of miR-29a-3p,up regulated the expression of INSR,PI3 K and AKT,and down regulated the m RNA expression of FOXO1,PEPCK and G6Pase(P < 0.05 or P < 0.01);Up regulation of INSR,p-PI3 K and p-AKT,down regulation of FOXO1,PEPCK and G6 Pase protein expression(P < 0.05 or P < 0.01).4.After injecting circPIK3R1 overexpression adenovirus into the tail vein of db /db mice and giving it by gavage for 14 days,compared with the model group,the fasting blood glucose of low,medium and high dose isoquercetin group decreased significantly(P < 0.05),while the fasting insulin content increased significantly(P < 0.05).5.HE staining showed that compared with the normal group,the shape of islet cells in the model group and empty plasmid adenovirus group changed,and the cell contour was unclear and irregular.Compared with the model group,different doses of isoquercetin group could significantly improve the morphology of pancreas;Immunohistochemical staining showed that compared with the normal group,the positive staining of Ins R protein in the model group decreased,while the positive staining of FOXO1 and PEPCK protein increased significantly(P < 0.01).Compared with the model group,the positive staining of Ins R protein in different doses of isoquercetin group increased,while the positive staining of FOXO1 and PEPCK protein decreased significantly(P < 0.01).6.Compared with the model group,low,medium and high doses of isoquercetin could up regulate Ins R,PI3 K and AKT,and down regulate the m RNA expression of FOXO1,PEPCK and G6Pase(P < 0.05 or P < 0.01);Up regulated INSR,p-PI3 K / PI3 K,p-AKT / AKT,down regulated the protein expression of FOXO1,PEPCK and G6Pase(P < 0.05 or P < 0.01).Conclusion:1.CircPIK3R1 and miR-29a-3p have targeted binding sites.2.Isoquercetin can ameliorate the glucose uptake of insulin resistant Hep G2 cells by interfering with circPIK3R1 transfection or co transfection of circPIK3R1 and miR-29a-3p.3.CircPIK3R1 may target the adsorption of miR-29a-3p to ameliorate IR,exert the synergistic effect of isoquercetin and circPIK3R1,jointly inhibit the overexpression of miR-29a-3p and activate its downstream PI3 K / AKT signal pathway,inhibit gluconeogenesis and ameliorate IR.4.Isoquercetin can regulate glucose metabolism and ameliorate pancreatic morphology in db / db circPIK3R1 overexpressing adenovirus mice.5.Isoquercetin can regulate the gene and protein expression levels of key signal molecules of PI3K/AKT pathway through circPIK3R1,and inhibit hepatic gluconeogenesis in db/db circPIK3R1 over expressing adenovirus mice.