The Effect Of MiR-29a And Cdc42 On Proliferation And Insulin Secretion In Islet β Cell

Background and objective:The glucose-stimulated induced insulin secretion(GSIS)is composed of two phases.Cell division control protein 42(Cdc42)play a importent role in the second stage of GSIS.Under physiological conditions,glucose regulates actin cytoskeletal rearrangement and stimulating insulin secretion by modulating Cdc42-GDP(inactive)and Cdc42-GTP(active).With the development of diabetes,pancreatic islet beta cells have different degrees of functional failure.Inhibition of Cdc42 leade to impaired insulin secretion.It remains to be explored whether Cdc42 can influence pancreaticβ-cells.MicroRNAs(miRNAs)is noncodingRNAs and short that size of approximately 22 nt.MiRNAs have been shown to be involved in the pathogenesis of diabetes,insulin resistance and affecting pancreatic β-cell function.Similar to Cdc42,miR-29 a is one of the abundantly expressed miRNAs in the beta cells of mouse and human pancreas.In in vitro experiments,miR-29 a is detected to negatively regulate the secretion of insulin because of overexpression of miR-29 a result in decrease GSIS levels in β-cell.In contrast,knocking out miR-29 a in mice lead to blood glucose levels increased and serum insulin levels decreased.The effect of miR-29 a on the secretion of insulin deserved our study.MiR-29 a has been shown to affect cell proliferation in many studies.Whether miR-29 a affects islet β cells remains to be explored.At the same time,Cdc42 was identified by the dual luciferase reporter assay as an in vitro direct target gene for miR-29 a in cancer stduies.MiR-29 a can negatively regulates Cdc42,overexpression of miR-29 a inhibits the expression of Cdc42.It is worth exploring further whether miR-29 a influences proliferation of islet cell and insulin secretion through negatively regulates Cdc42 in islet cells.Therefore,the purpose of this experiment was to investigate the effects of Cdc42 and miR-29 a on islet cell proliferation and insulin secretion.Methods:1、To observe the effect of miR-29 a on the biological function of MIN6 cells in vitro.Firstly miR-29 a mimics or miR-29a-inhibitor were transiently transfected into MIN6 cells and using q PCR detecte the expression level of MiR-29 a.Secondly proliferation of MIN6 cell was detected by CCK after improve or reduce miR-29a’s expression.Third under the stimulation of high glucose concentration or base glucose concentration,the secretion of insulin in MIN6 cell was detected by ELISA after increase or reduce the expression of MiR-29 a.2、To observe the effect of Cdc42 on the biological function of MIN6 cells in vitro.Firstly Cdc42-pc DNA3.1 or Cdc42-siRNA were transiently transfected into MIN6 cells and using q PCR or western blotting detect the expression level of Cdc42.Secondly proliferation of MIN6 cell was detected by CCK after improve or inhibitor Cdc42’s expression.Third under the stimulation of high glucose concentration or base glucose concentration,the secretion of insulin in MIN6 cell was detected by ELISA after improve or inhibitor the expression of Cdc42.3、Whether or not there is miR-29a-Cdc42 signal transduction pathway in the MIN6 cell.Firstly miR-29 a mimics or miR-29a-inhibitor were transiently transfected into MIN6 cells and the expression of Cdc42 was detected by q PCR and WB.Overexpression of miR-29 a and Cdc42 simultaneously reversed the inhibitory effect of miR-29a-mimics on proliferation of MIN6 cell and reversed the inhibitory effect of miR-29a-mimics on insulin secretion stimulated by high glucose concentrations..The effect of miR-29a-inhibitor on the proliferation of MIN6 cell was reversed and the effect of miR-29a-inhibitor on insulin secretion was stimulated by high glucose concentration.Result:1、MiR-29 a expression was increased in MIN6 cells after transfection with miR-29a-mimics(P<0.01).Over expression of miR-29 a can inhibit proliferation(P<0.01)and insulin secretion that under high glucose stimulation(P<0.01).Increased expression of MiR-29 a had no effect on insulin secretion under basal stimulation.After transfection of miR-29a-inhibitor,the expression of MiR-29 a in MIN6 cells decreased(P<0.01).And reducing the expression of MiR-29 a can promote the proliferation(P<0.01)and insulin secretion (P<0.01)that under high glucose stimulation.Inhibition of MiR-29 a expression has no effect on insulin secretion under base glucose concentration stimulation.2、Cdc42 expression was increased in MIN6 cells after transfection with Cdc42-pc DNA3.1.Over expression of Cdc42 can promote proliferation and insulin secretion that under high glucose stimulation.Increased expression of Cdc42 had no effect on insulin secretion under basal stimulation.After transfection of Cdc42-siRNA,the expression of Cdc42 in MIN6 cells decreased.And reducing the expression of Cdc42 can inhibit the proliferation and insulin secretion that under high glucose stimulation.Inhibition of Cdc42 expression has no effect on insulin secretion under base glucose concentration stimulation.3、After transfection of MiR-29a-mimics in MIN6 cells,the expression of Cdc42 at the protein level decreased(P<0.01).Comparing with overexpressing MiR-29 a group,the proliferation ability of MIN6 cell and the insulin secretion recovered(P<0.01)after the over-expression of MiR-29 a and Cdc42(P<0.01).After transfection of MiR-29a-inhibitor in MIN6 cells,the expression of Cdc42 at the protein level increased(P<0.01).While inhibiting the expression of MiR-29 a and Cdc42,the proliferation ability and the high glucose concentration stimulated insulin secretion of the MIN6 cell returned(P<0.01).Conclusion:1、Increased expression of Micro29 a can inhibit the proliferation of MIN6 cell lines and insulin secretion under high glucose stimulation.Decreased expression of Micro29 a can promote the proliferation of MIN6 cell lines and insulin secretion under high glucose stimulation.2、Increased Cdc42 expression can promote the proliferation of MIN6 cell and insulin secretion under high glucose stimulation.Decreased Cdc42 expression can inhibit the proliferation of MIN6 cell and insulin secretion under high glucose stimulation.3、MiR-29 a can affect the MIN6 proliferate and secretion of insulin by inhibiting the expression of Cdc42.