MiR-128 Regulates DJ-1 Expression And Affects The Biological Functions Of Ishikawa Cells Via PTEN/PI3K/Akt Pathway

Objective:To investigate whether miR-128 regulates the expression of DJ-1 protein through the PTEN/PI3K/Akt signaling pathway,thereby affecting the biological functions of Ishikawa endometrial cancer cells,such as proliferation,cell cycle,apoptosis,invasion and migration.Methods:1.The expressions of miR-128,PTEN mRNA,and DJ-1 mRNA in normal endometrial tissues,endometrial cancer tissues,ESC normal endometrial cells,and Ishikawa endometrial cancer cells were detected by Real time RT-PCR.Meanwhile,the protein expression levels of DJ-1,PTEN,and phosphorylated Akt(p-Akt)in the above tissues and cell lines were detected by Western blot.In addition,the correlation between miR-128 and DJ-1 or PTEN in endometrial carcinoma tissues and cell lines was statistically analyzed.2.To determine whether regulating miR-128 expression affects the PTEN/PI3K/Akt pathway and DJ-1 expression in Ishikawa cells,miR-128 mimics and miR-128 inhibitor were respectively transfected into Ishikawa endometrial cancer cells for 48 h,and the corresponding negative controls were set up.Subsequently,miR-128 expression was detected by Real time RT-PCR,and the protein expression levels of DJ-1,PTEN,and p-Akt were detected by Western blot.3.To observe whether PTEN silencing affects the expressions of miR-128,p-Akt,and DJ-1 in Ishikawa cells,PTEN siRNA was transfected into Ishikawa cells.Moreover,to observe whether the effect of miR-128 inhibitor on the expressions of p-Akt and DJ-1 was reversed by PTEN knockdown in Ishikawa cells,PTEN siRNA was co-transfected with miR-128 inhibitor into Ishikawa cells.48 h after the above transfection,the expression level of miR-128 was detected by Real time RT-PCR,and the protein expression levels of PTEN,p-Akt,and DJ-1 was detected by Western Blot.4.To observe whether inhibition of PI3K/Akt pathway affects the expressions of miR-128,PTEN,and DJ-1-1,and whether the effect of miR-128 mimics on the expressions of PTEN,p-Akt,and DJ-1 is reversed by LY294002 in Ishikawa cells,Ishikawa cells were incubated with PI3K/Akt pathway inhibitor LY294002 for 24 h,or Ishikawa cells were transfected with miR-128 mimics for 48 h and then pretreated with LY294002 for 24 h.Subsequently,the expression level of miR-128 was detected by Real time RT-PCR,and the expression protein levels of PTEN,p-Akt and DJ-1 were detected by Western Blot.5.To determine whether miR-128 further affects the biological functions of Ishikawa cells by activation of PTEN/PI3K/Akt signaling pathway and consequent upregulation of DJ-1 protein,Ishikawa cells were transfected with miR-128 mimics for 48 h and then incubated with 10 μM LY294002 or transfected with DJ-1 siRNA for 24 h.Subsequently,cell proliferation was detected by CCK-8 and colony formation assay.Cell cycle and apoptosis were detected by flow cytometry.Cell migration was detected by Wound healing assay.Cell invasion was detected by Transwell assay.Results:1.Compared with normal endometrial tissues,the expression levels of miR-128,DJ-1 mRNA and protein,and p-Akt in endometrial cancer tissues were significantly increased,while the expression levels of PTEN mRNA and protein were significantly decreased.Similarly,compared with ESC normal endometrial cells,the expressions of miR-128,DJ-1 mRNA and protein,and p-Akt in the Ishikawa endometrial cancer cells were also up-regulated,while the expressions of PTEN mRNA and protein were down-regulated.Interestingly,the expression of miR-128 was negatively correlated with the expression of PTEN,while positively correlated with the expression of DJ-1.2.After transfection of miR-128 mimics into Ishikawa cells,the expression of miR-128 was up-regulated,while the expression of PTEN protein was down-regulated,and p-Akt and DJ-1 proteins were up-regulated.In contrast,after transfection of miR-128 inhibitor into Ishikawa cells,the expression of miR-128 was down-regulated,accompanied by the increase of PTEN protein expression and the decrease of p-Akt and DJ-1 proteins.3.After transfection of PTEN siRNA into Ishikawa cells,the expression of PTEN protein was significantly down-regulated,while the expression of miR-128 was not affected,but p-Akt and DJ-1 proteins were significantly up-regulated.In addition,the up-regulation of PTEN protein and down-regulation of p-Akt and DJ-1 proteins by miR-128 inhibitor were inhibited by PTEN siRNA transfection,but the down-regulation of miR-128 by miR-128 inhibitor transfection was not affected by PTEN siRNA.4.After pretreatment Ishikawa cells with LY294002,the expressions of p-Akt and DJ-1 proteins were significantly down-regulated,but there was no significant change in the expression levels of miR-128 and PTEN.In addition,the up-regulation of p-Akt and DJ-1 proteins by miR-128 mimics transfection was inhibited by LY294002,but the up-regulation of miR-128 and down-regulation of PTEN protein by miR-128 mimics transfection were not affected by LY294002.5.After transfection of miR-128 mimics into Ishikawa cells,the cell proliferation and clone formation abilities were significantly enhanced,and the transition from G to S phase was promoted,and the cell apoptosis rate was significantly reduced,and the cell migration and invasion abilities were enhanced.Importantly,the above effects of miR-128 mimics were inhibited by LY294002 and DJ-1 siRNA,respectively.Conclusions:miR-128 up-regulates DJ-1 expression through activating PTEN/PI3K/Akt signaling pathway,and thus participates in the regulation of cell proliferation,cell cycle,apoptosis,and cell invasion and migration in endometrial cancer cells.