Effect of miR-128-2 silencing mediated by DNA methylation on the expression of DJ-1 in endometrial cancer cells
Objective To further clarify the important regulatory mechanism for the up-regulation of DJ-1 expression in EC cells.Methods 1.The levels of DNA methylation in the promoter region of miR-128-2 gene and the expression levels of miR-128-2,DJ-1 mRNA and DJ-1 protein in normal human endometrial epithelial cells(ESC)and four endometrial cancer cell lines were detected,and their differences and correlations in different endometrial cancer cell lines were compared and analyzed.2.MiR-128-2 mimic and miR-128-2 inhibitor were transfected into Ishikawa and SPEC-2 endometrial cancer cell lines,respectively.The expression levels of miR-128-2,DJ-1 mRNA and DJ-1 protein in the cells were measured to clarify whether miR-128-2 can regulate the expression of endogenous DJ-1.3.The recombinant luciferase reporter vectors(pGL3-DJ-1-3'UTR-wt containing wild-type DJ-1-3'UTR and pGL3-DJ-1-3'UTR-mut containing mutant DJ-1-3'UTR)were constructed.The vectors(DJ-1-3'UTR-wt and DJ-1-3'UTR-mut)were constructed to verify whether DJ-1 is a direct target gene of miR-128-2.4.Ishikawa and SPEC-2 endometrial cancer cells were pretreated with 5 μM methylation inhibitor 5-Aza-2'-deoxycytidine(5-Aza-dC)for 48 h or pretreated with 5-Aza-dC and then transfected with miR-128-2 inhibitor for 48 h.The changes of DNA methylation level of miR-128-2 gene promoter,the expression abundance of miR-128-2 and DJ-1 mRNA,and the expression level of DJ-1 protein were detected.In order to clarify whether the DNA methylation of miR-128-2 gene promoter in endometrial cancer cells silences the expression of miR-128-2 and up-regulates the expression of DJ-1.Results 1.Comparing with ESC cells,the degree of miR-128-2 promoter DNA methylation were significantly increased in in four different human EC cell lines(Ishikawa,RL95-2,KLE,and SPEC-2)(P<0.01);Meanwhile,the miR-128-2 expression was decresed,while the DJ-1 mRNA and protein expressions were up-regulated(P<0.01).Through correlation analysis,it was found that the methylation level of miR-128-2 was significantly inversely correlated with miR-128-2 expression(r =-0.9124,P<0.01),and correlated with the DJ-1 mRNA expression positively(r = 0.8304,P<0.01).In addition,the expression level of miR-128-2 was inversely correlated with that of DJ-1 mRNA(r =-0.8963,P<0.01).2.With miR-128-2 inhibitor transfecting in Ishikawa and SPEC-2 cells,DJ-1 mRNA and protein expression levels were significantly higher than those in the control group(P<0.01).While miR-128-2 mimic transfected cells,the expression levels of DJ-1 mRNA and protein were significantly down-regulated compared with the control group(P<0.01).3.After transfection of miR-128-2 mimic into cells,the luciferase activity of the recombinant reporter plasmid PGL3-DJ-1-3'UTR-wt was significantly reduced(P<0.01).However,miR-128-2 mimic had no effect on the luciferase activity of the recombinant reporter plasmid PGL3-DJ-1-3'UTR-mut(P>0.05).The results suggested that miR-128-2 might bind to the DJ-13'UTR directly.4.After pretreatment Ishikawa and SPEC-2 cells for 48 h with 5 μM 5-Aza-dC,the level of miR-128-2 promoter DNA methylation was significantly decreased(P<0.01);Meanwhile,the expression of miR-128-2 was increasd,at the same time DJ-1 mRNA/protein expressions were decreased(P<0.01).Interestingly,we found that the miR-128-2 up-regulation or the DJ-1 mRNA/protein down-regulation following 5-Aza-dC pretreatment could be reversed by miR-128-2 inhibitor(P<0.01),but the reduction of miR-128-2 methylation by 5-Aza-dC pretreatment was not affected by miR-128-2 inhibitor.Conclusion smiR-128-2 can directly target and negatively regulate DJ-1 expression in EC cells.DNA methylation-mediated silencing of miR-128-2 expression maybe a crucial regulatory mechanism for up-regulation of DJ-1 expression in EC cells.
miR-128-2DNA methylationDJ-1endometrial cancer cells5-Aza-dC
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