【摘要】 目的 在子宫内膜癌细胞系水平，进一步明确上调子宫内膜癌细胞内DJ-1表达的重要调控机制。方法 1.利用正常人子宫内膜上皮细胞(ESC)和4种子宫内膜癌细胞株，检测细胞中miR-128-2基因启动子区DNA甲基化的水平、miR-128-2、DJ-1 mRNA及DJ-1蛋白表达水平，比较分析它们在不同的子宫内膜癌细胞系中的差异变化及相关性。2.将miR-128-2 mimic和miR-128-2 inhibitor依次分别转染Ishikawa和SPEC-2子宫内膜癌细胞株，测定细胞中miR-128-2、DJ-1 mRNA及DJ-1蛋白表达水平的变化，以求阐明miR-128-2是否能调控内源性DJ-1表达。3.分别构建含野生型DJ-1-3’UTR的重组荧光素酶报告载体pGL3-DJ-1-3’UTR-wt和含有突变型DJ-1-3’UTR的重组荧光素酶报告载体pGL3-DJ-1-3’UTR-mut,验证DJ-1是否为miR-128-2直接靶基因。4.Ishikawa和SPEC-2子宫内膜癌细胞经5μM甲基化抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-dC)药物预处理48 h或5-Aza-dC预处理后再转染miR-128-2 inhibitor 48 h,随后检测miR-128-2基因启动子DNA甲基化水平的变化、miR-128-2和DJ-1 mRNA表达丰度的变化、DJ-1蛋白的表达水平变化，以求阐明子宫内膜癌细胞中miR-128-2基因启动子DNA甲基化是否沉默miR-128-2表达进而上调DJ-1表达。结果 1.与正常ESC相比较，Ishikawa、RL95-2、KLE及SPEC-2四种子宫内膜癌细胞中miR-128-2基因启动子区DNA甲基化水平均显著增加(P<0.01),同时miR-128-2表达明显下调(P<0.01),而DJ-1 mRNA及蛋白表达显著增加(P<0.01);通过相关性分析发现miR-128-2甲基化水平与miR-128-2表达具有显著负相关性(r=-0.9124,P<0.01),而与DJ-1 mRNA表达具有显著正相关性(r=0.8304,P<0.01);此外，miR-128-2表达与DJ-1 mRNA的表达表现为负相关性(r=-0.8963,P<0.01)。2.Ishikawa和SPEC-2细胞中转染miR-128-2 inhibitor后，DJ-1 mRNA及蛋白表达水平较对照组均有明显升高(P<0.01),而miR-128-2 mimic转染细胞后，DJ-1 mRNA及蛋白表达水平较对照组均出现明显下降(P<0.01)。3.miR-128-2 mimic转染细胞后，重组报告质粒pGL3-DJ-1-3’UTR-wt的荧光素酶活性大幅降低(P<0.01),但miR-128-2 mimic对重组报告质粒pGL3-DJ-1-3’UTR-mut的荧光素酶活性却无法产生作用(P>0.05),提示miR-128-2可直接与DJ-1 3’UTR靶向结合。4.经5μM甲基化抑制剂5-Aza-dC预处理48 h后，Ishikawa及SPEC-2细胞中miR-128-2甲基化水平均显著下降(P<0.01),miR-128-2表达均显著上升，而DJ-1 mRNA和蛋白表达水平均显著下降(P<0.01)。此外，5-Aza-dC预处理上调miR-128-2表达并下调DJ-1 mRNA和蛋白表达的作用可被miR-128-2 inhibitor所逆转(P<0.01),然而，5-Aza-dC预处理降低miR-128-2甲基化水平的作用却未能被miR-128-2 inhibitor影响。结论 子宫内膜癌细胞中DJ-1的表达可由miR-128-2直接靶向负调控；miR-128-2基因启动子区DNA甲基化致miR-128-2表达沉默可能是子宫内膜癌细胞中上调DJ-1表达的关键机制。更多还原

【Abstract】 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-1 3’ 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.更多还原