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-13’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。
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 5μM 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 5μM 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 upregulation 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.
作者
朱其舟
李慧娟
肖仲清
龙生根
陈和平
舒宽勇
Zhu Qizhou;Li Huijuan;Xiao Zhongqing;Long Shenggen;Chen Heping;Shu Kuanyong(Department of Gynecological Oncology,Jiangxi Maternal and Child Health Hospital Nanchang Jiangxi 330008;Medical Department,Jiangxi Maternal and Child Health Hospital,Nanchang Jiangxi 330008;Key Laboratory of Basic Pharmacology,School of Pharmaceutical Sciences,Jiangxi Medical College of Nanchang University,Nanchang Jiangxi 330006,P.R.China)
出处
《中国计划生育和妇产科》
2024年第4期66-71,共6页
Chinese Journal of Family Planning & Gynecotokology
基金
国家自然科学基金资助项目(项目编号:81760473)
江西省卫健委科技计划资助项目(项目编号:SKJP_220219589)。
