溃疡性结肠炎小鼠结肠组织中miR-21及JNK/AP-1信号通路相关标志物的表达

Expression of intestinal miR-21 and JNK/AP-1 signal pathway markers in mice with ulcerative colitis

目的探讨溃疡性结肠炎(UC)小鼠结肠组织中微小RNA-21(miR-21)及c-Jun氨基末端激酶(JNK)/转录激活蛋白1(AP-1)信号通路相关标志物的表达。方法将16只C57BL/6小鼠随机分为对照组和模型组各8只。模型组小鼠自由饮用2.5%葡聚糖硫酸钠水溶液6 d建立急性UC模型,对照组小鼠自由饮用双蒸水。观察小鼠一般情况,按Cooper法进行小鼠疾病活动度(DAI)评分。造模第11天处死小鼠,取结肠组织,量取结肠长度。RT-PCR法检测结肠组织中miR-21、JNK1、JNK2、c-Jun mRNA表达,Western blotting法检测结肠组织中p-JNK、p-Ser63、p-Ser73蛋白表达,采用染色质免疫共沉淀实验检测结肠组织中miR-21与c-Jun的相互作用。结果与对照组比较,模型组造模第2天开始DAI评分持续升高,直至实验结束;模型组小鼠结肠长度短于对照组(P<0.01)。与对照组比较,模型组结肠组织中miR-21相对表达量高(P<0.01),JNK1、JNK2 mRNA相对表达量低(P均<0.01),c-Jun mRNA相对表达量高(P<0.01),p-JNK、p-Ser63、p-Ser73蛋白相对表达量高(P均<0.01)。染色质免疫共沉淀实验结果显示在AP-1孔处并未出现条带,表明c-Jun蛋白并未与miR-21启动子区AP-1位点结合。结论UC小鼠结肠组织中miR-21呈高表达;JNK/AP-1信号通路相关标志物JNK1、JNK2呈低表达,c-Jun、p-JNK、p-Ser63、p-Ser73呈高表达。但JNK信号通路并未直接通过下游转录因子c-Jun调节miR-21的表达。

Objective To detect the expression of intestinal microRNA-21 and JNK/activator protein(AP)-1 signaling pathway markers in the colon tissues of mice with ulcerative colitis(UC).Methods Sixteen C57BL/6 mice were randomly divided into the control group and model group.The acute UC mice were induced by drinking 2.5%dextran sodium sulfate for 6 days,and mice in the control group drank double distilled water.The general condition of mice was observed and the disease activity(DAI)of mice was evaluated by Cooper method.On the 11th day,mice were executed,the colon tissues were taken,and the colon length was measured.The expression levels of miR-21,JNK1,JNK2,and c-Jun mRNA were detected by RT-PCR;the expression levels of p-JNK,p-Ser63,and p-Ser73 protein were measured by Western blotting.The interaction between miR-21 and c-jun was detected by chromatin immunoprecipitation.Results Compared with the control group,the DAI score of the model group increased continuously from the second day to the end of the experiment,the colon length of the model group was shorter(both P<0.01),the expression levels of microRNA-21 and c-Jun in the colon tissues were enhanced(both P<0.01),the expression of JNK1 and JNK2 decreased(both P<0.01),and the expression levels of p-JNK,p-Ser63 and p-Ser73 increased(all P<0.01).The results of chromatin immunocoprecipitation showed that there was no band in AP-1 pore,indicating that c-Jun protein did not bind to AP-1 site of miR-21 promoter.Conclusion The miR-21 is highly expressed,JNK1 and JNK2 are low expressec,and c-Jun,p-JNK,p-ser63 and p-ser73 are highly expressed in the colon tissues of UC mice,but JNK signal pathway does not directly regulate the expression of miR-21 through downstream transcription factor c-Jun.