miR-155通过Wnt/β-catenin信号通路影响溃疡性结肠炎结肠组织E-cadherin及snail的表达

Effect of miR-155 Mediated by Wnt/β-catenin Signaling Pathway on Experssion of E-cadherin and Snail in Ulcerative Colitis

目的通过检测小鼠结肠组织中miR-155、Wnt/β-catenin、E-cadherin及snail等相关指标变化,探讨miR-155通过激活Wnt/β-catenin信号通路介导上皮-间充质转化,影响溃疡性结肠炎(UC)的机制。方法将32只C57小鼠随机分为4组:正常组、模型组、miR-155抑制剂组、阴性对照组。除正常组外,其余各组予以3%葡聚糖硫酸钠(DSS)自由饮用1周制备UC模型,从第5天开始,miR-155抑制剂组及阴性对照组分别腹腔注射miR-155抑制剂或其阴性对照制剂(80 mg/kg),正常组和模型组予以等量磷酸盐缓冲液(PBS)腹腔注射,连续给药3 d。自造模第1天起,每天记录小鼠体质量变化、粪便性状、血便等情况。第8天处死全部小鼠,对小鼠进行疾病活动指数评分(DAI);测量结肠组织长度变化;HE染色法检测结肠组织病理结构变化;原位杂交技术检测结肠组织中miR-155含量;实时荧光定量PCR法、Western blot及免疫组化法检测结肠组织miR-155、Wnt1、β-catenin、Cyclin D1、E-cadherin及snail的表达量。结果与正常组相比,模型组结肠组织黏膜及黏膜下层可见大量炎性细胞浸润、杯状细胞减少且排列紊乱、黏膜缺损及溃疡形成等,DAI评分显著升高(P<0.01),结肠长度显著减少(P<0.01),结肠组织miR-155含量明显升高,Wnt1、β-catenin、Cyclin D1和snail mRNA及蛋白质表达水平均明显升高(均P<0.01),E-cadherin表达量则明显降低(P<0.01);然而,与模型组相比,miR-155抑制剂组结肠组织病变明显改善,DAI评分显著降低(P<0.01),结肠长度显著增加(P<0.01),结肠组织miR-155含量明显降低,Wnt1、β-catenin、Cyclin D1和snail mRNA及蛋白质表达水平均明显降低(均P<0.01),E-cadherin表达量则显著升高(P<0.05)。结论 miR-155可通过激活Wnt/β-catenin信号通路,下调E-cadherin并上调snail表达,介导上皮-间充质转化,加重溃疡性结肠炎。

Objective To detect changes of miR-155,Wnt/β-catenin,E-cadherin and snail in colon tissues of mice and to investigate the mechanism of miR-155 on epithelial-mesenchymal transition mediated by Wnt/β-catenin signaling pathway in ulcerative colitis(UC).Methods Thirty-two C57 mice were randomly divided into 4 groups:normal group,model group,miR-155 antagomir group and negative control group.Except the normal group,each group was given 3% dextran sulfate sodium(DSS)for one week to prepare the UC model.From the fifth day,the miR-155 antagomir group and the negative control group were intraperitoneally injected with miR-155 antagomir and the negative control preparations(80 mg/kg weight)and the equal volume of phosphate buffered saline(PBS)were administered intraperitoneally in the normal group and the model group for three consecutive days.From the first day of modeling,mouse body weight changes,fecal traits,blood stools,etc.were recorded daily.On the eighth day,all the mice were sacrificed,and the disease activity index score(DAI)was measured;the length of colon tissue was measured;hematoxylin-eosin(HE)staining method was used to detect the pathological changes of colon tissue;in situ hybridization was used to detect miR-155 content in colon tissue;Real-time PCR,Western blotting and immunohistochemistry(IHC)were used to detect the expression of miR-155,Wnt1,β-catenin,Cyclin D1,E-cadherin and snail in colon tissues.Results Compared with the normal group,HE in the model group showed infiltrations of a large number of inflammatory cells,goblet cell reduction and arrangement disorder,mucosal defect and ulcer formation in the mucosa and submucosa of the colon tissue,and the DAI score was significantly increased(P<0.01).The length of colon was significantly decreased(P<0.01),the content of miR-155 in colon tissue was significantly increased,and the expression levels of Wnt1,β-catenin,Cyclin D1 and snail mRNA and protein were significantly increased(P<0.01),but E-cadherin expression was significantly lower(P<0.01);however,compared with the model group,HE in the miR-155 antagomir group showed a significant improvement in colonic tissue lesions,a significant decrease in DAI score(P<0.01),and a significant increase in colon length(P<0.01).The content of miR-155 in colon tissue was significantly decreased,and the expression levels of Wnt1,β-catenin,Cyclin D1 and snail mRNA and protein were significantly decreased(P<0.01),and the expression of E-cadherin was significantly increased(P<0.05).Conclusion MiR-155 can down-regulate E-cadherin and up-regulate snail expression,activate epithelial-mesenchymal transition,and induce aggravation of UC by activating Wnt/β-catenin signaling pathway.