微小RNA-195靶向调控Delta样配体4抗结直肠癌分子机制研究

目的观察微小RNA(microRNA,miR)-195调控结直肠癌中Notch通路配体Delta样配体4(Dll4)表达,探讨其作用靶点,明确miR-195通过Notch通路抗结直肠癌的分子机制。方法收集北京大学人民医院胃肠外科2010年11月至2011年2月56例行结直肠癌根治术切除的标本,3、应用芯片技术筛选6例结直肠肿瘤组织和正常肠黏膜组织中micro-RNA的表达差异,用实时荧光定量聚合酶链反应(PCR)检测miR-195在组织中相对表达量,应用固定化蛋白质印迹法(Western blot)检测56例结直肠癌组织及其邻近正常肠黏膜中Notch通路中Dll4蛋白表达;采用双荧光素酶报告基因实验检测miR-195与Dll43’端非编码区(3’UTR)区的相互作用及活性,采用基因过表达miR-195(40 pmol/L)处理结肠癌细胞系SW480,运用流式细胞仪观察其对细胞凋亡的影响,Western blot检测通路相关蛋白Dll4、锯齿状蛋白1(Jagged1)、Notch受体胞内段(NICD)、细胞周期蛋白(Cyclin)D1、转录因子发状分裂相关增强子1(HES1)、B细胞淋巴瘤/白血病-2(bcl-2)、核因子-κB(NF-κB)的表达。组间比较采用方差分析(ANOVA),独立样本t检验比较蛋白表达量差异,统计学方法分别采用独立样本t检验、配对样本t检验及单因素方差分析。结果结直肠癌组织中miR-195表达水平明显低于正常肠黏膜,而Dll4蛋白表达水平高于正常肠黏膜,分别为正常肠黏膜的0.34倍(0.341±0.008)及1.92倍(1.922±0.003)(t=3.116、2.374,P<0.05),差异均有统计学意义,体外转入miR-195后,Dll4荧光素酶活性低于对照组(0.442±0.010、1.010±0.002,t=4.305,P<0.01),差异有统计学意义,miR-195和γ-分泌酶抑制剂(DAPT)都可阻断Notch1通路,抑制SW480细胞的增殖(3.1%比17.3%,t=4.232,P<0.01),差异有统计学意义,诱导其凋亡(13.7%比48.3%,t=8.355,P<0.01),两者具有协同作用(t=7.474,P<0.01),差异有统计学意义。同时Notch通路相关蛋白NICD、Hes-1随作用时间延长而表达下降。结论结直肠癌中miR-195及Dll4呈拮抗性表达,与其病理学特征密切相关,Dll4为miR-195调控的下游靶基因,miR-195通过负性调控Dll4/Notch信号通路抗结直肠癌。

Molecular mechanism of microRNA-195 negative regulation on Delta-like ligand 4/Notch transduction pathway in colorectal cancer

Objective To investigate the effect of microRNA(miRNA,miR)-195 on the expression of Delta-like ligand 4(Dll4),a ligand of Notch pathway,in colorectal cancer(CRC).Methods From November 2010 to February 2011,56 patients with CRC underwent radical resection in our department.MiRNA microarray was used to screen the differential expression of miR-195 in CRC tissues from 6 cases and normal intestinal mucosa tissues.Real-time quantitative polymerase chain reaction(PCR)was used to detect the relative expression of miR-195.Western blotting was used to detect the expression of miR-195.The expression of Dll4 protein in Notch pathway was detected in 56 cases of CRC tissues and adjacent normal intestinal mucosa.The interaction and activity of miR-195 and Dll43′untranslated regions(3′UTR)region were detected by double luciferase reporter gene assay.Colon cancer cell line SW480 was treated with miR-195(40 pmol/L),and its effect on cell apoptosis was observed by flow cytometry.The expression of Dll4,Jagged1,intracellular domain of notch(NICD),Cyclin D1,Hes1,B cell lymphoma/leukemia-2(bcl-2)and nuclear factor-κB(NF-κB)was detected by Western blotting.Results The expression level of miR-195 was significantly lower0.34 times(0.341±0.008)],and that of Dll4 was significantly higher1.92 times(1.922±0.003)]in CRC tissues than in normal intestinal mucosa(t=3.116,t=2.374,P<0.05,respectively).After miR-195 was transferred into SW480 cells in vitro,the luciferase activity of Dll4 decreased as compared with that in the control group(0.442±0.010,1.010±0.002,t=4.305,P<0.01).miR-195 and dapt could block Notch1 pathway,inhibit the proliferation(3.1%vs.17.3%,t=4.232,P<0.01)and induce apoptosis(13.7%vs.48.3%,t=8.355,P<0.01)of SW480 cells,and they had synergistic effects(t=7.474,P<0.01).At the same time,the expression of NICD and Hes-1 decreased with the time.Conclusion The expression of miR-195 and Dll4 is antagonistic in CRC,which is closely related to its pathological characteristics.Dll4 is the downstream target gene regulated by miR-195.miR-195 inhibits CRC by negatively regulating Dll4/Notch signaling pathway.