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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Dll4 3’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n 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n t检验、配对样本n t检验及单因素方差分析。n 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) (n t＝3.116、2.374，n 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，n t＝4.305，n 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%，n t＝4.232，n P<0.01)，差异有统计学意义，诱导其凋亡(13.7%比48.3%，n t＝8.355，n P<0.01)，两者具有协同作用(n t＝7.474，n 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。n 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。“,”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 Dll4 3′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 lower [0.34 times (0.341±0.008)], and that of Dll4 was significantly higher [1.92 times (1.922±0.003)] in CRC tissues than in normal intestinal mucosa (n t=3.116, n t=2.374, n P<0.05, respectively). After miR-195 was transferred into SW480 cellsn in vitro, the luciferase activity of Dll4 decreased as compared with that in the control group (0.442±0.010, 1.010±0.002, n t=4.305, n P<0.01). miR-195 and dapt could block Notch1 pathway, inhibit the proliferation (3.1% vs. 17.3%,n t=4.232, n P<0.01) and induce apoptosis (13.7% vs. 48.3%,n t=8.355, n P<0.01) of SW480 cells, and they had synergistic effects (n t=7.474, n P<0.01). At the same time, the expression of NICD and Hes-1 decreased with the time.n 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.