Objective: To investigate the role and mechanism of ring finger protein 152 (RNF152) in the development of colitis-associated colon cancer (CAC). Methods: CAC was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. Three different stages of mice during the development of colon cancer were obtained, named AD1, AD2 and AD3, respectively. A control group of mice without any treatment was set up as well. The expression of RNF152 in mouse colon tissues was measured by real-time quantitative polymerase chain reaction (RT-qPCR). The effects of RNF152 overexpression on apoptosis and nitric oxide (NO) induced apoptosis was examined by flow cytometry. The expressions of Bcl-2 and Bcl-XL were detected by western blot. Results: CAC was effectively induced by AOM and DSS in C57BL/6 mice. The tumor incidence rate of AD3 group was 100%. The whole genome expression microarray data from mouse AOM-DSS model indicated that the mRNA level of RNF152 was gradually decreased during the development of colon cancer. The RT-qPCR results showed that RNF152 mRNA level in AD3 was 1.23±0.18, higher than 0.52±0.08 in negative control (P<0.01). Flow cytometry analysis showed that overexpression of RNF152 increased the apoptosis of RKO cells (P<0.01). The apoptotic rate of RKO-RNF152 cells treated with NO donor DETA NONOate was (31.2±3.1)%, higher than (14.2±2.1)% in RKO-PCDB cells (P<0.001). Overexpression of RNF152 significantly decreased the protein expressions of Bcl-XL and Bcl-2. Conclusion: Downregulation of RNF152 may facilitate the development of CAC by inhibiting the cell apoptosis.
目的： 探讨环指蛋白152(RNF152)在结肠炎相关结肠癌(CAC)发生发展过程中的作用及其作用机制。 方法： 联合应用氧化偶氮甲烷(AOM)和葡聚糖硫酸钠(DSS)诱导C57BL/6小鼠，建立CAC发生发展4个阶段(分别为正常上皮组织、炎症恢复期、轻度不典型增生、腺癌)的小鼠模型。采用实时荧光定量聚合酶链反应(RT-qPCR)检测不同阶段小鼠结肠组织中RNF152 mRNA的表达。利用人结直肠癌RKO细胞建立稳定高表达RNF152的细胞系。采用流式细胞术检测RNF152高表达对细胞凋亡的影响，Western blot法检测凋亡相关蛋白Bcl-2和Bcl-XL的表达。使用NO自由基供体DETA NONOate处理细胞，比较RNF152高表达对NO诱导细胞凋亡的影响。 结果： AOM和DSS联合使用能有效地模拟人类CAC的病程，AD3组小鼠成瘤率为100%。CAC小鼠模型的结肠全基因组表达谱芯片显示，RNF152 mRNA的表达水平随着结肠癌的发生发展逐步降低。RT-qPCR检测结果显示，腺癌组结肠组织中RNF152 mRNA的表达水平为1.23±0.18，高于对照组结肠组织(0.52±0.08, P<0.01)。RNF152稳定高表达的RKO-RNF152细胞中，sub-G1期细胞比例[(3.6±0.4)%]高于RKO-PCDB细胞[(1.8±0.1)%, P<0.01]，Bcl-XL和Bcl-2的蛋白表达水平明显下调。DETA NONOate处理后，RKO-RNF152细胞的凋亡比例为(31.2±3.1)%，高于RKO-PCDB细胞[(14.2±2.1)%, P<0.001]。 结论： RNF152的表达降低与CAC的发生发展有关，可能通过抑制细胞凋亡促进了CAC的发生发展。.
Keywords: Apoptosis; Colonic neoplasm; NO; RNF152.