舒林酸对人结肠癌细胞miRNA-17和miRNA-21表达抑制作用机制探讨  

Effect of Sulindac on miRNA-17 and miRNA-21 expressions in human colon cancer cells

作　　者：刘军[1,2] 牟艳玲[1,2] 靳晶[3] 李军[1,2] 崔淑香[1,2] 

机构地区：[1]山东省医学科学院药物研究所.山东省罕少见病重点实验室,山东济南250062 [2]济南大学.山东省医学科学院医学与生命科学学院,山东济南250000 [3]山东大学药学院,山东济南250012

出　　处：《中华肿瘤防治杂志》2014年第7期485-489,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81072665);山东省自然科学基金(Y2008C137)

摘　　要：目的:探讨非甾体抗炎药舒林酸对miRNA-17(miR-17)和miRNA-21(miR-21)表达的抑制作用及其机制。方法:选取Balb/c(nu/nu)裸小鼠24只,建立人HT-29裸小鼠异种移植瘤模型,实验分为对照组、舒林酸高剂量组(50mg/kg)、舒林酸低剂量组(25mg/kg)和阿司匹林组(300mg/kg),每组6只,分组给药后取肿瘤组织。观察舒林酸组抑瘤作用并与阿司匹林组比较,蛋白质印迹法及免疫组织化学法分别检测肿瘤组织中NF-κB及相关蛋白表达水平,RT-PCR法检测miR-17和miR-21表达水平,染色质免疫共沉淀法检测NF-κB与miR-17和miR-21的启动子序列结合能力。结果:舒林酸50、25mg/kg组和阿司匹林组对肿瘤生长抑制率分别为56.29%、46.11%和20.36%,舒林酸组与阿司匹林组比较,差异有统计学意义,P<0.05。蛋白质印迹法检测结果显示,对照组、舒林酸25、50mg/kg和阿司匹林组p-NF-κB p65的表达水平分别为(108.08±8.1)%、(56.84±5.4)%、(31.25±3.1)%和(23.26±2.4)%,各组间比较差异有统计学意义,F=160.141,P<0.001。p-IκBα的表达水平各组间比较差异有统计学意义,F=42.620,P<0.001;IκBα的表达水平各组间比较差异有统计学意义,F=35.486,P<0.001。免疫组化结果显示,对照组NF-κB p65在细胞质和细胞核中都有明显表达,舒林酸组NF-κB p65的表达主要集中在细胞质,即舒林酸能抑制NF-κB p65由细胞质转入细胞核。舒林酸50、25mg/kg和阿司匹林组对miR-17的表达水平分别降低了74.80%、67.10%和19.90%,差异均有统计学意义,F=1 178.70,P<0.001;对miR-21的表达水平分别降低了74.90%、73.70%和39.40%,差异均有统计学意义,F=812.48,P<0.001。染色质免疫沉淀法检测结果显示,NF-κB p65能够识别miR-17及miR-21的启动子序列,并与之结合,调控靶基因的转录。结论:舒林酸降低IκBα的磷酸化,减少IκBα的降解,抑制NF-κB p65入核;减少核内NF-κB p65的磷酸化水平,从而抑制NF-κB活性。通过抑制NF-κB活性,舒林酸抑制NF-κOBJECTIVE： To research the mechanism of the nonsteroidal anti-inflammatory drug Sulindac on sup-pressing the expressions of miRNA-17 （miR-17） and miRNA-21 （miR-21） which would provide theory basis for clinic treatment of malignant tumor. METHODS： HT-29 xenograft models were established,and tumors were excised after ad-ministration to observe the inhibitory effect of sulindac and compared it with aspirin. NF-κB and related protein expression levels in tumor tissue were measured by Western blotting and IHC. The expressions of miR-17 and miR-21 were detected by RT-PCR. Chromatin immunoprecipitation was used to observe the combining capacity between NF-κB p65 and the pro-moters of miR-17, miR-21. RESULTS： The growth inhibition ratio of Sulindae on HT-29 xenografts at the dose of 50 mg/kg and 25 mg/kg were 56.29%（P〈0.05）,46. 11%（P〈0.05）vs aspirin（20.36%）,respectively. Compared with the control （108.08 ± 8.1 ）%, the activated form of phosphor-NF-κB was markedly reduced in the nucleus from the treat-ment groups（F 160. 141, P〈0. 001）. In addition,Sulindac prevented the translocation of NF-κB p65 to the nucleus by decreasing the phosphorylation of IκBα significantly versus the accumulation of IκBα（F= 42. 620, P〈0. 001;F=35. 486, P〈0. 001 ）. The level of miR-17 in Sulindac-treated HT-29 xenografts at the dose of 50 mg/kg,25 mg/kg and Aspirin treated at 300 mg/kg were significantly decreased by 74.80%,67.10% and 19.90%（F=1 178.70,P（0.001）,respec-tively;while the level of miR-21 in Sulindac-treated HT-29 xenografts at the dose of 50 mg/kg, 25 mg/kg and Aspirin-treated at 300 mg/kg were significantly decreased by 74.90%,73. 70% and 39.40%（F=812.48,P〈0. 001） vs control. Also NF-κB was found to bind the promoters of miR 17 and miR-21 to suppress their expression at the transcriptional lev-el. CONCLUSIONS： This study demonstrate that Sulindac can decrease phosphorylated IκBα and accumulate unphosphory-lated IκBα which retains NF-κB in the cytoplasm. Als

关 键 词：结肠肿瘤 舒林酸 NF—κB 微RNAS 

分 类 号：R735.35[医药卫生—肿瘤]