沉默信息调节因子1在结肠癌耐药中的作用及其机制研究  

Effects and mechanism of silent information regulator of transcription 1 in the drug-resistance of colonic cancer

作　　者：付强[1] 张永磊[1] 成静[3] 陈小兵[2] 谢建国[1] 罗素霞[2] 

机构地区：[1]河南省肿瘤医院胃肠肿瘤外科,郑州450008 [2]河南省肿瘤医院消化内科,郑州450008 [3]郑州大学附属郑州中心医院肿瘤内科,450002

出　　处：《中华消化外科杂志》2015年第3期221-229,共9页Chinese Journal of Digestive Surgery

摘　　要：目的探讨沉默信息调节因子1（SIRT1）在结肠癌耐药中的作用及其可能机制。方法回顾性分析2012年12月至2013年12月河南省肿瘤医院收治的25例5-氟尿嘧啶（5-Fu）化疗耐药结肠癌患者和30例化疗敏感患者的临床资料。收集患者手术切除的结肠癌标本进行研究。（1）采用免疫组织化学染色检测化疗耐药和化疗敏感结肠癌患者的结肠癌组织中SIR1T蛋白表达。RT—PCR检测结肠癌细胞HCT1l6（以下简称HCT116细胞）和耐药结肠癌细胞HCT116／5-FU（以下简称HCT116／5-FU细胞）中SIRT1 mRNA。Western blot检测2种细胞中SIRT1蛋白表达。（2）细胞分组：①将HCT116／5-FU细胞进行siRNA干扰：空白对照组（细胞不做任何处理）、空载体组（细胞转染对照siRNA）和SIRT1沉默组（细胞转染SIRT1 siRNA）。②将HCT116／5-FU细胞进行JNK蛋白抑制剂处理：SP600125组（浓度为30μmol／L的JNK蛋白抑制剂SP60012处理12h），DMSO组（0．1％DMSO处理12h）以及对照组（加入等量细胞培养液）。③将HCT116／5-FU细胞中SIRT147位点丝氨酸突变成丙氨酸或天冬氨酸，获得突变体分别为S47A组和S47D组，以未转染的野生型细胞作为S47野生型组，并以转染空载体apCMV-3Tag-3细胞为阴性对照，均采用8μmol／L的5-Fu干预12h。（3）分别采用0、1、10、50、100nmol／LSIRT1激动剂白藜芦醇和0、1、2、3、4、5ng／L SIRT1抑制剂尼克酰胺处理HTC116和HTC116／5-FU细胞。MTT法检测细胞增殖率。（4）流式细胞仪检测细胞凋亡。（5）RT—PCR检测相关基因的表达情况。（6）Westernblot检测各组细胞相关蛋白的表达情况。计数资料比较采用，检验，正态分布的计量资料以x±s表示，多组间比较采用单因素方差分析，多组间的两两比较采用LSD—t检验，两两比较采用t检验。结果（1）免疫组织化学染色检测结果显示：化疗敏感和化疗耐药结肠癌患者结肠癌组织中SIRTObjective To investigate the effects of mechanism of silent information regulator of transcrip- tion 1 （ SIRT1 ） in the drug-resistance of colonic cancer. Methods The clinical data of 25 colonic cancer patients with 5-Fu-resistance and 30 colonic cancer patients with chemosensitivity who were admitted to the Henan Tumor Hospital from December 2012 to December 2013 were retrospectively analyzed. The specimens of colonic cancer were collected for study. （ 1 ） The protein expression of SIRT1 in patients with drug-resistance or chemotherapeutic sensitivity was tested by immunohistochemical staining. The protein expression of SIRT1 in the HCT116 and HCT116/5-FU cells was detected by Western blot. （2）HCT116/5-FU cells were interfered by siRNA and divided into the blank control group （cells untreated）, the empty vector group （cells treated by siRNA） and the SIRT1 silence group （cells treated by SIRT1 siRNA）. The protein expression of the HCT116/5-FU cells were inhibited by the c-Jun N-terminal kinase （JNK） and then divided into the SP600125 group [ cells were treated by JNK sig- naling pathway inhibitor SP60012 （concentration： 30 μmol/L）for 12 hours ], the DMSO group [ cells were treated by DMSO （cells were treated by 0.1% DMSO for 12 hours] and the control group （cells were treated by cell culture media）. （3）Serine in the SIRT1 ser47 was mutated to alanine or aspartic acid, and mutations S47A （S47A group, serine to alanine） and S47D （S47D group, serine to aspartic acid） ; Untransfected HCT116/5-FU cells were in the S47 wild type group, and apCMV-3Tag-3 cells transfected by empty vector were served as negative control; all the HCT116/5-FU cells were interfered by 5-FU （ concentration： 8 μmol/L） for 12 hours. HTC116 cells and HTC116/5-FU cells were treated by SIRT1 inhibitor resveratrol at concentrations of 0, 1, 10, 50, 100 nmol/L and SIRT1 activator niacinamide at concentrations of 0, 1, 2, 3, 4, 5 ng/L. Cell proliferation was detected by MTT. （4） Cell apo

关 键 词：结肠肿瘤 沉默信息调节因子1 耐药 P-糖蛋白 

分 类 号：R735.35[医药卫生—肿瘤]