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

付强; 张永磊; 成静; 陈小兵; 谢建国; 罗素霞

doi:10.3760/cma.j.issn.1673-9752.2015.03.011

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 drugresistance of colonic cancer

摘要

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(5FU)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。RTPCR检测结肠癌细胞HCT116(以下简称HCT116细胞)和耐药结肠癌细胞HCT116/5FU(以下简称HCT116/5FU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HCT116/5FU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HCT116/5FU细胞进行JNK蛋白抑制剂处理：SP600125组(浓度为30 μmol/L的JNK蛋白抑制剂SP60012处理12 h)，DMSO组(0.1%DMSO处理12 h)以及对照组(加入等量细胞培养液)。③将HCT116/5FU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8 μmol/L的5FU干预12 h。(3)分别采用0、1、10、50、100 nmol/L SIRT1激动剂白藜芦醇和0、1、2、3、4、5 ng/L SIRT1抑制剂尼克酰胺处理HTC116和HTC116/5FU细胞。MTT法检测细胞增殖率。(4)流式细胞仪检测细胞凋亡。(5)RTPCR检测相关基因的表达情况。(6)Western blot检测各组细胞相关蛋白的表达情况。计数资料比较采用χ2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LSDt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SIRT1阳性表达率分别为16.7%(5/30)和92.0%(23/25)，两组比较，差异有统计学意义(χ²=30.965，P<0.05)。HCT116/5FU细胞中SIRT1 mRNA和蛋白相对表达水平分别为1.870±0.100和1.660±0.109，均显著高于HCT116细胞的1.000±0.070和1.000±0.050，两者比较，差异均有统计学意义(t=11.721，8.963，P<0.05)。(2)MTT检测细胞增殖率结果：HCT116/5FU细胞在0、1、10、50 nmol/L白藜芦醇处理下的细胞增殖率分别为100%±12%、105%±14%、129%±10%、144%±17%，均高于对应浓度处理的HCT116细胞增殖率41%±10%、49%±11%、74%±16%、105%±17%，两者比较，差异均有统计学意义(t=8.226，-7.236，6.673，3.510，P<0.05)。HCT116/5FU细胞在0、1、2 ng/L尼克酰胺处理下的细胞增殖率分别为87%±12%、78%±12%、69%±11%，均高于对应浓度处理的HCT116细胞增殖率36%±6%、32%±5%、30%±6%，两者比较，差异均有统计学意义(t=-8.593，-8.006，-7.000，P<0.05)。空白对照组、空载体组和SIRT1沉默组HCT116/5FU细胞增殖率分别为100%±8%、99%±9%、37%±6%，3组比较，差异有统计学意义(F=66.597，P<0.05)，SIRT1沉默组细胞增殖率低于空白对照组，两组比较，差异有统计学意义(t=10.113，P<0.05)。(3)流式细胞仪检测细胞凋亡结果：SIRT1沉默组、空载体组、空白对照组HCT116/5FU细胞的细胞凋亡率分别为60%±5%、36%±4%、35%±4%，3组比较，差异有统计学意义(F=36.549，P<0.05)；SIRT1沉默组的细胞凋亡率显著高于空白对照组和空载体组，两组比较，差异有统计学意义(t=-7.215，-7.084，P<0.05)。(4)RTPCR检测结果显示：SIRT1沉默组、空载体组和空白对照组HCT116/5FU细胞中Pgp mRNA的相对表达量分别为0.320±0.030、0.990±0.060、1.000±0.090，3组比较，差异有统计学意义(F=10.107，P<0.05)。SIRT1沉默组Pgp mRNA的相对表达量显著低于空白对照组，两组比较，差异有统计学意义(t=11.463，P<0.05)。SP600125组、DMSO组以及对照组HCT116/5FU细胞中Pgp mRNA的相对表达量分别为0.240±0.040、0.990±0.100、1.000±0.070，3组比较，差异有统计学意义(F=19.002，P<0.05)；SP600125组Pgp mRNA的相对表达量显著低于对照组，两组比较，差异有统计学意义(t=7.301，P<0.05)。(5)Western blot检测结果显示：空白对照组、空载体组、SIRT1沉默组HCT116/5FU细胞中pJNK蛋白的相对表达量分别为1.000±0.090、1.090±0.020、0.080±0.010，3组比较，差异有统计学意义(F=12.130，P<0.05)。5FU干预HCT116细胞中各磷酸化位点蛋白水平与SIRT1总磷酸化蛋白水平比值pSIRT1S27/TSIRT1、pSIRT1T530/TSIRT1、pSIRT1S47/TSIRT1分别为1.158±0.140、1.209±0.150、3.760±0.150；DMSO干预HCT116细胞分别为1.120±0.109、1.130±0.100、2.160±0.110，2种处理下细胞3个位点比较，差异均有统计学意义(F=9.763，10.261，P<0.05)。HCT116细胞5FU和DMSO干预pSIRT1S47/TSIRT1均高于HCT116/5FU细胞对应的0.940±0.040和1.121±0.110，两者比较，差异有统计学意义(t=14.721，21.335，P<0.05)。(6)S47野生型组、阴性对照组以及突变体S47A组和S47D组细胞增殖率分别为41%±31%、39%±4%、64%±2%和26%±5%，4组比较，差异有统计学意义(F=6.371，P<0.05)。
结论:SIRT1促进结肠癌耐药细胞的增殖，并通过JNK信号通路上调Pgp的表达，从而增强结肠癌耐药性。SIRT1 S47位点为HCT116/5FU细胞耐药的关键性位点。

Abstract: Objective:To investigate the effects of mechanism of silent information regulator of transcription 1 (SIRT1) in the drugresistance of colonic cancer.
Methods:The clinical data of 25 colonic cancer patients with 5Furesistance 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 SIRT1in patients with drugresistance or chemotherapeutic sensitivity was tested by immunohistochemical staining. The protein expression of SIRT1 in the HCT116 and HCT116/5FU cells was detected by Western blot. (2)HCT116/5FU 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 HCT116/5FU cells were inhibited by the cJun Nterminal kinase (JNK) and then divided into the SP600125 group ［cells were treated by JNK signaling 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 HCT116/5FU cells were in the S47 wild type group, and apCMV-3Tag-3 cells transfected by empty vector were served as negative control; all the HCT116/5FU cells were interfered by 5FU (concentration: 8 μmol/L) for 12 hours. HTC116 cells and HTC116/5FU 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 apoptosis was detected by flow cytometry. (5)The expressions of related genes were detected by realtime PCR . (6)The expressions of related proteins were detected by western blot. The count data were analyzed using the chisquare test. The measurement data with normal distribution were presented as X±s. The comparison among groups were analyzed using the oneway analysis of variance and LSDt test. The pairwise comparisons were analyzed using the t text.
Results:(1)The results of immunohistochemical staining were as follows. The positive expressions of SIRT1 in patients with chemotherapeutic sensitivity and drugresistance were 16.7%(5/30) and 92.0%(23/25), respectively, with significant difference (χ²=30.965, P<0.05). The relative mRNA and protein expressions of SIRT1 in HCT116/5FU cells with drugresistance were 1.870±0.100 and 1.660±0.109, which were significantly higher than 1.000±0.070 and 1.000±0.050 in HCT116/5FU cells without drugresistance (t=11.721, 8.963, P<0.05). (2)The results of MTT were as follows. The proliferation rates of HCT116/5FU cells treated by resveratrol at concentrations of 0, 1, 10, 50 nmol/L were 100%±12%,105%±14%,129%±10% and 144%±17%, which were significantly higher than 41%±10%, 49%±11%, 74%±16% and 105%±17% of HCT116 cells which were treated by reseratrol at the same contrations (t=8.226,-7.236, 6.673, 3.510, P<0.05). The proliferation rates of HCT116/5FU cell treated by niacinamide at concentrations of 0, 1, 2 ng/L were 87%±12%, 78%±12%, 69%±11%, which were significantly higher than 36%±6%, 32%±5%, 30%±6% of HCT116 cells which were treated by niacinamide at the same concentrations (t=-8.593,-8.006,-7.000, P<0.05). The proliferation rates of HCT116/5FU cells in the blank control group, the empty vector group and the SIRT1 silence group were 100%±8%, 99%±9%, 37%±6%, with significant differences among the 3 groups (F=66.597, P<0.05), and the proliferation rate of HCT116/5FU cells in the SIRT1 silence group was significantly lower than that in the blank control group (t=10.113, P<0.05).(3) The results of flow cytometry were as follows. The apoptotic rates of HCT116/5FU cells in the SIRT1 silence group, the empty vector group and the blank control group were 60%±5%, 36%±4%, 35%±4%, with significant differences among the 3 groups (F=36.549, P<0.05), and the apoptotic rates of HCT116/5FU cells in the SIRT1 silence group were significantly higher than that in the blank control group and the empty vector group (t=-7.215,-7.084, P<0.05). (4)The results of RTPCR were as follows. The relative expression rates of Pgp mRNA in the HCT116/5FU cells in the SIRT1 silence group, the empty vector group and the blank control group were 0.320±0.030, 0.990±0.060, 1.000±0.090, with significant differences among the 3 groups (F=10.107, P<0.05), and the relative expression rate of Pgp mRNA in the SIRT1 silence group was significantly lower than that in the blank control group (t=11.463, P<0.05). The relative expression rates of Pgp mRNA in the HCT116/5FU cells in the SP600125 group, the DMSO group and the control group were 0.240±0.040, 0.990±0.100, 1.000±0.070, with significant difference among the 3 groups (F=19.002, P<0.05), and the relative expression rates of Pgp mRNA in the SP600125 group was significantly lower than that in the control group (t=7.301, P<0.05). (5) The results of western blot were as follows. The relative expression rates of pJNK protein in the HCT116/5FU cells in the blank control group, the empty vector group and the SIRT1 silence group were 1.000±0.090, 1.090±0.020, 0.080±0.010, with significant difference among the 3 groups (F=12.130, P<0.05). The ratios of pSIRT1S27/TSIRT1, pSIRT1T530/TSIRT1, pSIRT1S47/TSIRT1 were 1.158±0.140, 1.209±0.150, 3.760±0.150 in HCT116 cells treated by 5FU, and 1.120±0.109, 1.130±0.100, 2.160±0.110 in HCT116 cells treated by DMSO, with significant differences (F=9.763, 10.261, P<0.05). The ratios of pSIRT1S47/TSIRT1 in HCT116 cells treated by 5FU and DMSO were 3.760±0.150 and 2.160±0.110, which were significantly higher than 0.940±0.040 and 1.121±0.110 in HCT116/5FU cells (t=14.721, 21.335, P<0.05).(6) The proliferation rates of HCT116/5FU cells in the S47 wild type group, the negative control group, the S47A group and the S47D group were 41%±31%, 39%±4%, 64%±2% and 26%±5%, with significant differences among the 4 groups (F=6.371, P<0.05).
Conclusions:SIRT1 promotes the proliferation of drugresistant colonic cancer cells and increases the expression of Pgp via JNK signaling pathway, there by enhances cellular drug resistance. SIRT1 S47 is the critical site for 5FUresistance in HCT116/5FU cells.

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