靶向热休克蛋白-27基因沉默对5-氟尿嘧啶诱导SW480细胞凋亡的影响及机制  

Effect of HSP27 gene silencing on apoptosis of SW480 cells induced by 5-FU and its mechanism

作　　者：黄崇杰[1] 刘长宝[1] 胡万乐[1] 蔡锚[1] 

机构地区：[1]温州医科大学附属第二医院肛肠外科,浙江温州325027

出　　处：《温州医科大学学报》2016年第12期873-877,共5页Journal of Wenzhou Medical University

基　　金：温州市科技局科研基金资助项目(Y20140107)

摘　　要：目的:通过RNA干扰抑制热休克蛋白-27(HSP27)基因的表达,探讨沉默该基因对5-氟尿嘧啶(5-FU)诱导大肠癌SW480细胞凋亡的影响及机制。方法:将细胞分为正常对照组(Normal组)、阴性si RNA转染组(NC组)、HSP27-siRNA转染组(siRNA组)、单纯5-FU组(5-FU组)、5-FU+阴性siRNA转染组(NC+5-FU组)、5-FU+HSP27-siRNA转染组(si RNA+5-FU组),通过脂质体LipofectamineTM 2000将HSP27-siRNA导入SW480细胞,CCK-8法检测靶向HSP27的siRNA和5-FU对SW480细胞增殖的抑制作用,流式细胞技术观察转染后细胞在5-FU作用下的早期凋亡情况,Western blot方法检测凋亡相关蛋白Active-casepase-3、Active-caspase-9及细胞色素C(Cytc)的表达。结果:CCK-8法检测结果显示siRNA+5-FU组细胞增殖抑制率明显高于同期siRNA组和5-FU组,表明联合应用抑制率明显升高(P<0.05)。流式细胞仪检测结果表明siRNA组和5-FU组均可诱导SW480细胞凋亡,两者联合应用细胞早期凋亡率明显升高,与Normal组及NC组比较,差异均有统计学意义(均P<0.05);Western blot检测结果显示siRNA+5-FU组Active-casepase-3、Active-caspase-9、Cytc蛋白相对表达水平均高于siRNA组和5-FU组,差异均有统计学意义(均P<0.05)。结论:靶向HSP27-siRNA能有效抑制大肠癌SW480细胞HSP27蛋白的表达,上调Active-casepase-3、Active-caspase-9、Cytc蛋白的表达,增强5-FU诱导SW480细胞凋亡,逆转肿瘤细胞对5-FU的耐药。Objective： To explore the effect and mechanism of 5-FU-induced apoptosis of colon cancer SW480 cells with HSP27 silenced by siRNA interference. Methods： Six groups of cells were used in this part of research, including Normal control group （Normal group）, negative siRNA group （NC group）, HSP27 siRNA group （siRNA group）, 5-FU group, 5-FU with negative siRNA group （NC＋5-FU group）, 5-FU with HSP27 siR- NA group （siRNA＋5-FU group）. HSP27-siRNA was transfected into SW480 cells by lipofectamine 2000. CCK-8 method was used to detect the siRNA targeted on HSP27 and 5-FU＇s inhibition effect on SW480 cell proliferation. Early apoptotic rate was measured with flow cytometry technique, and Western blot analysis was applied to de- tect the expression of apoptosis-related protein ofActive-casepase-3, Active-caspase-9 and Cytc. Results： Result detected by CCK-8 showed that the cell proliferation inhibition rate of siRNA＋5-FU group was obviously higher than that of 5-FU group, which indicated that combined use of 5-FU and siRNA could significantly improve the inhibition rate （P〈0.05）. Result detected with flow cytometry （FCM） showed that both siRNA group and 5-FU group could induce SW480 cell apoptosis, and the combined use of 5-FU and siRNA could greatly improve the early apoptotic rate. Comparing the siRNA and 5-FU group with Normal and NC group, the difference had statis- tically significant （P〈0.05）. Western blot analysis showed that the expression level of Active-casepase-3, Active- caspase-9 and Cytc in combination group were higher than that of siRNA group and 5-FU group, the difference had statistically significant （P〈0.05）. Conclusion： HSP27-siRNA can significantly inhibit HSP27 expression in SW480 cells, and improve the expression of protein Active-casepase-3, Active-caspase-9 and Cytc, then enhance the apoptosis-inducing effect of 5-FU on SW480 cells, and reverse the drug resistance of SW480 cells to 5-FU.

关 键 词：结直肠肿瘤 热休克蛋白-27 RNA干扰 5-氟尿嘧啶 细胞凋亡 

分 类 号：R735.34[医药卫生—肿瘤]