摘要
目的:探讨过表达Twist1对结直肠癌SW620细胞多药耐药性(MDR)的影响及其可能的机制。方法:利用过表达Twist1的慢病毒和阴性对照病毒转染结直肠癌SW620细胞,分别为实验组(SW620-Twist1)和对照组(SW620-NC),采用RT-qPCR和Western blot技术检测两组细胞Twist1基因及耐药基因ABCB1、ABCG2 mRNA和蛋白表达; CCK8法测定5-氟尿嘧啶(5-FU)和奥沙利铂(OXA)对两组细胞在24、48及72 h生长抑制率的影响;流式细胞仪检测两组细胞的肿瘤干细胞标记物CD133、CD44表达差异。结果:SW620-Twist1组与SW620-NC组相比,Twsit1基因mRNA和蛋白显著升高,差异具有统计学意义(P<0. 01),提示过表达Twist1基因的SW620细胞系构建成功; 5-FU和OXA两种药物在48、72 h时对细胞的抑制率均明显降低(48 h,P<0. 05; 72 h,P<0. 01); ABCB1、ABCG2基因mRNA及蛋白的表达量均明显升高(P<0. 01); CD133及CD44表达量均显著增高(P<0. 01)。结论:过表达Twist1可促进直肠癌SW620细胞耐药指标ABCB1、ABCG2表达,使肿瘤细胞获得MDR,该现象可能与促进干性获得有关。
Objective: To investigate the effect of overexpression of Twist1 on multidrug resistance( MDR) in colorectal cancer SW620 cells and its possible mechanism. Methods: The colorectal cancer SW620 cells were transfected with Twist1 overexpressing lentivirus and negative control virus,which were called experimental group( SW620-Twist1) and control group( SW620-NC) respectively. The protein and mRNA expression of Twist1、ABCB1 and ABCG2 in two groups were detected by Western blot and RT-qPCR,respectively. The growth inhibitory rate of 5-Fluorouracil( 5-FU) and Oxaliplatin( OXA) at 24,48 and 72 h in two groups were determined by CCK8 assay. Cancer stem cell markers CD133,CD44 expression levels were detected by Flow cytometry. Results: The experimental group was compared with the control group,the expressions of Twsit1 gene mRNA and protein increased significantly( P<0. 01),Twist1 gene overexpressing SW620 cell line was successfully constructed. The inhibition rates of cell proliferation of the three chemotherapeutic drugs at 48 h and 72 h were significantly decreased( 48 h,P < 0. 05; 72 h,P < 0. 01). The mRNA and protein expression levels of ABCB1,ABCG2 were significantly increased( P<0. 01). The expression of CSCs markers CD133 and CD44 increased significantly( P<0. 01). Conclusion: Overexpression of Twist1 can promote the expression of ABCB1 and ABCG2 genes in SW620 cells and make the tumor cells acquire MDR. This phenomenon may be related to the promotion of stemness.
引文
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