论文部分内容阅读
目的 :探讨核仁蛋白PES1(pescadillo homolog 1)对结肠癌细胞恶性表型的影响。方法 :构建针对PES1基因的sh RNA干扰质粒,将其转染结肠癌HCT116细胞,经G418筛选耐药细胞克隆,蛋白质印迹法验证靶基因的沉默效果。采用MTT法、平板克隆形成实验、裸鼠成瘤实验、Transwell迁移及侵袭实验和FCM法分别检测沉默PES1表达对HCT116细胞增殖、克隆形成、体内成瘤、体外迁移和侵袭以及凋亡的影响,并采用实时荧光定量PCR和蛋白质印迹法检测细胞增殖和凋亡相关蛋白的表达水平。结果 :成功获得PES1稳定低表达的结肠癌HCT116细胞。沉默PES1表达后,HCT116细胞的增殖、克隆形成和裸鼠体内成瘤能力均明显降低(P值均<0.01),细胞迁移和侵袭能力明显减弱(P值均<0.01),细胞凋亡水平明显提高(P<0.05)。PES1表达被抑制后,HCT116细胞中细胞周期蛋白D1(cyclin D1)和Bcl-2蛋白表达水平明显降低(P值均<0.01),而Bcl-2相互作用杀伤蛋白(Bcl-2 interacting killer,BIK)、prune同源蛋白2(prune homolog 2,PRUNE2)和基质金属蛋白酶组织抑制因子3(tissue inhibitor of matrix metalloproteinase 3,TIMP3)的m RNA表达水平明显升高(P值均<0.01)。结论 :PES1可能对结肠癌细胞的多种恶性表型起促进作用。
Objective: To investigate the effect of pescadillo homolog 1 on the malignant phenotype of colon cancer cells. Methods: The sh RNA interference plasmid targeting PES1 gene was constructed and transfected into HCT116 colon cancer cells. The drug-resistant cell clone was screened by G418 and the silencing effect of target gene was verified by Western blotting. The effects of silencing PES1 expression on the proliferation, colony formation, in vivo tumorigenesis, in vitro migration, invasion and apoptosis of HCT116 cells were detected by MTT, plate clone formation assay, nude mouse tumorigenicity assay, Transwell migration and invasion assay and FCM method respectively. Real-time fluorescence quantitative PCR and Western blotting were used to detect the expression of cell proliferation and apoptosis related proteins. Results: The stable and low expression of PES1 colon cancer HCT116 cells. The silencing of PES1 expression, HCT116 cell proliferation, colony formation and tumor formation in nude mice were significantly reduced (P values were <0.01), cell migration and invasion were significantly weakened (P value <0.01), the level of apoptosis was significantly Increase (P <0.05). After PES1 expression was inhibited, the expressions of cyclin D1 and Bcl-2 protein in HCT116 cells were significantly decreased (all P <0.01), whereas Bcl-2 interacting killer (BIK ), Prune homolog 2 (PRUNE2) and tissue inhibitor of matrix metalloproteinase 3 (TIMP3) mRNA expression levels were significantly increased (P <0.01). Conclusion: PES1 may promote a variety of malignant phenotypes of colon cancer cells.