沉默PES1基因表达对结肠癌细胞恶性表型的抑制作用

Inhibitory effects of silencing PES1 gene expression on the malignant phenotypes of colon cancer cells

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 effects of nucleolar protein PES1 （pescadillo homolog 1） on the malignant phenotypes of colon cancer cells. Methods： The plasmid combined with specific shRNA targeting PES1 gene was constructed and transfected into colon cancer HCT1 16 cells. The silencing of PES1 gene expression was confirmed by Western blotting. After PES1 gene was knocked down, MTT assay,colony formation assay, tumor xenograft in mice assay, Transwel1 migration and invasion assays as well as flow cytometry were performed to detect the proliferation, colony formation, xenograft formation in vivo, migration, invasion and apoptosis of HCT116 cells, respectively. Meanwhile, the expression levels of proliferation- and apoptosis-related fators were detected by real-time fluorescent quantitative-PCR and Western blotting. Results： The colon cancer HCT11 6 cells with stable low expression of PES1 were successfully established. The deficiency of PES1 gene expression resulted in decreased proliferation and colony formation in vitro （both P 〈 0.01）, lowered xenograft growth in nude mice （P 〈 0.01）, diminished migration and invasion abilities （both P 〈 0.01）, and increased apoptosis （P 〈 0.05）. Furthermore, the protein levels of cyclin D1 and Bcl-2 were decreased （both P 〈 0.01）, but the mRNA expression levels of Bcl-2 interacting killer （BIK）, prune homolog 2 （PRUNE2） and tissue inhibitor of matrix metalloproteinase 3 （TIMP3） were increased （all P 〈 0.01） in HCT116 cells with low expression of PES1. Conclusion： PES1 may contribute to promoting the malignant phenotypes of colon cancer cells