Yu Jing, Chen Kui-Sheng, Li Ya-Nan, Yang Juan, Zhao Lu
Department of Pathology, First Affiliated Hospital of Zhengzhou University and Henan Key Laboratory for Tumor Pathology, Zhengzhou, China.
Asian Pac J Cancer Prev. 2012;13(8):4147-51. doi: 10.7314/apjcp.2012.13.8.4147.
The current study was conducted to explore the inhibitory effects of a small interfering RNA (siRNA) on 3-phosphoinositide-dependent protein kinase 1 (PDK1) expression in esophageal cancer 9706 (EC9706) cells and the influence on their biological behavior. After transfection of a synthesized PDK1 siRNA, PDK1 mRNA and protein expression and the phosphorylation level of the downstream Akt protein were assessed using RT-PCR and Western blot analysis. Proliferation, apoptosis, cell invasion and in vivo tumor formation capacity were also investigated using MTT, flow cytometry, Transwell invasion trials, and nude mouse tumor transplantation, respectively. PDK1 siRNA effectively suppressed PDK1 mRNA and protein expression, and down-regulated the phosphorylation level of the Akt protein in the EC9706 cells (P<0.05). It also inhibited cell proliferation and invasion, and promoted apoptosis; such effects were particularly obvious at 48 h and 72 h after transfection (P<0.05). Growth of transplanted tumors was inhibited in nude mice, with decreased PDK1 expression in tumor tissues. PDK1 may be closely correlated with proliferation, apoptosis and invasion of esophageal cancer cells and thus may serve as an effective target for gene therapy.
本研究旨在探讨小干扰RNA(siRNA)对食管癌9706(EC9706)细胞中3-磷酸肌醇依赖性蛋白激酶1(PDK1)表达的抑制作用及其对细胞生物学行为的影响。转染合成的PDK1 siRNA后,采用逆转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹分析评估PDK1 mRNA和蛋白表达以及下游Akt蛋白的磷酸化水平。分别采用MTT法、流式细胞术、Transwell侵袭实验和裸鼠肿瘤移植实验研究细胞增殖、凋亡、侵袭及体内成瘤能力。PDK1 siRNA能有效抑制EC9706细胞中PDK1 mRNA和蛋白表达,并下调Akt蛋白的磷酸化水平(P<0.05)。同时抑制细胞增殖和侵袭,促进细胞凋亡;转染后48 h和72 h这些作用尤为明显(P<0.05)。裸鼠移植瘤生长受到抑制,肿瘤组织中PDK1表达降低。PDK1可能与食管癌细胞的增殖、凋亡和侵袭密切相关,有望成为基因治疗的有效靶点。