Wang Jianqiang, Lao Lijun, Zhao Hui, Huang Yuan
Department of Gastroenterology, Second Hospital of Nanchang University Nanchang 330006, China.
Department of General Surgery, First Affiliated Hospital, Liaoning Medical University Jinzhou 121001, China.
Int J Clin Exp Med. 2014 Feb 15;7(2):348-55. eCollection 2014.
This study aimed to investigate the effects of serine threonine kinase Pim-3 on the growth of HepG2 cells and to explore the role of STAT3 signaling pathway.
Synthetic Pim-3shRNA and negative control shRNA were independently transfected into HepG2 cells in the presence of Lipofectamine(TM) 2000. Cells were divided into 4 groups: Pim-3 shRNA group, negative control group, liposome control group, and blank control group. Flow cytometry was performed to detect the apoptosis of these cells; RT-PCR was employed to detect the mRNA expression of Pim-3; Western blot assay was done to measure the protein expression of Pim-3, STAT3, pSTAT3(Tyr705), Bcl-Xl, Bad and pBad(Ser112).
When compared with blank control group, liposome group and negative control group, the apoptosis index increased and the protein expression of Pim-3, pSTAT3(Tyr705), Bcl-Xl and pBad(Ser112) and the Pim-3 mRNA expression reduced in the Pim-3 shRNA group, but the protein expression of STAT3 and Bad was comparable among groups.
Pim-3 shRNA may down-regulate pSTAT3(Tyr705) and pBad(Ser112) protein expression to inhibit the proliferation of liver cancer cells and Pim-3 may serve as a target for the treatment of liver cancer.
本研究旨在探讨丝氨酸苏氨酸激酶Pim-3对肝癌细胞HepG2生长的影响,并探究信号转导和转录激活因子3(STAT3)信号通路的作用。
在Lipofectamine™ 2000存在的情况下,将合成的Pim-3短发夹RNA(shRNA)和阴性对照shRNA分别转染至HepG2细胞中。细胞分为4组:Pim-3 shRNA组、阴性对照组、脂质体对照组和空白对照组。采用流式细胞术检测细胞凋亡情况;运用逆转录-聚合酶链反应(RT-PCR)检测Pim-3的信使核糖核酸(mRNA)表达;通过蛋白质免疫印迹法检测Pim-3、STAT3、磷酸化STAT3(Tyr705)、Bcl-Xl、Bad和磷酸化Bad(Ser112)的蛋白表达。
与空白对照组、脂质体组和阴性对照组相比,Pim-3 shRNA组细胞凋亡指数升高,Pim-3、磷酸化STAT3(Tyr705)、Bcl-Xl和磷酸化Bad(Ser112)的蛋白表达降低,Pim-3的mRNA表达减少,但各组间STAT3和Bad的蛋白表达相当。
Pim-3 shRNA可能通过下调磷酸化STAT3(Tyr705)和磷酸化Bad(Ser112)蛋白表达来抑制肝癌细胞增殖,Pim-3可能成为肝癌治疗的靶点。