摘要
目的检测MAP4K4对肝癌细胞生物学活性的影响并初步探讨其作用机制。方法选取四种肝癌细胞(Hep3B,HepG2,SMMC7721,Huh-7)采用实时定量PCR和Western blot方法分别检测MAP4K4 mRNA和蛋白水平的表达。设计靶向MAP4K4的特异性siRNA核苷酸序列质粒转染组及空载体质粒转染组,用Lipofectamine 2000法分别转染到MAP4K4蛋白表达活性最强的肝癌细胞,QRT-PCR和Western blot方法检测干扰效率,并对转染后肝癌细胞进行WST-8细胞增殖实验,平板克隆形成实验,PI染色法细胞周期检测,Annexin V法细胞凋亡检测;对干扰组及对照组细胞进行基因芯片检测,以进一步探究MAP4K4基因在肝癌细胞中可能的作用机制。结果 MAP4K4在各细胞株的表达依次为:HepG2>Hep3B>Huh-7>SMMC7721;MAP4K4的干扰效率达50%以上;MAP4K4基因敲减后细胞生长减慢;平板克隆形成能力减弱;细胞周期阻滞于S、G2期;细胞凋亡明显增加;MAP4K4基因敲减后,其上游的基因:TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、MyD88、IRAK、TRAF6、TIRAP的表达明显下调,下游基因TAK1(MAP3K7)和MKK3的表达也明显下调。结论 MAP4K4基因敲减可以阻滞细胞周期的进程,抑制肝癌细胞的恶性增殖,其机制可能是通过NF-κB信号转导途径、JNK信号转导途径发挥作用。
Objective To study the preliminary mechanism of the influence of MAP4K4 on biological activity（cell proliferation,cell clone,cell cycle and cell apoptosis） of hepatoma carcinoma cell（HCC）.Methods The expressions of MAP4K4 protein in HepG2,Hep3B,Huh7 and SMMC-7721 were detected to select the highest expressed cell for RNAi experiment.siRNA targeted MAP4K4（0-HepG2-pGCSil-U6,1-pSilMAP4K4-1,2-pSilMAP4K4-2,3-pSilMAP4K4-3） was transfected into hepatoma carcinoma cell and the interfering efficiency was detected through QRT-PCR and Western blot： Biological activity of HCC was analyzed by WST-8 cell proliferation test,flat plate clone formation test,cell cycle test and apoptosis test.To further explore the possible mechanism of MAP4K4 in HCC,the different expression genes between the highest-transfection group and the control group were researched by TOLL-like receptor signaling associated gene chip.Results The expression level of MAP4K4 in cell lines was： HepG2 Hep3BHuh-7 SMMC7721.The inhibition rate of MAP4K4 siRNA exceeded 50%.After interference,the cell proliferation and cloning efficiencies were decreased obviously compared to the control group.Cell cycle was blocked at S、G2 phase and the percentage of apoptotic was increased.The upstream genes（TLR3,TLR4,TLR5,TLR6,TLR7,TLR8,MyD88,IRAK,TRAF6,TIRAP） and downstream genes（MAP3K7 and MKK3）were down-regulated markedly.Conclusion MAP4K4 gene knockdown could arrest cell cycle and then inhibit proliferation of HCC.The mechanism might be associated with NF-κB and JNK signal pathway.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2012年第2期140-145,共6页
Cancer Research on Prevention and Treatment
基金
江西省自然科学基金资助项目(2009GZY0161)
