HCVC蛋白调控肝门部胆管癌细胞中NF-κB活性的研究

The hepatitis C virus core protein promotes NF-κB activity in QBC939 cell line

目的 探讨HCVC蛋白在肝门部胆管癌细胞中对核转录因子κB(NF κB)调控作用。方法 利用稳定表达HCVC蛋白的QBC939细胞株 (QBC93 9HCVC+) ,通过NF κB报道基因分析法、凝胶迁移率分析 (EMSA)检测HCVC蛋白增强肝门部胆管癌细胞NF κB的DNA结合活性和反式激活活性。RT PCR、Westernblot检测HCVC蛋白对核转录因子κB抑制蛋白α(IκBα)mRNA及 p5 0、p6 5、IκBα蛋白表达及IκBα蛋白磷酸化的影响。 结果 ①EMSA结果显示QBC93 9HCVC+细胞系中NF κB相对信号密度明显比QBC939细胞高。②将pNF κB lun表达质粒转染稳定表达HCVC蛋白胆管癌细胞系中 ,通过单光子检测仪检测荧光素酶活性 ,结果显示QBC93 9HCVC+细胞中活化NF κB为 12 .8倍 ,而QBC939细胞中NF κB为 2 .6倍。③RT PCR显示IκBαmRNA在QBC93 9HCVC+细胞和QBC939细胞中的表达无明显差异 ;Westernblot结果显示稳定表达HCVC蛋白的QBC93 9HCVC+细胞株的胞核中的p5 0、p6 5蛋白高水平表达 ,而未转染HCVC蛋白的QBC939细胞仅检测出极低水平的p5 0、p6 5蛋白。在QBC93 9HCVC+细胞胞浆中IκBα蛋白低水平表达 ,QBC939细胞高水平表达 ,但IκBα蛋白磷酸化水平则相反。结论 HCVC蛋白通过增加IкBα降解激活的NF

Objective To elucidate the mechanisms by which HCV C protein activates NF-κB in hilar cholangiocarcinoma cells. Methods QBC 939 HCV C + cells of expressing HCV C protein devived from QBC939 cells were used as a model. The NF-κB trans-activity and NF-κB-DNA binding activity were studies by transient transfection, reporter gene assay and electrophoretic mobility shift assay. IκBα mRNA, p50, p65, IκBα proteins and phosphoration protein expressions were studied by RT-PCR and western blot analysis. Results The NF-κB trans-activity and NF-κB DNA binding activity were greater in QBC 939 HCV C + cell compared with QBC939. High levels of nucleus protein of p50 and p65 were detected in QBC 939 HCV C + cells, the expression levels of IκBα protein in the QBC939HCV C + cells were much lower than that in the QBC939 cell, whereas IκBα mRNA did not change. Conclusion It is possible that NF-κB overexpression accompanied by dysregulation of IκBα may play a role in the hilar cholangiocarcinogenesis of HCV infection。