肝癌患者核因子-κB异常表达及转录干预对多药耐药的逆转效果

Abnormal expression of circulating nuclear factor-κB in hepatocellular carcinoma and reversal of multidrug resistance through intervening its gene transcription

目的分析肝病患者核因子-κB（NF-κB）及P-糖蛋白（P-gp）表达并探讨干扰NF-κB基因转录对肝癌多药耐药（MDR）逆转效果与机制。方法以酶联免疫吸附法定量检测294例肝病患者血NF-κB和P-gp浓度；培养肝癌HepG2细胞，加入阿霉素、以特异性小干扰RNA（siRNA）下调NF-κB基因转录，以Western印迹检测蛋白表达，以荧光定量聚合酶链反应分析基因转录水平。结果从慢性肝炎、肝硬化到肝癌的进展过程中NF-κB[（0．55±0．27）、（0．76±0．74）、（2．76±3．42）μg/L]和P-gP[（6．80±3．92）、（7．22±4．95）、（12．44±9．63）μg/L]呈渐进性高表达。肝癌组NF-κB和P-gP水平显著高于肝硬化、慢性肝炎和对照组（P〈0．001）。肝癌患者伴HBV感染与NF-κB（t=2．14，P〈0．05）和P-gP（t=5．50，P〈0．001）表达明显相关；介入化疗用药次数或时间延长均显著升高[46例肝癌3次及以上介入化疗后NF-κB达（5．75±5．60）μg/L]；肝癌术后均显著下降（P〈0．001）。肝癌HepG2细胞加阿霉素后培养，NF-κB和P-gP表达增强；siRNA干预HepG2细胞或阿霉素处理后的耐药HepG：细胞，NF-κB和P-gP表达均明显减低，使HepG2细胞对阿霉素更敏感。结论NF-κB和P-gP参与肝癌MDR形成，干预NF-κB活化可抑制P-gp表达，有助于逆转肝癌MDR。

Obiective To investigate the expressions of nuclear factor-κB （NF-κB） and P- glycoprotein （P-gp） in patients with liver diseases and the effects and mechanism of intervening NF-κB activation on multiple drug resistance （MDR） reversal. Methods The levels of circulating NF-κB and P-gp expression were quantitatively detected in 294 patients with liver diseases by using enzyme-linked immunosorbent assay. NF-κB gene transcription in human HepG2 cells was intervened by specific siRNA with/without doxorubicin treatment. Levels of protein or gene expression were analyzed by Western blotting or fluorescence quantitative polymerase chain reaction （PCR）. Results The dynamic increase of circulating NF-κB and P-gp expressions were observed during patients from chronic hepatitis to cirrhosis to hepatocellu｜ar carcinoma （HCC） development. The levels of serum NF-κB and P-gp expression in HCC were significantly higher （P 〈 0. 001 ） than those in cirrhosis, chronic hepatitis, and normal controls. The expression levels of serum NF-κB and P-gp were significantly associated with HBV infection （ P 〈 0. 05 ）, elevated after interventional chemotherapy with higher frequency or prolonged therapy, and significantly decreased in the post-operative HCC （P 〈 0. 001 ）. Both expressions in HepG2 cells were significantly higher after adding doxorubicin in the cell cultures. After the cells were transfected with siRNA, the NF-κB expression was down-regulated at mRNA or protein level with higher sensitivity to doxorubicin. Conclusion The over-expressions of NF-κB and P-gp are closely associated with MDR formation; intervention of NF-κB activation can inhibit P-gp expression, and enhance the sensitivity to anti-cancer drug and reverse MDR of HCC.