蛋白激酶B活化诱导骨桥蛋白表达与肝癌细胞骨转移间的关系

Correlation of protein kinase B-induced osteopontin expression with bone metastasis of hepatocellular carcinoma

目的:研究蛋白激酶B(AKT)活化诱导骨桥蛋白(OPN)表达在肝细胞肝癌(HCC)骨转移中的作用及其机制。方法:收集40例HCC骨转移灶及其原发灶标本,并另选取22例无转移的HCC组织标本作为对照,行免疫组化染色检测AKT、OPN表达水平;同时用miR-21表达载体转染人HCC细胞系BEL-7402,诱导AKT磷酸化激活,同时用AKT特异性抑制剂(MK-2206)抑制AKT磷酸化激活,分别观察AKT磷酸化水平(p-AKT)与OPN表达间的关系;采用OPN特异性小干扰RNA(OPN-siRNA)沉默BEL-7402细胞OPN表达,并用MTT法检测其对细胞增殖的影响。结果:在发生HCC骨转移灶中,p-AKT和OPN阳性率分别为85.00%(34/40)和77.50%(31/40),明显高于其相应的HCC原发灶组织(p-AKT为60.00%;OPN为55.00%,P<0.05)及无转移的HCC组织(p-AKT为54.55%;OPN为36.36%,P<0.01);BEL-7402细胞转染miR-21诱导p-AKT上调后,OPN表达升高并促进肿瘤细胞增殖加速;反之,MK-2206抑制AKT磷酸化后,BEL-7402细胞OPN表达下降,肿瘤细胞增殖速率降低;采用特异siRNA沉默OPN表达,同样可抑制肿瘤细胞增殖。结论:HCC细胞OPN高表达与AKT信号通路的激活有关,AKT磷酸化水平的升高能诱导OPN表达,进而促进HCC细胞的增殖和转移,提示检测p-AKT与OPN的表达水平对于HCC恶性生物学行为的判断具有潜在的应用价值。

Objective： To investigate the effect and mechanism of protein kinase B （AKT）-induced osteopontin （OPN） expression on bone metastasis of hepatocellular carcinoma （HCC）. Methods： Tumor specimens including the prima- ry lesions and matched metastatic lesions from 40 cases of HCC with bone metastasis, and tumor specimens from 22 cases of HCC without metastasis were investigated. The expression levels of AKT and OPN were detected by immunohistochemi- cal staining. In addition, HCC cell line BEL-7402 was transfected with miR-21 to induce the phosphorylation of AKT, or treated with the AKT inhibitor MK-2206 to reduce the phosphorylation of AKT, and then the relationship between AKT phosphorylation and OPN expression was investigated. Small interfering RNA of OPN （OPN-siRNA） was used to silence OPN expression, and the effect of OPN expression on HCC cell proliferation was evaluated. Results： The positive expres- sion rates of phosphorylated AKT （p-AKT） and OPN in bone metastatic lesions were significantly higher than that in HCC primary lesions of cases with bone metastasis （p-AKT： 85.00% vs 60.00%; OPN： 77.50% vs 55.00%; P〈0.05） and HCC specimens without metastasis （p-AKT： 85.00% vs 54.55%; OPN： 77.50% vs 36.36%; P〈0.01）. After up-regulation of p- AKT induced by tranfection of miR-21, the expression level of OPN was increased in BEL-7402 cells, thereafter the pro- liferation of BEL-7402 ceils was promoted. In contrast, the phosphorylation of AKT was down-regulated by MK-2206, the expression of OPN was decreased and cell growth of BEL-7402 cells was inhibited. Moreover, the silence of OPN expres- sion by siRNA also resulted in inhibition of cancer cell proliferation. Conclusions： The high expression of OPN in HCC isclosely correlated with the activation of AKT signaling pathway, and detecting the expression levels of p-AKT and OPN may have potential value for estimating the malignant biological behavior of HCC.