Raf激酶抑制蛋白与肝细胞癌侵袭转移的关系

Expression of Raf kinase inhibitor protein and its significance in invasion and metastasis of hepatocellular carcinoma

目的 探讨Raf激酶抑制蛋白（RKIP）与肝细胞癌（HCC）侵袭转移的关系.方法 采用逆转录聚合酶链反应（RT-PCR）方法检测HCC、癌旁组织及正常肝组织中RKIP mRNA的表达,采用免疫组织化学和Western blot法检测不同肝组织中RKIP、p65和磷酸化细胞外调节蛋白激酶（pERK）蛋白的表达,分析RKIP表达与HCC临床病理学特征的关系及其与p65和pERK表达的相关性.结果 HCC、癌旁组织和正常肝组织中RKIP mRNA的表达量分别为1.357±0.569、1.512±0.623和1.550±0.426,组问差异无统计学意义（P〉0.05）.HCC、癌旁组织和正常肝组织中,RKIP蛋白的表达量分别为0.579±0.380、1.178±0.659和1.115±0.442,p65蛋白的表达量分别为0.830±0.376、0.630±0.337和0.466±0.345,pERK蛋白的表达量分别为1.023±0.478、0.605±0.367和0.461±0.293.免疫组织化学结果显示,HCC、癌旁组织和正常肝组织中,RKIP的阳性表达率分别为22.2%、86.0%和93.8%,p65的阳性表达率分别为73.6%、56.0%和37.5%;pERK的阳性表达率为65.3%、38.0%和31.3%.RKIP蛋白在HCC组织中的表达显著低于癌旁组织及正常肝组织,而p65和pERK在HCC组织中的表达均显著高于癌旁组织及正常肝组织（均P〈0.05）.RKIP蛋白表达水平与HCC分化程度和有尤门静脉或胆管癌柃情况有关（P〈0.05）,p65蛋白表达水平与HCC分化程度和有无门静脉或胆管癌栓有关,pERK蛋白表达水平与HCC分化程度、有无门静脉或胆管癌栓和肝内或淋巴结转移情况有关（均P〈0.05）.HCC组织中RKIP的表达与pERK的表达相关（P=0.04）,但与p65的表达无关（P=0.143）.结论 RKIP表达的减少或缺失、p65和pERK的高表达与HCC的发生发展、侵袭转移密切相关.RKIP可能通过下调pERK的表达抑制HCC的侵袭转移.

Objective To investigate the expression of RKIP, p65 and pERK in hepatoceUular carcinoma （ HCC） and their correlation with invasion and metastasis of HCC. Methods Reverse transcription polymerase chain reaction （RT-PCR） was used to detect the expression of RKIP mRNA. The expression levels of RKIP, p65 and pERK proteins in HCC tumor and peritumoral tissues were determined by immunohistochemistry and Western blot analysis. Statistical analysis was performed to determine the relationship between their expression and clinicopathological parameters. Results RKIP protein expression level （RKIP/actin） was 0.579 ±0.380 in HCCs, 1. 178 ±0.659 in peritumoral tissues and 1.115 ±0.442 in normal liver tissues. The pERK protein level was 1. 023 ± 0. 478, 0. 605 ± 0. 367 and 0. 461 ±0. 293, p65 protein level was 0. 83 ± 0.376, 0.63 ± 0.337 and 0.466 ± 0. 345, respectively. Immunohistochemistry analysis showed that the RKIP positive rates in HCCs, peritumoral tissues and normal liver tissues, were 22.2% , 86.0% , and 93.8% , positive rates of p65 were 73. 6% , 56. 0% and 37. 5% , positive rates of pERK were 65.3%, 38. 0% and 31.3%, respectively. Statistical analysis revealed that there was a significant difference in RKIP protein expression levels （P 〈 0.05）, but no significant difference in RKIP mRNA expression levels （P〉0.05） among HCC tumors, peritumoral tissues and normal liver tissues. The p65-positive and pERK-positive rates were higher in tumor tissues than that in peritumoral tissues and in normal liver tissues （P 〈 0.05 ） , but RKIP-positive rates were lower in tumor tissues than that in paritumoral tissues and normal liver tissues （P 〈0.05）. RKIP protein expression levels were significantly lower in HCCs with intrahepatic or lymphatic metastasis than that in without. The RKIP positive rates in moderately and well differentiated HCCs were significantly higher than that in poorly differentiated HCCs. There was a relationship between RKIP and pERK expressions （P=0.04） , but RKIP expression was not correlated with p65 expression in HCCs （P = 0. 143）. Conclusions Our findings indicate that the down-regulation of RKIP expression may serve as a predictive marker for HCC development, progression and metastasis, which may contribute to the elevated ERK activity. The inhibiting effect of RKIP on invasion and metastasis of liver cancer cells may be due to the down-regulation of pERK expression rather than p65 expression.