乙型肝炎病毒X蛋白和环氧合酶-2与乙型肝炎相关肝癌微血管生成和转移的关系及其可能机制

The expression of hepatitis B virus X protein and cyclooxygenase-2 in hepatitis B virus-related hepato- cellular carcinoma： correlation with microangiogenesis and metastasis, and what is the possible mechanism

目的 探讨乙型肝炎病毒X蛋白（HBX）、环氧合酶-2（COX-2）在乙型肝炎相关性肝细胞肝癌（HCC）组织微血管生成和转移中的关系及其可能的调节机制. 方法选择84例乙型肝炎相关性肝细胞肝癌组织和22例非乙型肝炎相关性肝细胞肝癌组织,免疫组织化学法检测HBX、COX-2及血管内皮细胞表面抗原（CD34）的表达,光镜下记录微血管计数（MVD）.Spearman相关性分析HBX、COX-2与乙型肝炎相关性HCC组织微血管生成及转移中的关系; Western blot和RT-PCR检测稳定转染HBX肝癌细胞HepG2（HepG2-X）中COX-2的变化;酶联免疫吸附法检测HepG2-X培养上清液中前列腺素E2（PGE2）表达水平及不同浓度（10、30、50 μ mol/L和70 μmol/L）COX-2选择性抑制剂塞来昔布作用后PGE2的表达水平;锥虫蓝拒染法检测细胞生长速度.结果 乙型肝炎相关性HCC组织中HBx、COX-2均高表达,HBx阳性表达组中COX-2阳性率为88.87%（55/62）,明显高于HBx阴性组的31.82%（7/22,x2=27.188,P＜0.01）和非乙型肝炎相关性HCC组40.91%（9/22,x2=20.453,P＜0.01）;HBx阴性表达的乙型肝炎相关性HCC组和非乙型肝炎相关性HCC组中COX-2阳性表达率差异无统计学意义（x2=0.393,P=0.531）; HBx和COX-2在转移组表达高于无转移组（P＜0.01）;HBx及COX-2的阳性表达组平均MVD值均明显高于HBx阴性组和非乙型肝炎相关性HCC组（P＜0.05）,且转移组MVD高于无转移组（P＜0.01）;门静脉侵犯组高于非侵犯组（P＜0.01）;Spearman相关性分析结果提示HBx、COX-2在乙型肝炎相关性HCC组织微血管生成及转移中的表达呈正相关（Rs=0.568,P＜0.01）;在HepG2-X细胞中,COX-2蛋白及mRNA表达水平与转染空载体的对照肝癌细胞HepG2 （空载体转染HepG2细胞,HepG2-PC）相比,均显著提高,并且细胞培养上清液中PGE2表达水平也显著提高;和转染空载体的对照细胞相比,塞来昔布对转染HBx基因的细胞分泌PGE2具有更强的抑制作用.结论 HBx、COX-2在乙型肝炎相关性HCC中有较高的表达水平,二者呈正相关,与HCC微血管生成和侵袭转移密切相关.COX-2可能是HBx促使肝癌组织微血管生成和转移的一个重要中间分子,其可能的调节机制是通过HBx、COX-2及PGE2作为信号转导通路在HCC的浸润与迁移,在微血管生成与转移的过程中发挥双重作用.

Objective To investigate the expression of HBx and COX-2 in hepatitis B virus-related hepatocellular carcinoma, and Its correlation with microangiogenesis and metastasis, and possible mechanism.Methods Immunohistochemistry was used to detect the expression of hepatitis B virus X, cyclooxygenase-2 and CD34 in hepatitis B virus related hepatic carcinoma and 22 non-HBV related hepatic carcinoma tissues.The expression of hepatitis B virus x protein and cyclooxygenase-2 in hepatitis B virus-related hepatocellular carcinoma correlated with microangiogenesis and metastasis was tested by Spearman correlation analysis.The expression of COX-2 in HepG2-X was detected by Western blot and RT-PCR. PGE2 was detected by ELISA in clear supernatant liquid of HepG2-X. Trypan blue exclusion was performed to examine the inhibitory effects of COX-2 selective inhibitor （celecoxib）. Results In Hepatitis B carcinoma tissue, HBx and COX-2 were expressed at high level. The positive rate of COX-2 expression was 88.87% （55/62） in HBx positive expression group, which was significantly higher than that of the positive expression 31.82% （7/22,x2 = 27.188, P 〈 0.01） in HBx negative expression group and 40.91% （9/22, x2 = 20.453, P 〈 0.01） in nonHBV related hepatic carcinoma tissues, but it had no statistical difference （ x2 = 0.393, P = 0.53l）between the HBx negative expression group and non-HBV related hepatic carcinoma tissue group. The expressions of HBx and COX-2 in metastasis group were higher than that in non- metastasis group （P〈0.01）, MVD in HBx or COX-2 positive expression group was significantly higher than that in negative expression group and nonHBV related hepatic carcinoma tissues （P〈0.01）. MVD with metastasis was higher than that without metastasis （P 〈 0.01） and MVD with portal vein invasion was higher than that without invasion （P〈0.05）.Spearman correlation analysis showed that the expression of COX-2 was significantly correlated with the expression of HBx （Rs = 0.568, P 〈 0.01）. Celecoxib suppressed the growth of both cells in a dose-dependent manner. HepG2-X was significantly susceptible to celecoxib as compared to the HepG2-PC cells. COX-2 protein and mRNA were upregulated in HepG2-X cells than in HepG2-PC. Moreover, PGE2 was upregulated in clear supernatant liquid of HepG2-X than in HepG2-PC. Conclusion The expressions of HBx and COX-2 were higer in HBV-related hepatocellular carcinoma. COX-2 was significanfiy correlated with HBx in HCC and it could be a key factor involved in HBx contributed hepatocellular carcinoma＇s microangiogenesis and metastasis. The possible mechanism of the dual effects might be through HBx,COX-2 and PGE2 pathways in infiltration involved metastasis and microangiogenesis involved metastasis.