黏附分子CD44的表达及其糖基化与肝癌转移的相关性  

Relationship between CD44 expression or glycosylation and hepatocellular carcinoma metastasis

作　　者：陈冰琳[1] 郭坤[2] 刘银坤[2] 

机构地区：[1]复旦大学生物医学研究院癌症研究中心,上海200032 [2]复旦大学附属中山医院肝癌研究所

出　　处：《中华肝脏病杂志》2011年第12期898-903,共6页Chinese Journal of Hepatology

基　　金：基金项目：国家重点基础研究发展规划（2011CB910604）

摘　　要：目的探讨CD44S及其变异体CD44v分子的表达和其糖基化与肝癌细胞侵袭转移的关系。方法用免疫组织化学法、量子点、RT—PCR、Westernblot、细胞免疫荧光染色、甲基化特异性聚合酶链反应等技术检测转移与非转移陛肝癌组织、不同转移潜能人肝癌细胞株中CD44S及其变异体CD44v的定位与表达；并应用多重凝集素印迹法检测这些细胞株中CD44v6的糖基化差异。组间均数比较应用方差分析及t检验，多组间等级资料的比较采用Wilcoxon秩和检验，各组间率的比较采用x2检验。结果免疫组织化学结果显示，CD44S蛋白定位以细胞质为主；CD44v3、CD44v4／5蛋白定位于细胞膜与细胞质；而CD44v6主要定位于细胞膜。组织芯片结果显示，相对于CD44S及其他CD44v，CD44v6在转移组的表达水平高于非转移组（阳性率为75％与46%），差异具有统计学意义（X。=8．828，P〈0．05）；量子点检测（t=2．392，P〈0．05）与血清学检测（t=2．56，P〈0．05）也证实这一结果。Westernblot结果显示，CD44v6的表达与肝癌细胞转移潜能呈正相关。此外，在MHCC97L、MHCC97H肝癌细胞中CD44v6基因启动子发生不完全甲基化，而在Hep3B细胞中则发生完全甲基化。而且，相对于Hep3B细胞，MHCC97L及MHCC97H细胞中CD44v6蛋白对朝鲜槐凝集素、黑接骨木凝集素及麦胚凝激素的亲和力较高。结论在CD44S及其变异体CD44v中，CD44v6蛋白的高表达与肝癌转移潜能的增强呈正相关；其高表达可能与基因启动子低甲基化有关。此外，CD44v6蛋白唾液酸寡糖链的增加可能与肝癌细胞转移潜能增高有关。Objective To explore the relationship of CD44 expression or glycosylation and hepatocellular carcinoma （HCC） metastasis. Methods IHC, Quantum dots dtection, RT-PCR, Western blot, Cellular immune fluorescence and MS-PCR were used to identify CD44 expression in HCC samples and a series of human HCC cell lines with different metastatic potentials. Lectin array was used to reveal the relationship of CD44v6 glycosylation and human HCC metastasis. Results Immunohistochemistry analysis showed that CD44v6 was mainly distributed on the cell membrane, while CD44S immunoreactivity was prominently in the cytoplasm, CD44v3 and CD44v4/5 were in cytoplasm on membrane. Among CD44S and those CD44 variants, only the expression of CD44v6 was higher in metastasis HCC samples as compared to that in non-metastasis group （x2 = 8.828, P 〈 0.05）. This result was also re-confirmed by the result of Quantum dots（t= 2.392, P〈 0.05） and serum detection （t= 2.56, P〈 0.05）. We found completely methylation ofCD44v6 gene in Hep3B and incomplete methylation in MHCC97H and MHCC97L cell lines with metastatic potentials. The lectin affinity assay indicated that lectin MAL, SNA and WGA showed more affinity to MHCC97H and MHCC97Lcell lines than that of the non-metastatic Hep3B cell lines. Conclusions CD44v6 over-expression presents a positive correlation with HCC metastatic potential, which may be associated with DNA methylation level in promoter sequence. The increasing sialic acid modified glycan of CD44v6 might be related to HCC metastatic ability.

关 键 词：癌 肝细胞 肿瘤转移 糖基化 CD44V6 DNA甲基化 

分 类 号：R735.7[医药卫生—肿瘤]