肝癌转移相关的骨桥蛋白表达及其糖基化的改变

目的 检测骨桥蛋白(OPN)在不同转移潜能肝癌细胞株及肝癌组织中的蛋白表达水平及其糖基化水平,探讨OPN糖基化改变与肝癌转移的相关性及其意义. 方法 用免疫组织化学和Western blot法检测人肝癌组织(非转移组6例、转移组7例)及不同转移潜能人肝癌细胞株(L02、Hep3B、MHCC97L、MHCC97H、HCCLM3、HCCLM6)中OPN蛋白水平;采用免疫沉淀技术纯化肝癌组织中的OPN蛋白,并采用多重凝集素印迹技术检测转移与非转移肝癌组织中OPN糖基化水平差异.数据统计采用t检验和方差分析.结果 OPN在不同转移潜能肝癌细胞株的表达差异具有统计学意义(F=5.04,P＜0.01).在肝癌组织中,转移组OPN蛋白表达水平明显高于非转移组(t=2.447,P＜0.05),相对吸光度值分别为0.69±0.21和0.45±0.14.免疫沉淀技术成功纯化肝癌组织中的OPN蛋白,后续的凝集素印迹结果显示:与非转移组相比,转移组OPN蛋白对凝集素朝鲜槐、红腰果E型、蔓陀罗、刀豆素A的亲和力较低(P值均＜0.05).结论 OPN蛋白表达水平与肝癌转移潜能增强呈正相关;OPN蛋白的α2,3-唾液酸、平分型GlcNAc、多天线、偏二天线的糖链、高甘露糖型N-糖等糖基化水平改变可能与肝癌转移潜能增高有关.

Identification of metastasis-related osteopontin expression and glycosylation in hepatocellular carcinoma

Objective To test expression level and glycosylation level of OPN in HCC cell lines with different metastatic potential and HCC tissues,and investigate the correlation between the glycosylation change and the liver cancer transporting as well as its significance.Methods The level of OPN expression in liver cancer tissue(6 cases of non-metastasis and 7 cases of metastasis)as well as HCC cell lines with different metastatic potential (L02,Hep3B,MHCC97L,MHCC97H,HCCLM3,HCCLM6)was identified by immunohistochemistry and Western Blot,and then OPN was purified from HCC tissues by immunoprecipitation,followed by glycosylation detection of OPN from non-metastatic and metastatic HCC tissues by multiple lectin blot.Data were analyzed by t-test and variance analysis.Results Different levels of OPN expression were observed in HCC cell lines with different metastatic potential (F =5.04,P =0.008).Additionally,OPN expression level in HCC tissues with metastasis was higher than that in non-metastasis group (t =2.447,P ＜ 0.05).Relative optical density value was 0.69 ± 0.21 and 0.45 ± 0.14 respectively.OPN in liver cancer tissue was successfully purified using immunoprecipitation.Followed lectin blotting result showed that OPN protein in metastasis group showed lower affinity to MAL,PHAE,DSA,ConA as compared with that in non-metastasis group (P ＜ 0.05).Conclusions The expression of OPN was positively correlated with the enhanced metastasis potential of HCC.OPN from metastasic HCC tissues presented lower level of some specific glycan structures such as α2,3- sialic acid,bisecting GlcNAc,biantennary,mutiantennary and high mannose type N-glycan structure.This study not only indicates the role of OPN in HCC metastasis for the first time,but also provide experimental support for the mechanism of the function of OPN in the transportation of liver cancer cells as well as offer potential target for clinical treatment.