肝癌细胞中骨桥蛋白基因表达调控的分子机制研究

Transcriptional Regulation of Human Osteopontin in Hepatocellular Cacinoma Cells

骨桥蛋白（osteopontin,OPN）是人体内一种重要的分泌蛋白,在细胞黏附、迁移、机体免疫等作用中发挥重要的生理功能。近年来的研究发现,OPN在恶性肿瘤组织中参与诱导细胞外基质的降解及重构,对肿瘤细胞的侵袭能力有重要调控作用。此外,OPN在肝癌及其他多种肿瘤类型中表达均显著上调,但调控机制尚未完全阐明。该研究从肝癌细胞株中克隆了OPN基因SPP1的启动子序列p SPP1,尝试鉴定肝癌中SPP1的主要调控机制。截断突变体及点突变体分析发现,完整的区域（–24~–17）是p SPP1转录活性所必需的,外源表达AP-1转录因子的c-jun亚基,能够显著提高这一区域的转录活性,然而,c-fos没有这种作用效果。凝胶迁移实验进一步证明AP-1转录因子与p SPP1的直接相互作用。利用实时定量PCR和Western blot还发现,外源表达c-jun能够显著提高内源性OPN的表达水平。以上实验结果揭示,AP-1转录因子介导的转录调控很可能是肝癌细胞OPN表达调控的关键机制,这一研究发现有望为肝癌诊断及治疗提供新的思路。

Osteopontin（OPN） is a secreted glycophosphoprotein which is important in cell adhesion, migration, body immune, etc. Recent studies recognized the essential role of OPN in carcinogenesis and tumor metastasis through regulating extracellular cell matrix（ECM） degradation and remodeling. OPN is significantly up-regulated in multiple cancers including hepatocellular carcinoma（HCC）, and is relevant with the malignancy. However, the molecular mechanism of OPN regulation remains largely illusive. In our study, in order to investigate the regulation mechanism of OPN gene SPP1 in hepatocellular carcinoma, the full-length promoter（p SPP1） was cloned. Deletion and point mutation analysis demonstrated that –24~–17 region is essential for the transcriptional activity of p SPP1. Overexpression of AP-1 factor subunit c-jun, but not c-fos, can significantly enhance the transcriptional activity, and EMSA assay proved the direct binding of c-jun to p SPP1. Moreover, overexpression of c-jun can effectively induce endogenous OPN expression, which is demonstrated by both Western blot and q RT-PCR assay.Taken together, our f indings recognize AP-1 factor as a key mechanism of OPN regulation in HCC cell, which brings new insights into HCC diagnosis and gene therapy.