HGF/c-met通路对胃癌细胞肝素酶表达的调控及其分子机制研究

Literatures and our previous studies have shown that expression of heparanase is closely associated with invasion and metastasis of gastric cancer,and also significantly positively related to c-met,which demonstrates that HGF/c-met may involve in the expression regulation of heparanase in gastric cancer. Literatures reported that HGF/c-met could phosphorylate and degrade IκB,promote nuclear translocation of NF-κB, and activate the downstream target gene.Our results of previous experiments have shown that intervention of NF-κB can significantly affect promoter activity of heparanase and expression of heparanase.Bioinformatics predicts that there are several binding sites of NF-κB on promoter of heparanase,which demonstrates that HGF/c-met could activate NF-κB and promote its nuclear translocation to bind to promoter of heparanase and regulate expression of heparanase.On this occasion, activated heparanase can further degrade extracellular matrix and release HGF,form a positive feedback control mechanism to maintain and amplify the effect of invasion and metastasis mediated by heparanse.Based on above hypothesis, this study will illustrate the molecular mechanism that HGF/c-met regulates expression of heparanase and its role in invasion and metastasis of gastrci cancer using luciferase experiment,EMSA and ChIP, which will provide a new theoretical basis for intervening gastric cancer through targeting heparanase.

文献及我们的研究表明，肝素酶的表达与胃癌侵袭转移密切相关,且肝素酶的表达亦与c-met呈明显正相关，提示HGF/c-met通路可能参与了胃癌细胞肝素酶的表达调控。文献报道HGF/c-met通路可以通过磷酸化降解IκB，促进NF-κB的核转位，激活下游的靶基因。课题组前期实验结果表明，干预NF-κB可以明显影响肝素酶启动子的活性及肝素酶蛋白的表达，生物信息学预测发现在肝素酶启动子上存在NF-κB的结合位点，提示HGF/c-met可通过活化NF-κB，促进其核转位而结合肝素酶启动子，调控肝素酶表达，而肝素酶又通过降解细胞外基质，促进HGF的释放，从而形成一种正向反馈调控通路，维持并放大肝素酶所致的肿瘤侵袭转移效应。本课题拟采用双荧光素酶实验、EMSA和ChIP等分子生物学技术，阐明HGF/c-met调控肝素酶表达的分子机制及在肿瘤侵袭转移中的作用，为以肝素酶为靶点的胃癌干预提供新的理论依据。

既往研究表明，肝素酶的表达与胃癌侵袭转移密切相关，然而胃癌细胞肝素酶的表达调控机制并不清楚。课题组前期研究发现，HGF/c-met表达与胃癌侵袭转移密切相关，并且与肝素酶的表达存在明显正相关，提示HGF可能参与了肝素酶的表达调控。我们课题组拟通过qPCR和western blot实验研究HGF是否可上调胃癌细胞肝素酶表达，检测HGF对NF-κB信号活化及肝素酶表达的影响，通过生物信息学预测肝素酶启动子上存在NF-κB的结合位点，ChIP实验证实p65与肝素酶启动子结合情况。结果发现，HGF与肝素酶在胃癌组织高表达且具有明显正相关；HGF可通过激活NF-κB信号促进胃癌细胞肝素酶的表达；生物信息学预测发现p65在肝素酶启动子存在结合位点，ChIP实验进一步证实p65可直接与肝素酶启动子结合，HGF可通过活化NF-κB信号促进p65结合肝素酶启动子而促进肝素酶转录表达，进而促进胃癌的侵袭转移；进一步研究发现，肝素酶的表达可促进ECM降解和HGF的释放，从而形成一种正向反馈调控通路，维持并放大HGF和肝素酶促胃癌侵袭转移效应。本项目阐明了HGF与肝素酶表达的正向反馈调控机制及在肿瘤侵袭转移中的作用，为以肝素酶为靶点的胃癌干预提供新的理论依据。