C-反应蛋白参与HBV复制及相关HCC发生的机制研究

The main reason of the occurrence and progression of HBV related HCC is chronic inflammation caused by persistent chronic HBV infection and viral replication. It may be the foundation of HBV replication and the occurrence and progression of HCC, through the interaction of key molecules with ROS, MAPKs, NF- kappa B and interferon signal pathway, HBV encoded protein activated IL-6-STAT3 signaling pathway associated with host inflammation.To find out the key proteins in the signaling pathway. Then to study the function of these key protein molecules combined with molecular biology and cell biology. It has important significance to explain the pathogenesis of HBV infection and find new therapeutic targets.. C reactive protein (CRP) is a kind of acute phase reactive protein, which is produced by the liver and significantly correlated with inflammation, infection and tissue injury, is the key effector molecules of IL-6-STAT3 signaling pathway related with inflammation.The IL-6-STAT3 signaling pathway is linked to multiple signaling pathways via CRP, it not only has the function of immune regulation, but also plays an important role in the occurrence and Progression of tumor.It has been proved that CRP is closely related to the degree of inflammation and the occurrence and progress of hepatic fibrosis and HCC of fatty liver, chronic hepatitis C, chronic hepatitis B, and so on. What is more interesting is that our previous studies have found that CRP can regulate the replication of HBV by affecting the expression of STAT3, NF- kappa B and interferon related genes, to promote the progress of HBV related HCC. Accordingly, we speculate that CRP may participate in the interaction between signaling pathways related to the occurrence and development of disease between HBV infection. However, the mechanism of CRP interacts with relative signaling pathways involved in the regulation of HBV replication and promotes the occurrence of HBV related HCC has not yet been studied in depth. Based on these, in this study, we will use HepG2 and HepG2.215 cell lines and transplanted tumor nude mice model, through importing CRP wild type and promoter or DNA binding domain mutant overexpression plasmid or CRP specific siRNA, use gene chip technology quickly and effectively to screen of key signaling pathways and key proteins interacting with CRP and draw out the interaction network of protein with CRP; then the functional proteins are validated and studied function. Finally, these proteins are associated with the function of CRP in order to elucidate the mechanism of CRP involved in HBV replication and the development of HCC. The purpose of this study is to provide theoretical basis for searching for molecular markers of new early screening HBV related HCC and drug targets of new treatment for HBV related diseases.

HBV激活炎症信号通路IL-6-STAT3，并与ROS、MAPKs、NF-κB和干扰素等通路相互作用是HBV复制及相关HCC发生的基础。CRP是IL-6-STAT3通路的关键效应分子。我们发现CRP可通过STAT3、NF-κB及干扰素相关基因调控HBV复制和相关HCC发生。推测：CRP可能是IL-6-STAT3与ROS等通路发生相互作用的关键节点分子。但目前哪些通路及关键点蛋白与CRP存在相互作用及其机制仍未研究。故拟：1）利用HepG2和HepG2.215细胞系及其移植瘤裸鼠模型，分别导入CRP过表达质粒或特异性siRNA后，采用基因芯片技术筛选出与CRP存在相互作用的关键信号通路和节点蛋白，并绘制出相互作用网络图；2）对筛选出的关键点蛋白进行验证和功能研究，最终将这些信号通路及关键点蛋白质组分与CRP的功能联系起来，以期阐明CRP参与HBV复制及相关HCC发生、进展的部分机制。

持续的HBV感染和病毒高复制状态导致的慢性炎症是HBV相关HCC发生、进展的主要原因。HBV编码蛋白通过激活宿主炎症相关的信号通路- IL-6-STAT3信号通路，并通过关键节点蛋白质分子与ROS、MAPKs、NF-κB和干扰素等信号通路相互作用可能是HBV复制及相关HCC发生、进展的基础。寻找出IL-6-STAT3信号通路中与ROS、MAPKs、NF-κB和干扰素等信号通路相互作用的关键节点蛋白质分子，然后结合分子生物学及细胞生物学方法对这些关键点蛋白质分子进行功能研究，对于阐明HBV感染相关疾病的发病机理和寻找新的治疗药物靶点具有重要的意义。课题组利用HepG2和HBV复制细胞模型HepG2.215细胞系及其移植瘤裸鼠模型，通过分别导入CRP野生型及启动子或DNA结合域突变型过表达质粒或CRP特异性siRNA后，采用基因芯片技术筛选出与CRP存在相互作用的关键信号通路和信号通路中关键节点蛋白质。体内实验中发现：CRP在HepG2.215细胞中的表达显著高于HepG2细胞，CRP基因过表达显著增强细胞CRP蛋白和CRPmRNA、HBVcccDNA、HBVpgRNA及HBVcAg定量表达，沉默CRP基因明显抑制上述指标表达水平；干扰CRP基因表达可显著影响HepG2.2.15细胞HBsAg、HBeAg、HBV-mRNA、HBVDNA以及炎症因子、凋亡及DNA损伤相关因子表达。体外实验中发现：CRP基因沉默组对小鼠肿瘤大小、重量、体积及生长曲线的影响较阴性对照组均显著性下降，并可促进瘤组织细胞凋亡；CRP基因沉默组HBsAg、HBeAg、HBcAg、HBxAg及HBVcccDNA及HBVpgRNA的表达较阴性对照组被明显抑制；TNF-a、NF-κB、IL-6、STAT3的表达也被显著抑制。信号转导基因芯片结果显示：CRP主要参与调控的信号通路与IL-6-STAT3信号通路与NF-κB信号通路相关。整体研究结果表明：高CRP水平不但可促进HBV复制，而且参与了HCC的增殖、凋亡、迁移细胞过程，其机制可能与CRP参与调控IL-6-STAT3信号通路与NF-κB信号通路相关。初步临床应用研究表明血清hs-CRP还具有HCC预测诊断价值，为hs-CRP作为一种新的预测HCC发生的临床诊断指标研究奠定了基础。