K19蛋白通过Rap1通路调控肝癌细胞生物学行为的分子机制研究

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world,accounting for an estimated 600,000 deaths annually. HCC is characterized by active metastasis and insufficient apoptosis, which account for rapid recurrence and poor survival. Therefore, identifying molecules that can suppress metastasis and promote apoptosis may provide novel therapies for treatment of HCC. .Keratin (K) 19, which is considered to be a biliary/hepatic progenitor cell marker, has attracted attention as a useful predictive marker for detecting the more aggressive HCCs, because tumors with K19 expression have a poorer prognosis and higher rates of recurrence. K19 was well correlated with clinicopathologic features of tumor aggressiveness. Interesting, HCCs expressing K19 proteins are characterized by increased telomere length, increased expression of shelterin complex proteins, and increased chromosomal instability compared to conventional HCCs..Rap1 (repressor/activator protein), a member of telomeres shelterin complex, has been shown to be an essential modulator of biological function in hepatocellular carcinoma. High expression of Rap1 were significantly associated with a more aggressive tumor biological behavior in HCC. In previous study, we found knockdown of Rap1 by microRNA (miRNA) interference enhanced signifi¬cantly apoptosis and 5-fluorouracil (5-FU) chemosensitivity in HepG2 cell line by downregulating nuclear factor-κB p65 (NF-κB p65) expression and upregulating inhibitor of NF-κB (IκB) expression. However, the molecular mechanism of K19 in aggressive biological behaviour of HCC has not been fully clarified, the effect of K19 on Rap1/ NF-κB signal in HCC remains unclear..In this study, we will first investigate the roles of K19 in tumor growth, metastasis, and apoptosis of HCC in vitro and vivo. Gain- and loss-of-function studies of the K19 are conducted by transfected a K19 expression plasmid or a K19 siRNA vector into human HCC cell lines. The potential role of K19 in HCC metastasis is investigated with transwell assays and wound healing assays in vitro. Its role on apoptosis is detected by the activity of caspase-3, flow cytometry with Tunel staining.To investigate the possible relationship between the K19 and Rap1/ NF-κB signalling pathway, we will examine the expression of Rap1, NF-κB p65, and IκB in the HCC cell lines transfected a K19 expression plasmid or a K19 siRNA vector and control empty vector. To further examine the role of Rap1 in K19-related HCC progression, we will investigate whether knockdown of Rap1 in HCC cell lines offset the effect of K19. The role of Rap1 in K19-related HCC progression is evaluated with transwell assays, wound healing assays, the activity of caspase-3, and flow cytometry with Tunel staining..Our study would provide additional insight into mechanisms of K19 in HCC. We speculate that inhibition of K19 expression in liver tumor cells might be a novel strategy for the treatment of HCC.

原发性肝细胞癌(HCC)的侵袭、转移、凋亡障碍等恶性生物学行为是导致其预后差的重要原因之一。抑制/激活蛋白（Rap1）是一种端粒结合蛋白，我们的研究表明Rap1可通过NF-κB（NF-kappa B）转录因子调控肝癌生物学行为，在HCC发展中起着重要的作用。新近研究表明肝癌组织中Rap1的表达和肝组细胞标记物角蛋白19（K19）表达成正相关，我们进一步敲除肝癌细胞株HepG2中K19的表达引起了Rap1表达下降，提示K19是调控Rap1表达的重要因子。但K19蛋白如何通过Rap1的信号通路调控HCC生物学行为尚不明确。因此本课题设想通过分子、细胞和动物水平进一步研究K19蛋白对HCC生物学行为的调节，阐明K19对HCC生物学行为调控中Rap1相关的作用机制，本研究有望发现调控HCC生物学行为的关键靶点分子，为开发新的靶向药物、提高HCC的综合治疗效果提供重要的实验依据。

肝细胞癌（HCC）是消化道肿瘤中的常见类型之一。在HCC中肿瘤细胞的形成与肝前体细胞密切相关。HPC的早期发展阶段会出现特异性的分子标记物：细胞角蛋白19（K19）。K19阳性的HCC具有早期转移、低度分化分化与高侵袭性等生物学特性。另一方面，抑制/激活蛋白（Rap1）是一种端粒结合蛋白，K19与Rap1表达成正相关，我们早期的研究表明Rap1可通过NF-κB（NF-kappa B）转录因子调控肝癌生物学行为，在HCC发展中起着重要的作用。但K19蛋白如何通过Rap1的信号通路调控HCC生物学行为尚不明确。本项目通过在K19表达干预前后，从体外（细胞学实验）和体内（皮下荷瘤模型/肝转移模型）等不同水平研究K19对肝癌增殖、侵袭、迁移和凋亡等生物学行为的影响，阐述K19对Rap1信号转导通路的调控、进而影响肝癌细胞生物学行为的具体分子机制。研究结果显示：. K19在HCC中对肿瘤细胞的增殖、侵袭与迁移具有促进作用。K19可引起Rap1、NF-κB p65表达上调。在外源性K19高表达的细胞株，敲除Rap1可阻断K19诱导的肿瘤细胞增殖、侵袭、迁移。. NF-κB抑制剂SN50亦可抑制K19诱导的肿瘤细胞增殖、侵袭、迁移。动物实验亦证明K19对肿瘤的生长与转移具有促进作用，SN50可抑制肿瘤细胞的生长及转移。以上研究结果提示：K19通过Rap1/NF-κB通路对HCC细胞的增殖、侵袭与迁移起着重要的促进作用。上述分子机制的研究为开发新的靶向药物、提高HCC的综合治疗效果提供重要的实验依据。