NEDD9促进肝细胞肝癌侵袭转移的作用研究DIXDC1在肝细胞肝癌中的表达及其与预后的相关性

发布时间：2018-03-16 11:20

本文选题：NEDD9　切入点：肝细胞肝癌　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：目的肝细胞肝癌是目前全球主要的恶性肿瘤之一,尽管目前肝癌的诊断及治疗手段的进步已使更多患者获得了延长生命的机会,但是肝癌术后的复发转移仍严重影响着患者的预后。NEDD9是一种与实体肿瘤转移密切相关的细胞内支架蛋白。最近研究发现NEDD9在肝细胞肝癌标本中表达上调,但NEDD9在肝细胞肝癌中的具体作用仍未阐明。因而我们通过体内外实验,探索NEDD9在肝细胞肝癌侵袭转移方面的具体作用以及可能的潜在机制。方法在肝癌细胞株以及140对肝细胞肝癌标本中,采用qPCR、Western blot以及免疫组化等多种方式检测NEDD9的表达情况,并结合患者临床病理资料分析NEDD9与患者临床特征以及预后的相关性。分组分析非侵袭性肝癌以及侵袭性肝癌中NEDD9的表达差异。构建NEDD9敲减以及过表达细胞株,通过MTS细胞增殖实验Transwell细胞迁移及侵袭实验以及裸鼠体内脾脏注射-肝转移模型,观察NEDD9对肿瘤增殖、侵袭、转移能力的影响。运用qPCR、Western blot、免疫荧光、免疫组化等多种方法初步探究NEDD9在促进肝细胞肝癌侵袭转移方面的潜在机制。结果qPCR以及Western blot检测发现NEDD9在肝癌细胞株中的表达较正常肝细胞株中表达上调。140对肝细胞肝癌标本通过免疫组化检测发现NEDD9在肝细胞肝癌中表达升高,且与患者的血清AFP浓度(p=0.008)、肿瘤癌栓(p=0.005)和肿瘤TNM病理分期(p=0.014)有明显相关性。根据肝癌患者是否合并肿瘤癌栓将患者分成非侵袭性肝癌组以及侵袭性肝癌组,发现两组之间NEDD9表达存在差异。结合肝癌患者的随访数据,采用Kaplan-Meier生存分析发现NEDD9高表达者较低表达者有更短的无瘤生存期和总生存期,COX多因素回归分析提示NEDD9是肝癌术后复发的独立危险因素。成功构建NEDD9过表达慢病毒质粒,并在肝癌细胞株HCC-LM3和Huh7中成功构建NEDD9敲减以及过表达细胞株。MTS细胞增殖实验提示NEDD9敲减能明显抑制肝癌细胞的生长。Transwell细胞迁移及侵袭实验提示,NEDD9敲减能明显抑制肝癌细胞的迁移及侵袭能力,NEDD9过表达能增强肝癌细胞株的迁移及侵袭能力。裸鼠体内脾脏注射-肝转移模型发现,NEDD9过表达组裸鼠较对照组在肝脏表面形成更多的转移结节。机制方面,我们发现在肝癌细胞以及肝癌组织中,NEDD9能反向调控E-cadherin的表达,FAK参与了 NEDD9对E-cadherin表达的调控。结论NEDD9在肝癌细胞株及肝癌组织中均表达上调。NEDD9与肝癌临床的侵袭转移特性有一定的相关性。NEDD9能明显增强肝癌细胞的侵袭及转移能力。NEDD9能反向调控肝癌中E-cadherin的表达,可能是NEDD9促进肝癌侵袭转移的潜在机制。目的肝细胞肝癌术后复发严重影响着肝癌患者的预后,肿瘤标记物作为一种从分子生物学角度参与评估肿瘤患者预后的新方法也具有潜在的重要意义。DIXDC1是一种含有DIX结构域的支架蛋白,作为Wnt信号通路的正向调控因子参与神经系统的发育。尽管DIXDC1已经在某些恶性肿瘤中进行了研究,但是目前在肝细胞肝癌中尚无任何的相关报道,因而我们本次研究目的主要是探究DIXDC1在肝癌中的表达及其临床意义。方法通过生物信息学的方法从Oncomine数据库中检索关于DIXDC1在肝细胞肝癌中表达情况的数据。采用qPCR、Western blot等方法检测25对新鲜肝细胞肝癌标本中DIXDC1的表达情况。进一步在140对肝细胞肝癌标本中,通过免疫组化的方法评估DIXDC1的表达,并结合患者临床病理资料分析DIXDC1与患者临床病理特征以及预后的相关性。结果Oncomine数据库中三个独立的检测数据提示,肝细胞肝癌中DIXDC1的mRNA表达水平较正常肝组织表达下调。在25对新鲜肝细胞肝癌组织中,通过qPCR以及Western blot检测发现DIXDC1在大部分肝癌组织中表达下降。在140对肝细胞肝癌标本中,通过免疫组化评估发现DIXDC1在肝细胞肝癌中低表达,且与肿瘤大小(p=0.024)、肿瘤细胞分化程度(p0.001)、肿瘤癌栓(p=0.019)、肿瘤TNM病理分期(p=0.019)及肝癌巴塞罗那临床分期(p=0.008)等患者疾病进展相关指标有密切关系。更重要的是,Kaplan-Meier生存分析发现DIXDC1低表达患者较高表达者有更短的无瘤生存期和总生存期,COX多因素回归分析提示DIXDC1是肝癌患者总生存期的独立预后因子。结论我们首次揭示DIXDC1在肝细胞肝癌中表达下调。低表达DIXDC1与患者的临床疾病进展状况以及较差的预后有明显相关性。DIXDC1可作为一个新的肿瘤标记物来预测肝细胞肝癌患者术后的临床转归情况。
[Abstract]:Objective: hepatocellular carcinoma is one of the major malignant tumor in the world at present, despite the current diagnosis and treatment for advanced HCC, progress has been made more patients get a chance to prolong the life, but the postoperative recurrence and metastasis of hepatocellular carcinoma still seriously affects the prognosis of patients with.NEDD9 is a scaffold protein is closely related to the metastasis and tumor cells. Recent studies have found that upregulation of NEDD9 expression in hepatocellular carcinoma tissues, but not the specific role of NEDD9 in hepatocellular carcinoma. So we clarify the in vitro and in vivo, to explore the potential mechanism of NEDD9 in hepatocellular carcinoma invasion and metastasis of the specific role and possible. Methods qPCR in HCC cell lines and 140 of liver cells hepatocellular carcinoma specimens, the expression of Western, blot, immunohistochemistry and other methods for detection of NEDD9, and combined with clinical data of patients with NEDD9 and patients with clinical analysis The bed characteristics and prognosis. Subgroup analysis of non invasive hepatocellular carcinoma and invasion of hepatocellular carcinoma NEDD9 expression difference. Construction of NEDD9 knockdown and overexpression cell lines by MTS cell proliferation assay of Transwell cell migration and invasion assay and nude mice spleen injection - liver metastasis model, observation of NEDD9 on tumor proliferation, invasion, influence transfer ability. Using qPCR, Western blot, immunofluorescence, immunohistochemistry and other methods of preliminary inquiry of NEDD9 in promoting hepatocellular carcinoma metastasis potential mechanism of invasion. The expression of qPCR and Western blot detection found NEDD9 in hepatocellular carcinoma cell lines compared with the normal liver cells up regulate the expression of.140 in hepatocellular carcinoma specimens by immunohistochemical detection showed that the expression of NEDD9 in hepatocellular carcinoma and increased serum AFP concentration in patients with tumor thrombi (p=0.008), (p=0.005) and tumor TNM staging (p=0.014) have significant correlation. According to the liver cancer patients with tumor thrombi were divided into non invasive HCC group and invasive HCC group, found that NEDD9 expression differences between the two groups. With the follow-up data of patients with hepatocellular carcinoma, using Kaplan-Meier survival analysis showed high expression of NEDD9 in tumor free survival and overall survival were lower the expression of a shorter, multi factor COX regression analysis showed that NEDD9 was independent risk factors of recurrence of hepatocellular carcinoma after operation. The successful construction of NEDD9 lentiviral expression plasmid, and the HCC-LM3 and Huh7 in hepatocellular carcinoma cell line was successfully constructed and knockdown of NEDD9 expression cell line.MTS cell proliferation assay suggested that NEDD9 knockdown could inhibit hepatocellular carcinoma cells the growth of.Transwell cell migration and invasion assay indicated that knockdown of NEDD9 could inhibit the migration and invasion of hepatocellular carcinoma cells, overexpression of NEDD9 can enhance the migration of hepatocellular carcinoma cell lines Migration and invasion. Nude mice spleen injection - liver metastasis model of nude mice found that overexpression of NEDD9 group compared with the control group formed more metastatic nodules on the surface of the liver. The mechanism, we found that in liver cancer cells and tumor tissues, NEDD9 expression can reverse the regulation of E-cadherin, FAK is involved in the regulation of NEDD9 on the expression of E-cadherin. Conclusion NEDD9 expression in hepatocellular carcinoma cell lines and tissues by.NEDD9 and liver cancer clinical characteristics of the invasion and metastasis of.NEDD9 have certain correlation can significantly enhance HCC cell invasion and metastasis ability of.NEDD9 can reverse regulation expression of E-cadherin in hepatocellular carcinoma, NEDD9 may be a potential mechanism to promote the invasion and metastasis of hepatocellular carcinoma. Objective: hepatocellular carcinoma recurrence after severe affects the prognosis of patients with hepatocellular carcinoma, tumor marker as a is also from the perspective of molecular biology in a new method for evaluating the prognosis of patients with tumor .DIXDC1 has a very important meaning potential is a scaffold protein containing DIX domain, as a positive regulator of Wnt signaling pathway in the development of the nervous system. Although DIXDC1 has been studied in some malignant tumors, but in hepatocellular carcinoma is seldom reported any, so we study the main purpose is to the expression of DIXDC1 in hepatocellular carcinoma and its clinical significance. Methods by bioinformatics methods from Oncomine database retrieval on the DIXDC1 expression data in hepatocellular carcinoma. The detection of Western qPCR, blot of 25 DIXDC1 fresh liver specimens. The expression of a further 140 in hepatocellular carcinoma specimens that expression was assessed using immunohistochemistry DIXDC1, and combined with the analysis of DIXDC1 and clinical pathological characteristics and prognosis of patients with clinical pathological data Correlation. The results of three independent test data in the Oncomine database indicated that DIXDC1 in hepatocellular carcinoma mRNA expression was downregulated in normal liver tissue. 25 of fresh liver tissues by qPCR and Western blot assay showed that DIXDC1 decreased in most liver tissues. The expression in 140 of hepatocellular carcinoma specimens in the immunohistochemical evaluation found that low expression of DIXDC1 in hepatocellular carcinoma, and tumor size (p=0.024), the degree of differentiation of tumor cells (p0.001), tumor thrombi (p=0.019), tumor TNM staging (p= 0.019) and Barcelona liver cancer clinical staging (p=0.008) related indicators in patients with disease more importantly, Kaplan-Meier survival analysis showed disease-free survival and overall survival of patients with low expression of DIXDC1 high expression had shorter, COX regression multivariate analysis showed that DIXDC1 was suffering from liver cancer Independent prognostic factor for total survival. Conclusion we first revealed the expression of DIXDC1 in hepatocellular carcinoma. Downregulation of.DIXDC1 had obvious correlation can be used as a new tumor marker to predict the clinical patients with hepatocellular liver cancer after operation and outcomes of clinical disease progression in patients with low DIXDC1 expression status and poor prognosis.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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