本文選題：NEDD9　切入點(diǎn)：肝細(xì)胞肝癌　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的肝細(xì)胞肝癌是目前全球主要的惡性腫瘤之一,盡管目前肝癌的診斷及治療手段的進(jìn)步已使更多患者獲得了延長生命的機(jī)會,但是肝癌術(shù)后的復(fù)發(fā)轉(zhuǎn)移仍嚴(yán)重影響著患者的預(yù)后。NEDD9是一種與實(shí)體腫瘤轉(zhuǎn)移密切相關(guān)的細(xì)胞內(nèi)支架蛋白。最近研究發(fā)現(xiàn)NEDD9在肝細(xì)胞肝癌標(biāo)本中表達(dá)上調(diào),但NEDD9在肝細(xì)胞肝癌中的具體作用仍未闡明。因而我們通過體內(nèi)外實(shí)驗(yàn),探索NEDD9在肝細(xì)胞肝癌侵襲轉(zhuǎn)移方面的具體作用以及可能的潛在機(jī)制。方法在肝癌細(xì)胞株以及140對肝細(xì)胞肝癌標(biāo)本中,采用qPCR、Western blot以及免疫組化等多種方式檢測NEDD9的表達(dá)情況,并結(jié)合患者臨床病理資料分析NEDD9與患者臨床特征以及預(yù)后的相關(guān)性。分組分析非侵襲性肝癌以及侵襲性肝癌中NEDD9的表達(dá)差異。構(gòu)建NEDD9敲減以及過表達(dá)細(xì)胞株,通過MTS細(xì)胞增殖實(shí)驗(yàn)Transwell細(xì)胞遷移及侵襲實(shí)驗(yàn)以及裸鼠體內(nèi)脾臟注射-肝轉(zhuǎn)移模型,觀察NEDD9對腫瘤增殖、侵襲、轉(zhuǎn)移能力的影響。運(yùn)用qPCR、Western blot、免疫熒光、免疫組化等多種方法初步探究NEDD9在促進(jìn)肝細(xì)胞肝癌侵襲轉(zhuǎn)移方面的潛在機(jī)制。結(jié)果qPCR以及Western blot檢測發(fā)現(xiàn)NEDD9在肝癌細(xì)胞株中的表達(dá)較正常肝細(xì)胞株中表達(dá)上調(diào)。140對肝細(xì)胞肝癌標(biāo)本通過免疫組化檢測發(fā)現(xiàn)NEDD9在肝細(xì)胞肝癌中表達(dá)升高,且與患者的血清AFP濃度(p=0.008)、腫瘤癌栓(p=0.005)和腫瘤TNM病理分期(p=0.014)有明顯相關(guān)性。根據(jù)肝癌患者是否合并腫瘤癌栓將患者分成非侵襲性肝癌組以及侵襲性肝癌組,發(fā)現(xiàn)兩組之間NEDD9表達(dá)存在差異。結(jié)合肝癌患者的隨訪數(shù)據(jù),采用Kaplan-Meier生存分析發(fā)現(xiàn)NEDD9高表達(dá)者較低表達(dá)者有更短的無瘤生存期和總生存期,COX多因素回歸分析提示NEDD9是肝癌術(shù)后復(fù)發(fā)的獨(dú)立危險(xiǎn)因素。成功構(gòu)建NEDD9過表達(dá)慢病毒質(zhì)粒,并在肝癌細(xì)胞株HCC-LM3和Huh7中成功構(gòu)建NEDD9敲減以及過表達(dá)細(xì)胞株。MTS細(xì)胞增殖實(shí)驗(yàn)提示NEDD9敲減能明顯抑制肝癌細(xì)胞的生長。Transwell細(xì)胞遷移及侵襲實(shí)驗(yàn)提示,NEDD9敲減能明顯抑制肝癌細(xì)胞的遷移及侵襲能力,NEDD9過表達(dá)能增強(qiáng)肝癌細(xì)胞株的遷移及侵襲能力。裸鼠體內(nèi)脾臟注射-肝轉(zhuǎn)移模型發(fā)現(xiàn),NEDD9過表達(dá)組裸鼠較對照組在肝臟表面形成更多的轉(zhuǎn)移結(jié)節(jié)。機(jī)制方面,我們發(fā)現(xiàn)在肝癌細(xì)胞以及肝癌組織中,NEDD9能反向調(diào)控E-cadherin的表達(dá),FAK參與了 NEDD9對E-cadherin表達(dá)的調(diào)控。結(jié)論NEDD9在肝癌細(xì)胞株及肝癌組織中均表達(dá)上調(diào)。NEDD9與肝癌臨床的侵襲轉(zhuǎn)移特性有一定的相關(guān)性。NEDD9能明顯增強(qiáng)肝癌細(xì)胞的侵襲及轉(zhuǎn)移能力。NEDD9能反向調(diào)控肝癌中E-cadherin的表達(dá),可能是NEDD9促進(jìn)肝癌侵襲轉(zhuǎn)移的潛在機(jī)制。目的肝細(xì)胞肝癌術(shù)后復(fù)發(fā)嚴(yán)重影響著肝癌患者的預(yù)后,腫瘤標(biāo)記物作為一種從分子生物學(xué)角度參與評估腫瘤患者預(yù)后的新方法也具有潛在的重要意義。DIXDC1是一種含有DIX結(jié)構(gòu)域的支架蛋白,作為Wnt信號通路的正向調(diào)控因子參與神經(jīng)系統(tǒng)的發(fā)育。盡管DIXDC1已經(jīng)在某些惡性腫瘤中進(jìn)行了研究,但是目前在肝細(xì)胞肝癌中尚無任何的相關(guān)報(bào)道,因而我們本次研究目的主要是探究DIXDC1在肝癌中的表達(dá)及其臨床意義。方法通過生物信息學(xué)的方法從Oncomine數(shù)據(jù)庫中檢索關(guān)于DIXDC1在肝細(xì)胞肝癌中表達(dá)情況的數(shù)據(jù)。采用qPCR、Western blot等方法檢測25對新鮮肝細(xì)胞肝癌標(biāo)本中DIXDC1的表達(dá)情況。進(jìn)一步在140對肝細(xì)胞肝癌標(biāo)本中,通過免疫組化的方法評估DIXDC1的表達(dá),并結(jié)合患者臨床病理資料分析DIXDC1與患者臨床病理特征以及預(yù)后的相關(guān)性。結(jié)果Oncomine數(shù)據(jù)庫中三個獨(dú)立的檢測數(shù)據(jù)提示,肝細(xì)胞肝癌中DIXDC1的mRNA表達(dá)水平較正常肝組織表達(dá)下調(diào)。在25對新鮮肝細(xì)胞肝癌組織中,通過qPCR以及Western blot檢測發(fā)現(xiàn)DIXDC1在大部分肝癌組織中表達(dá)下降。在140對肝細(xì)胞肝癌標(biāo)本中,通過免疫組化評估發(fā)現(xiàn)DIXDC1在肝細(xì)胞肝癌中低表達(dá),且與腫瘤大小(p=0.024)、腫瘤細(xì)胞分化程度(p0.001)、腫瘤癌栓(p=0.019)、腫瘤TNM病理分期(p=0.019)及肝癌巴塞羅那臨床分期(p=0.008)等患者疾病進(jìn)展相關(guān)指標(biāo)有密切關(guān)系。更重要的是,Kaplan-Meier生存分析發(fā)現(xiàn)DIXDC1低表達(dá)患者較高表達(dá)者有更短的無瘤生存期和總生存期,COX多因素回歸分析提示DIXDC1是肝癌患者總生存期的獨(dú)立預(yù)后因子。結(jié)論我們首次揭示DIXDC1在肝細(xì)胞肝癌中表達(dá)下調(diào)。低表達(dá)DIXDC1與患者的臨床疾病進(jìn)展?fàn)顩r以及較差的預(yù)后有明顯相關(guān)性。DIXDC1可作為一個新的腫瘤標(biāo)記物來預(yù)測肝細(xì)胞肝癌患者術(shù)后的臨床轉(zhuǎn)歸情況。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Ali Raza;Gagan K Sood;;Hepatocellular carcinoma review:Current treatment,and evidence-based medicine[J];World Journal of Gastroenterology;2014年15期

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本文編號：1619689