本文選題：Nrf1α　切入點：肝細胞癌　出處：《重慶大學》2016年博士論文　論文類型：學位論文

【摘要】：肝細胞癌(Hepatocellular carcinoma, HCC)作為全球高發(fā)惡性腫瘤之一,嚴重威脅人類健康,而我國更是肝細胞癌高發(fā)國家。跨膜轉(zhuǎn)錄因子Nrf1 (nuclear factor-erythroid 2 related factor 1)屬于堿性亮氨酸拉鏈CNC-bZIP (cap'n'collar basic-region leucine zipper)亞家族成員,通過調(diào)控抗氧化基因、蛋白酶體基因的轉(zhuǎn)錄,保護細胞免受氧化應激影響,維持機體內(nèi)平衡。而Nrfl肝臟特異敲除的小鼠誘發(fā)原發(fā)性肝細胞癌,為了更深入揭示Nrfl與肝細胞癌的發(fā)病機制,我們選擇人肝癌細胞HepG2作為研究對象,觀察Nrfl a (Nrfl全長亞型)對肝癌細胞HepG2惡性行為的影響。而且Nrf1a是否發(fā)揮腫瘤抑制因子的作用也未見報道。因此我們將研究聚焦于此,探討Nrf1α在肝細胞癌發(fā)生發(fā)展中的生物學功能及其機制,為Nrfl缺失誘發(fā)肝細胞癌的發(fā)病機理、臨床病理及預后判斷提供新的思路。研究方法與結(jié)果：① TALEN (transcription activator-like effector nuclease)介導同源雙敲除Nrfl a肝癌細胞株的建立通過TaqMan探針和DNA測序技術分別檢測Nrf1的兩種全長亞型Nrf1α和TCF11,發(fā)現(xiàn)TCF11亞型在不同肝癌細胞中明顯低表達,而正常肝細胞Nrf1α和TCF11表達豐度接近一致。因此,我們選擇低表達TCF11的HepG2細胞作為研究對象。接下來構建TALEN介導敲除Nrf1α的HepG2細胞株,根據(jù)TALEN敲基因原理,我們分別設計TALEN左臂不PTALEN右臂,經(jīng)過質(zhì)粒共轉(zhuǎn)染細胞,細胞測序驗證,單克隆篩選,成功獲得同源雙敲除Nrf1α-/-的HepG2細胞株,命名為HEA157(Nrf1α-/-)。為了對照驗證,我們設計了沉默Nrf1的慢病毒載體,篩選出沉默Nrf1最有效的shRNA靶序列,建立穩(wěn)定敲減Nrfl細胞株,命名為HepG2-shNrf1。最后,我們設計了Nrf1 a超表達慢病毒載體,轉(zhuǎn)染HEA157(Nrf1α-/-)細胞,獲得穩(wěn)定異源恢復表達Nrf1α的細胞株,命名為HEA157Nrf1α。② TALEN介導敲除Nrf1α對肝癌細胞生物學行為的影響MTS、細胞克隆形成實驗檢測細胞增殖,結(jié)果均提示在Hep(G2細胞中敲除Nrf1α可促進細胞增殖。流式細胞儀測定細胞凋亡,結(jié)果顯示,HEA157(Nrf1α-/-)細胞晚期凋亡率明顯降低,但早期凋亡并沒有明顯變化。細胞劃痕及Transwell侵襲遷移實驗觀察細胞運動能力,實驗結(jié)果顯示,敲除Nrf1α后肝癌細胞遷移及侵襲能力均顯著增強(p0.05)。綜合實驗結(jié)果分析,Nrf1α能夠?qū)Ω伟┘毎飳W行為進行調(diào)節(jié),通過調(diào)控肝癌細胞增殖和EMT發(fā)生增強肝癌細胞遷移侵襲能力。促進肝癌細胞體外惡性轉(zhuǎn)化。③敲除Nrf1α調(diào)控肝癌細胞遷移侵襲的機制探究檢測4HEA157(Nrfla-/-)細胞中與腫瘤轉(zhuǎn)移相關基因的表達,發(fā)現(xiàn)MMP-9、 MMP-17表達顯著升高,上皮細胞標志物CDH1表達顯著下調(diào)(p0.01)。間質(zhì)細胞標志物CDH2表達顯著上調(diào)(p0.01)。Western Blot分析CDH1、CDH2、SNAI1、 SNAI2、Vimentin的表達也證實敲除Nrf1α促進肝癌細胞發(fā)生上皮間質(zhì)轉(zhuǎn)化。通過建立裸鼠皮下移植瘤模型,發(fā)現(xiàn)HEA157(Nrf1α-/-)細胞形成的移植瘤生長速度更快,在注射細胞后的20-35天,移植瘤呈現(xiàn)指數(shù)生長。且最終形成的移植瘤體積更大,并伴隨潰爛出血。同時發(fā)現(xiàn)HEA157(Nrf1α-/-)組小鼠肝臟出現(xiàn)大量轉(zhuǎn)移癌結(jié)節(jié)。免疫組化檢測模型鼠移植瘤Nrf1、CDH1、CDH2的表達,發(fā)現(xiàn)HEA157(Nrf1α-/-)實驗組Nrf1表達減少,CDH1表達下調(diào),CDH2表達上調(diào)。Real-time PCR檢測結(jié)果與免疫組織化學結(jié)果一致。實時定量PCR實驗,Western Blot及免疫組織化學方法檢測發(fā)現(xiàn)Nrfl在HCC組織中的表達與病理嚴重程度呈現(xiàn)相關性,高分化HCC組織Nrfl高表達,低分化HCC組織Nrf1低表達,且癌旁組織表達豐度高于癌組織。通過高通量表達譜測序及信號通路聚類分析,發(fā)現(xiàn)敲除Nrfα激活HepG2細胞PTEN-PI3K/AK T信號通路。進一步實驗驗證敲除Nrf1α, PTEN表達下調(diào),PIK3CA、 PIK3CB表達上調(diào),AKT2總蛋白表達并未發(fā)生明顯變化,但p-AKT2T308和p-AKT2S473磷酸化水平加強。啟動子分析和雙熒光素酶檢測結(jié)果提示Nrf1α與PTEN、 CDH1存在直接作用關系。因此,我們認為下調(diào)Nrfα表達激活PTEN-PI3K/AK T信號通路,促進AKT的活化并促進PI3K/AKT下游靶基因表達。結(jié)論：①利用TALEN基因定點編輯技術,成功構建敲除Nrf1α亞型的HepG2肝癌細胞株HEA157(Nrf1α-/-)。②Nrf1α缺失促進肝癌細胞EMT發(fā)生,并導致裸鼠皮下移植瘤肝臟轉(zhuǎn)移,促進肝癌細胞惡性遷移侵襲能力顯著增強。③Nrf1在HCC組織中的表達與病理嚴重程度呈現(xiàn)相關性,高分化HCC組織Nrfl高表達,低分化HCC組織Nrf1低表達,且癌旁組織表達豐度高于癌組織。④下調(diào)Nrfα表達激活PTEN-PI3K/AKT信號通路,促進AKT的活化并促進PI3K/AKT下游靶基因表達。
[Abstract]:Hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) as one of the world's most common malignant tumor, a serious threat to human health, and our country is a high incidence of hepatocellular carcinoma. The transmembrane transcription factor Nrf1 (nuclear factor-erythroid 2 related factor 1) belongs to the basic leucine zipper (cap'n'collar CNC-bZIP basic-region leucine zipper) subfamily, through the regulation of antioxidant gene proteasome gene transcription, and protect cells from oxidative stress, maintain body balance. Nrfl liver specific knockout mice induced in primary hepatocellular carcinoma, in order to further reveal the pathogenesis of Nrfl and hepatocellular carcinoma, we selected human liver cancer cell HepG2 as the research object, observe the Nrfl a (full length Nrfl subtype) effect on hepatocellular carcinoma cells HepG2. The malignant behavior of tumor suppressor and whether Nrf1a play a role has not been reported. So we will research The focus in this study, Nrf1 alpha biological function and its mechanism in the development of hepatocellular carcinoma. The pathogenesis of Nrfl deletion induced hepatocellular carcinoma, clinical pathology and prognosis to provide new ideas and research methods. Results: TALEN (transcription activator-like effector nuclease) mediated by homologous double knockout Nrfl a hepatocellular carcinoma cell line Nrf1 were detected by TaqMan probe and DNA sequencing of two full-length isoforms of Nrf1 alpha and TCF11, found that TCF11 isoforms in different hepatoma cells was significantly lower expression, and normal liver cells Nrf1 alpha and TCF11 expression abundance in close agreement. Therefore, we choose the low expression of TCF11 HepG2 cells as the research the next object. Construction of TALEN mediated knock HepG2 cell lines except Nrf1 alpha, according to the TALEN gene knockout principle, we design the TALEN left arm right arm of PTALEN respectively, after plasmid co transfection, cell test In order to verify, monoclonal screening, successful homologous double knockout Nrf1 alpha - HepG2 cell line, named HEA157 (Nrf1 alpha -). In order to verify the control, we design a lentiviral vector of Nrf1 silencing, selected shRNA target sequence the most effective silencing of Nrf1, to establish a stable Nrfl knockdown cell lines, named HepG2-shNrf1. finally, we design a Nrf1 a lentivirus expression vector, transfected HEA157 (Nrf1 alpha -) cells, stable heterologous expression of Nrf1 restored cell line, named HEA157Nrf1. The alpha TALEN mediated knockdown effect of Nrf1 alpha on hepatocellular carcinoma cell biology for MTS cell clone formation assay were used to detect proliferation the results showed that, in Hep cells (G2 knockout Nrf1 alpha can promote cell proliferation. Apoptosis was determined by flow cytometry. The results showed that HEA157 (Nrf1 alpha -) late apoptotic cells were significantly decreased, but the early apoptosis cells row did not change significantly. Mark and Transwell invasion and migration experimental observation of cell movement ability, the experimental results show that knockdown of hepatoma cell migration and invasion of Nrf1 alpha were significantly increased (P0.05). A comprehensive analysis of the experimental results, Nrf1 was able to regulate the biological behavior of hepatocellular carcinoma cells, through the regulation of liver cancer cell proliferation and EMT enhanced the migration and invasion of hepatocellular carcinoma cells promote the malignant transformation of hepatocellular carcinoma cells in vitro. The Nrf1 alpha knockout mechanism. The regulation of liver cancer cell migration and invasion of detection of 4HEA157 (Nrfla-/-) expression in cells and tumor metastasis related genes MMP-9, MMP-17 expression was significantly increased, the epithelial cell marker CDH1 expression was significantly reduced (P0.01). The mesenchymal cell marker CDH2 expression significantly up regulation of.Western Blot CDH1 (P0.01) analysis, CDH2, SNAI1, SNAI2, Vimentin expression also confirmed that knockdown of Nrf1 alpha promotes HCC cells epithelial mesenchymal transition through the establishment. The model of nude mice, found that HEA157 (Nrf1 alpha -) cells to form tumor growth faster, in the 20-35 days after the injection of tumor cells, tumor showed exponential growth. The tumor volume and the final form of the larger, and with ulceration bleeding. At the same time that HEA157 (Nrf1 alpha - / -) mice liver a large number of nodules of metastatic carcinoma. Immunohistochemical detection of rat model of transplanted tumor of Nrf1, CDH1, CDH2 expression, HEA157 (Nrf1 alpha -) decreased Nrf1 expression in the experimental group, the expression of CDH1 and CDH2 expression of.Real-time PCR detection results and immunohistochemical results. Real time quantitative PCR experiments, Western Blot and immunohistochemical detection chemical analysis showed that the Nrfl in the HCC tissues and pathological severity were associated with high expression of high differentiation, HCC Nrfl, HCC Nrf1 low expression in low differentiation tissues, paracancerous tissues and cancer tissues. The expression abundance was higher than that of high throughput Expression of signal pathway of sequencing and clustering analysis, found that knockdown of Nrf alpha activation of HepG2 cells PTEN-PI3K/AK T signal pathway. Further experimental verification of knockdown of Nrf1 alpha, PTEN expression, PIK3CA expression, PIK3CB, total AKT2 protein expression did not change significantly, but the p-AKT2T308 and p-AKT2S473 phosphorylation enhanced promoter analysis and dual luciferase. Test results shows that the Nrf1 alpha and PTEN, there is a direct effect between CDH1. Therefore, we believe that the down-regulation of Nrf alpha expression and activation of PTEN-PI3K/AK T signaling pathway to promote AKT activation and promote PI3K/AKT downstream target gene expression. Conclusion: the TALEN gene editing technology, the successful construction of knockout Nrf1 isoform HepG2 in hepatocellular carcinoma cell lines HEA157 (Nrf1 alpha -). The Nrf1 alpha deletion to promote cancer cell EMT, and lead to liver metastasis of nude mice, promote cancer cell migration and invasion were malignant The Nrf1 in HCC increased. The expression and pathological severity were associated with high expression of high differentiation, HCC Nrfl, HCC Nrf1 low expression in low differentiation tissues, paracancerous tissues and cancer tissues. The expression abundance was higher than that of the down-regulation of Nrf alpha expression and activation of PTEN-PI3K/AKT signaling pathway, promoting the activation of AKT and promote the expression of downstream target genes of PI3K/AKT.

【學位授予單位】：重慶大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.7
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本文編號：1634785