转录因子Nrf1α管控肝癌细胞恶性行为及其分子机制研究

发布时间：2018-03-19 15:03

  本文选题：Nrf1α　切入点：肝细胞癌　出处：《重庆大学》2016年博士论文　论文类型：学位论文

【摘要】：肝细胞癌(Hepatocellular carcinoma, HCC)作为全球高发恶性肿瘤之一,严重威胁人类健康,而我国更是肝细胞癌高发国家。跨膜转录因子Nrf1 (nuclear factor-erythroid 2 related factor 1)属于碱性亮氨酸拉链CNC-bZIP (cap'n'collar basic-region leucine zipper)亚家族成员,通过调控抗氧化基因、蛋白酶体基因的转录,保护细胞免受氧化应激影响,维持机体内平衡。而Nrfl肝脏特异敲除的小鼠诱发原发性肝细胞癌,为了更深入揭示Nrfl与肝细胞癌的发病机制,我们选择人肝癌细胞HepG2作为研究对象,观察Nrfl a (Nrfl全长亚型)对肝癌细胞HepG2恶性行为的影响。而且Nrf1a是否发挥肿瘤抑制因子的作用也未见报道。因此我们将研究聚焦于此,探讨Nrf1α在肝细胞癌发生发展中的生物学功能及其机制,为Nrfl缺失诱发肝细胞癌的发病机理、临床病理及预后判断提供新的思路。研究方法与结果：① TALEN (transcription activator-like effector nuclease)介导同源双敲除Nrfl a肝癌细胞株的建立通过TaqMan探针和DNA测序技术分别检测Nrf1的两种全长亚型Nrf1α和TCF11,发现TCF11亚型在不同肝癌细胞中明显低表达,而正常肝细胞Nrf1α和TCF11表达丰度接近一致。因此,我们选择低表达TCF11的HepG2细胞作为研究对象。接下来构建TALEN介导敲除Nrf1α的HepG2细胞株,根据TALEN敲基因原理,我们分别设计TALEN左臂不PTALEN右臂,经过质粒共转染细胞,细胞测序验证,单克隆筛选,成功获得同源双敲除Nrf1α-/-的HepG2细胞株,命名为HEA157(Nrf1α-/-)。为了对照验证,我们设计了沉默Nrf1的慢病毒载体,筛选出沉默Nrf1最有效的shRNA靶序列,建立稳定敲减Nrfl细胞株,命名为HepG2-shNrf1。最后,我们设计了Nrf1 a超表达慢病毒载体,转染HEA157(Nrf1α-/-)细胞,获得稳定异源恢复表达Nrf1α的细胞株,命名为HEA157Nrf1α。② TALEN介导敲除Nrf1α对肝癌细胞生物学行为的影响MTS、细胞克隆形成实验检测细胞增殖,结果均提示在Hep(G2细胞中敲除Nrf1α可促进细胞增殖。流式细胞仪测定细胞凋亡,结果显示,HEA157(Nrf1α-/-)细胞晚期凋亡率明显降低,但早期凋亡并没有明显变化。细胞划痕及Transwell侵袭迁移实验观察细胞运动能力,实验结果显示,敲除Nrf1α后肝癌细胞迁移及侵袭能力均显著增强(p0.05)。综合实验结果分析,Nrf1α能够对肝癌细胞生物学行为进行调节,通过调控肝癌细胞增殖和EMT发生增强肝癌细胞迁移侵袭能力。促进肝癌细胞体外恶性转化。③敲除Nrf1α调控肝癌细胞迁移侵袭的机制探究检测4HEA157(Nrfla-/-)细胞中与肿瘤转移相关基因的表达,发现MMP-9、 MMP-17表达显著升高,上皮细胞标志物CDH1表达显著下调(p0.01)。间质细胞标志物CDH2表达显著上调(p0.01)。Western Blot分析CDH1、CDH2、SNAI1、 SNAI2、Vimentin的表达也证实敲除Nrf1α促进肝癌细胞发生上皮间质转化。通过建立裸鼠皮下移植瘤模型,发现HEA157(Nrf1α-/-)细胞形成的移植瘤生长速度更快,在注射细胞后的20-35天,移植瘤呈现指数生长。且最终形成的移植瘤体积更大,并伴随溃烂出血。同时发现HEA157(Nrf1α-/-)组小鼠肝脏出现大量转移癌结节。免疫组化检测模型鼠移植瘤Nrf1、CDH1、CDH2的表达,发现HEA157(Nrf1α-/-)实验组Nrf1表达减少,CDH1表达下调,CDH2表达上调。Real-time PCR检测结果与免疫组织化学结果一致。实时定量PCR实验,Western Blot及免疫组织化学方法检测发现Nrfl在HCC组织中的表达与病理严重程度呈现相关性,高分化HCC组织Nrfl高表达,低分化HCC组织Nrf1低表达,且癌旁组织表达丰度高于癌组织。通过高通量表达谱测序及信号通路聚类分析,发现敲除Nrfα激活HepG2细胞PTEN-PI3K/AK T信号通路。进一步实验验证敲除Nrf1α, PTEN表达下调,PIK3CA、 PIK3CB表达上调,AKT2总蛋白表达并未发生明显变化,但p-AKT2T308和p-AKT2S473磷酸化水平加强。启动子分析和双荧光素酶检测结果提示Nrf1α与PTEN、 CDH1存在直接作用关系。因此,我们认为下调Nrfα表达激活PTEN-PI3K/AK T信号通路,促进AKT的活化并促进PI3K/AKT下游靶基因表达。结论：①利用TALEN基因定点编辑技术,成功构建敲除Nrf1α亚型的HepG2肝癌细胞株HEA157(Nrf1α-/-)。②Nrf1α缺失促进肝癌细胞EMT发生,并导致裸鼠皮下移植瘤肝脏转移,促进肝癌细胞恶性迁移侵袭能力显著增强。③Nrf1在HCC组织中的表达与病理严重程度呈现相关性,高分化HCC组织Nrfl高表达,低分化HCC组织Nrf1低表达,且癌旁组织表达丰度高于癌组织。④下调Nrfα表达激活PTEN-PI3K/AKT信号通路,促进AKT的活化并促进PI3K/AKT下游靶基因表达。
[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.

【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
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本文编号：1634785