Caveolin-1基因敲除对化学诱导肝癌小鼠体内核心岩藻糖基化的影响
发布时间：2019-01-29 20:46
【摘要】：背景:窖蛋白-1(Caveolin-1,Cav-1)是胞膜窖的主要结构和功能成分,参与调控胆固醇转运、细胞内吞、信号转导以及肿瘤转移等重要生物学过程。Caveolin-1在癌症发生发展过程中的作用具有组织特异性,发挥抑癌作用还是促癌作用也一直存在争论。原发性肝癌,简称肝癌(liver cancer),是我国最常见的恶性肿瘤之一,在全球其致死率高居癌症中第三位。有文献报道,Cav-1能够促进肝癌细胞的侵袭和转移,并与其恶性程度有关。核心岩藻糖基化是重要的蛋白质翻译后修饰形式之一。核心岩藻糖基化主要由核心岩藻糖基转移酶8(α1,6-fucosyltransferase,FUT8/Fut8)催化完成,即岩藻糖通过α1-6糖苷键与N-聚糖核心的N-乙酰葡糖胺(GlcNAc)相连。肝癌等恶性肿瘤发生发展过程中,核心岩藻糖基化异常高表达,但其发生和作用机制不明。经典Wnt信号通路在细胞的分化、增值、凋亡以及细胞的癌变肿瘤侵袭等病理过程中起了重要的调控作用,其核心因子β-catenin与TCF/LEF结合促进下游靶基因转录。生物信息预测表明,核心岩藻糖基转移酶(Fut8)启动子上游存在TCF/LEF结合序列,这提示:核心岩藻糖基化水平可能通过Wnt/β-catenin信号通路调控,Fut8基因可能是Wnt/β-catenin信号通路的靶基因。本实验室前期研究结果和文献报道显示,在肝癌细胞中,Cav-1过表达参与Wnt/β-catenin信号通路活化,增加细胞核内β-catenin的富集,上调Fut8基因的转录活性,促进核心岩藻糖基化水平升高。但在整体水平上Cav-1的表达及Wnt/β-catenin信号通路,对核心岩藻糖基转移酶Fut8表达、核心岩藻糖基化水平影响及机制研究,尚未见报道。目的:在整体水平上,探讨Cav-1基因敲除对小鼠体内肝癌组织及血清核心岩藻糖基转移酶Fut8基因表达和核心岩藻糖基化水平的影响及其作用机制。方法:以Cav-1基因敲除(KO)以及野生(WT)型C57BL/6J雄性小鼠为研究对象,注射化学试剂二乙基亚硝胺联合灌喂四氯化碳/乙醇至24周;组织病理分析确认诱导小鼠肝癌模型建立;通过Western-blot、Real Time PCR、Lectin-blot以及MALDI-TOF/TOF质谱等方法,分析比较各时期小鼠肝/肝癌组织和血清Fut8等基因和蛋白表达及核心岩藻糖基化水平。结果:1)在化学诱导0周时,与WT小鼠相比,KO小鼠肝脏组织中Fut8表达、血清中核心岩藻糖基化水平均显著下降。2)在化学诱导24周时,与0周时正常相比,WT小鼠和KO小鼠的肝癌组织中β-catenin和Fut8表达、血清中核心岩藻糖基化水平均显著升高;但KO小鼠的显著降低WT小鼠的水平。3)在化学诱导0-24周期间,WT小鼠的Cav-1、β-catenin和N-cadherin表达逐渐显著上升、E-cadherin表达逐渐显著下降;但这些蛋白在KO小鼠中的水平显著低于在WT小鼠中的水平。这暗示Cav-1可能影响上皮-间充质细胞转化(Epithelial Mesenchymal Transition,EMT)的发生,促进与E-cadherin结合的β-catenin积累。结论:在化学诱导小鼠肝癌发生过程中,敲除Cav-1基因降低小鼠Fut8的表达及血清中核心岩藻糖基化水平,其作用机制可能与影响EMT及Wnt/β-catenin通路有关。本研究提供了体内证据支持以上结论。
[Abstract]:BACKGROUND: The cellar protein-1 (Cav-1) is the main structure and functional component of the cell membrane cellar, and is involved in the regulation of the important biological processes such as cholesterol transport, endocytosis, signal transduction and tumor metastasis. The role of Caveolin-1 in the course of the development of cancer is of tissue-specific, the role of cancer-inhibiting, and the role of promoting cancer has been debated. Primary liver cancer, referred to as liver cancer, is one of the most common malignant tumors in our country, and is the third of the most common cancer in the world. It is reported that Cav-1 can promote the invasion and metastasis of liver cancer cells and is related to the degree of malignancy. Glycosylation of core rock is one of the most important post-translational modifications. The core-rock-algae glycosylation is mainly catalyzed by the core-rock-alginate-transferase 8 (FUT8/ Fut8), that is, the rock-algae sugar is linked to the N-glycosaminoglycan (GlcNAc) of the N-glycan core through the 1-6 sugar-binding key. In the development of malignant tumor such as liver cancer, the abnormal glycosylation of the core rock is highly expressed, but the mechanism of its occurrence and action is unknown. The classical Wnt signaling pathway plays an important role in the process of cell differentiation, value-added, apoptosis, and the invasion and other pathological processes of the cells, and the core factor of the classical Wnt signaling pathway is to promote the transcription of the downstream target gene in combination with the TCF/ LEF. The prediction of biological information indicates that there is a TCF/ LEF binding sequence in the upstream of the core-rock-alginate-transferase (Fut8) promoter, which suggests that the level of glycosylation of the core-rock-algae may be regulated by the Wnt/ HCO3-catenin signal pathway, and the Fut8 gene may be the target gene of the Wnt/ HCO3-catenin signal pathway. In the early stage of this lab, the results of the earlier study and the literature report show that, in the liver cancer cell, the overexpression of Cav-1 is involved in the activation of the Wnt/ P-cattenin signal pathway, increasing the enrichment of the intracellin-cattenin, and increasing the transcription activity of the Fut8 gene and promoting the increase of the glycosylation level of the core rock. But at the whole level, the expression of Cav-1 and the Wnt/ I-cattenin signal pathway, the expression of the core-rock-alginate-glycosyltransferase (Fut8), the influence on the level of the sylation of the core-rock and the mechanism, have not been reported. Objective: To study the effect of Cav-1 gene knockout on the expression of the Fut8 gene and the level of the glycosylation of the core rock and the mechanism of its action on the whole level. Methods: Cav-1 gene knockout (KO) and wild (WT) C57BL/ 6J male mice were used as the research object, and the chemical reagent was injected with the chemical reagent, diethylnitrosamine combined with carbon tetrachloride/ ethanol to 24 weeks, and the pathological analysis was performed to confirm the establishment of the mouse liver cancer model; by Western-blot and Real Time PCR, Lectin-blot and MALDI-TOF/ TOF mass spectrometry (MALDI-TOF/ TOF) mass spectrometry (MALDI-TOF/ TOF) mass spectrometry (MALDI-TOF/ TOF) were used to analyze the expression of gene and protein of liver/ liver cancer tissue and serum Fut8 and the glycosylation of core rock. Results: 1) In the 0-week chemical induction, the expression of Fut8 in the liver of KO mice and the level of the glycosylation of the core rock in the serum of KO mice decreased significantly. The levels of Cav-1, P-catenin and N-cadherin in WT mice were significantly increased during the 0-24 weeks of chemical induction. The expression of E-cadherin decreased significantly; however, the levels of these proteins in KO mice were significantly lower than in WT mice. 杩欐殫绀篊av-1鍙兘褰卞搷涓婄毊-闂村厖璐ㄧ粏鑳炶浆鍖，
本文编号：2417848
[Abstract]:BACKGROUND: The cellar protein-1 (Cav-1) is the main structure and functional component of the cell membrane cellar, and is involved in the regulation of the important biological processes such as cholesterol transport, endocytosis, signal transduction and tumor metastasis. The role of Caveolin-1 in the course of the development of cancer is of tissue-specific, the role of cancer-inhibiting, and the role of promoting cancer has been debated. Primary liver cancer, referred to as liver cancer, is one of the most common malignant tumors in our country, and is the third of the most common cancer in the world. It is reported that Cav-1 can promote the invasion and metastasis of liver cancer cells and is related to the degree of malignancy. Glycosylation of core rock is one of the most important post-translational modifications. The core-rock-algae glycosylation is mainly catalyzed by the core-rock-alginate-transferase 8 (FUT8/ Fut8), that is, the rock-algae sugar is linked to the N-glycosaminoglycan (GlcNAc) of the N-glycan core through the 1-6 sugar-binding key. In the development of malignant tumor such as liver cancer, the abnormal glycosylation of the core rock is highly expressed, but the mechanism of its occurrence and action is unknown. The classical Wnt signaling pathway plays an important role in the process of cell differentiation, value-added, apoptosis, and the invasion and other pathological processes of the cells, and the core factor of the classical Wnt signaling pathway is to promote the transcription of the downstream target gene in combination with the TCF/ LEF. The prediction of biological information indicates that there is a TCF/ LEF binding sequence in the upstream of the core-rock-alginate-transferase (Fut8) promoter, which suggests that the level of glycosylation of the core-rock-algae may be regulated by the Wnt/ HCO3-catenin signal pathway, and the Fut8 gene may be the target gene of the Wnt/ HCO3-catenin signal pathway. In the early stage of this lab, the results of the earlier study and the literature report show that, in the liver cancer cell, the overexpression of Cav-1 is involved in the activation of the Wnt/ P-cattenin signal pathway, increasing the enrichment of the intracellin-cattenin, and increasing the transcription activity of the Fut8 gene and promoting the increase of the glycosylation level of the core rock. But at the whole level, the expression of Cav-1 and the Wnt/ I-cattenin signal pathway, the expression of the core-rock-alginate-glycosyltransferase (Fut8), the influence on the level of the sylation of the core-rock and the mechanism, have not been reported. Objective: To study the effect of Cav-1 gene knockout on the expression of the Fut8 gene and the level of the glycosylation of the core rock and the mechanism of its action on the whole level. Methods: Cav-1 gene knockout (KO) and wild (WT) C57BL/ 6J male mice were used as the research object, and the chemical reagent was injected with the chemical reagent, diethylnitrosamine combined with carbon tetrachloride/ ethanol to 24 weeks, and the pathological analysis was performed to confirm the establishment of the mouse liver cancer model; by Western-blot and Real Time PCR, Lectin-blot and MALDI-TOF/ TOF mass spectrometry (MALDI-TOF/ TOF) mass spectrometry (MALDI-TOF/ TOF) mass spectrometry (MALDI-TOF/ TOF) were used to analyze the expression of gene and protein of liver/ liver cancer tissue and serum Fut8 and the glycosylation of core rock. Results: 1) In the 0-week chemical induction, the expression of Fut8 in the liver of KO mice and the level of the glycosylation of the core rock in the serum of KO mice decreased significantly. The levels of Cav-1, P-catenin and N-cadherin in WT mice were significantly increased during the 0-24 weeks of chemical induction. The expression of E-cadherin decreased significantly; however, the levels of these proteins in KO mice were significantly lower than in WT mice. 杩欐殫绀篊av-1鍙兘褰卞搷涓婄毊-闂村厖璐ㄧ粏鑳炶浆鍖，
本文编号：2417848
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