Caveolin-1基因敲除對化學(xué)誘導(dǎo)肝癌小鼠體內(nèi)核心巖藻糖基化的影響
發(fā)布時(shí)間：2019-01-29 20:46
【摘要】：背景:窖蛋白-1(Caveolin-1,Cav-1)是胞膜窖的主要結(jié)構(gòu)和功能成分,參與調(diào)控膽固醇轉(zhuǎn)運(yùn)、細(xì)胞內(nèi)吞、信號轉(zhuǎn)導(dǎo)以及腫瘤轉(zhuǎn)移等重要生物學(xué)過程。Caveolin-1在癌癥發(fā)生發(fā)展過程中的作用具有組織特異性,發(fā)揮抑癌作用還是促癌作用也一直存在爭論。原發(fā)性肝癌,簡稱肝癌(liver cancer),是我國最常見的惡性腫瘤之一,在全球其致死率高居癌癥中第三位。有文獻(xiàn)報(bào)道,Cav-1能夠促進(jìn)肝癌細(xì)胞的侵襲和轉(zhuǎn)移,并與其惡性程度有關(guān)。核心巖藻糖基化是重要的蛋白質(zhì)翻譯后修飾形式之一。核心巖藻糖基化主要由核心巖藻糖基轉(zhuǎn)移酶8(α1,6-fucosyltransferase,FUT8/Fut8)催化完成,即巖藻糖通過α1-6糖苷鍵與N-聚糖核心的N-乙酰葡糖胺(GlcNAc)相連。肝癌等惡性腫瘤發(fā)生發(fā)展過程中,核心巖藻糖基化異常高表達(dá),但其發(fā)生和作用機(jī)制不明。經(jīng)典Wnt信號通路在細(xì)胞的分化、增值、凋亡以及細(xì)胞的癌變腫瘤侵襲等病理過程中起了重要的調(diào)控作用,其核心因子β-catenin與TCF/LEF結(jié)合促進(jìn)下游靶基因轉(zhuǎn)錄。生物信息預(yù)測表明,核心巖藻糖基轉(zhuǎn)移酶(Fut8)啟動子上游存在TCF/LEF結(jié)合序列,這提示:核心巖藻糖基化水平可能通過Wnt/β-catenin信號通路調(diào)控,Fut8基因可能是Wnt/β-catenin信號通路的靶基因。本實(shí)驗(yàn)室前期研究結(jié)果和文獻(xiàn)報(bào)道顯示,在肝癌細(xì)胞中,Cav-1過表達(dá)參與Wnt/β-catenin信號通路活化,增加細(xì)胞核內(nèi)β-catenin的富集,上調(diào)Fut8基因的轉(zhuǎn)錄活性,促進(jìn)核心巖藻糖基化水平升高。但在整體水平上Cav-1的表達(dá)及Wnt/β-catenin信號通路,對核心巖藻糖基轉(zhuǎn)移酶Fut8表達(dá)、核心巖藻糖基化水平影響及機(jī)制研究,尚未見報(bào)道。目的:在整體水平上,探討Cav-1基因敲除對小鼠體內(nèi)肝癌組織及血清核心巖藻糖基轉(zhuǎn)移酶Fut8基因表達(dá)和核心巖藻糖基化水平的影響及其作用機(jī)制。方法:以Cav-1基因敲除(KO)以及野生(WT)型C57BL/6J雄性小鼠為研究對象,注射化學(xué)試劑二乙基亞硝胺聯(lián)合灌喂四氯化碳/乙醇至24周;組織病理分析確認(rèn)誘導(dǎo)小鼠肝癌模型建立;通過Western-blot、Real Time PCR、Lectin-blot以及MALDI-TOF/TOF質(zhì)譜等方法,分析比較各時(shí)期小鼠肝/肝癌組織和血清Fut8等基因和蛋白表達(dá)及核心巖藻糖基化水平。結(jié)果:1)在化學(xué)誘導(dǎo)0周時(shí),與WT小鼠相比,KO小鼠肝臟組織中Fut8表達(dá)、血清中核心巖藻糖基化水平均顯著下降。2)在化學(xué)誘導(dǎo)24周時(shí),與0周時(shí)正常相比,WT小鼠和KO小鼠的肝癌組織中β-catenin和Fut8表達(dá)、血清中核心巖藻糖基化水平均顯著升高;但KO小鼠的顯著降低WT小鼠的水平。3)在化學(xué)誘導(dǎo)0-24周期間,WT小鼠的Cav-1、β-catenin和N-cadherin表達(dá)逐漸顯著上升、E-cadherin表達(dá)逐漸顯著下降;但這些蛋白在KO小鼠中的水平顯著低于在WT小鼠中的水平。這暗示Cav-1可能影響上皮-間充質(zhì)細(xì)胞轉(zhuǎn)化(Epithelial Mesenchymal Transition,EMT)的發(fā)生,促進(jìn)與E-cadherin結(jié)合的β-catenin積累。結(jié)論:在化學(xué)誘導(dǎo)小鼠肝癌發(fā)生過程中,敲除Cav-1基因降低小鼠Fut8的表達(dá)及血清中核心巖藻糖基化水平,其作用機(jī)制可能與影響EMT及Wnt/β-catenin通路有關(guān)。本研究提供了體內(nèi)證據(jù)支持以上結(jié)論。
[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. 榪欐殫紺篊av-1鍙兘褰卞搷涓婄毊-闂村厖璐ㄧ粏鑳?yōu)铦{鍖，
本文編號：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. 榪欐殫紺篊av-1鍙兘褰卞搷涓婄毊-闂村厖璐ㄧ粏鑳?yōu)铦{鍖，
本文編號：2417848
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