LPLUNC1抑制NF-κB和Stat3信号通路的激活抵抗促炎因子IL-6促进的鼻咽癌发生发展

发布时间：2018-06-13 20:31

本文选题：鼻咽癌 + LPLUNC1　；参考：《中南大学》2012年博士论文

【摘要】：[研究背景] 慢性炎症是肿瘤的第七大生物学特征,慢性炎症在触发肿瘤发生、促进肿瘤发展中起着非常重要的作用；炎症诱导的肿瘤称为炎症相关性肿瘤。鼻咽癌是一种具有明显地区差异的恶性肿瘤。鼻咽由于生理和解剖位置的特性,鼻咽上皮细胞时刻受到各种外源病原微生物如EBV和有毒化学物质等损害,使得鼻咽粘膜保护机制容易被破坏,极易导致鼻咽慢性炎症,因而有利于诱导鼻咽癌发生发展,因此鼻咽癌也是一种炎症相关性肿瘤。然而,鼻咽慢性炎症如何诱发鼻咽癌发生发展,尤其是在此过程中激活的免疫细胞如巨噬细胞、鼻咽上皮细胞充当何种角色,目前不是很清楚。LPLUNC1基因是我室克隆的鼻咽癌表达下调的基因。现有研究表明LPLUNC1基因编码的蛋白是一种分泌性蛋白,大量存在于鼻咽和呼吸道分泌物、灌洗液和痰液中；LPLUNC1蛋白可借助其BPI结构域行使宿主防御功能,具有杀菌抗炎的作用,而BPI结构域可以抑制肿瘤生长与转移。因此,我们对LPLUNC1基因在鼻咽慢性炎症促进鼻咽癌发生发展过程中的作用及其相关分子机制进行了探讨。 [炎症因子刺激鼻咽癌上皮细胞参与炎症反应] 为尽可能的获取炎症时各种促炎因子,我们用低浓度LPS(10ng/ml,近似于细菌脂多糖的生理浓度,如细菌感染时)刺激巨噬细胞,qRT-PCR检测发现低浓度LPS可明显上调促炎因子TNF-α、IL-6、IL-1β和IL-8基因表达,ELISA检测显示在巨噬细胞上清培养液中含有大量的促炎因子TNF-α、IL-6、IL-1β和IL-8。因此,为能更好的在体外模拟炎症环境,反映慢性炎症时鼻咽癌细胞的状态,我们收集低浓度LPS刺激和未刺激巨噬细胞培养上清作为条件培养基(分别以D-THP-LPS, D-THP表示)处理鼻咽癌5-8F细胞,检测促炎因子对5-8F细胞的影响。结果显示,无论在mRNA水平还是蛋白水平,鼻咽癌5-8F细胞经D-THP-LPS条件培养基处理24小时后,细胞中促炎因子TNF-α、IL-6、IL-1β和IL-8表达明显增加,D-THP-LPS条件培养基可促进鼻咽癌5-8F细胞增殖,激活NF-κB和Stat3信号通路。说明炎症微环境下鼻咽癌上皮细胞可因炎性因子的刺激而激活NF-κB和Stat3信号通路,自分泌促炎因子,维持和扩大局部炎症反应,从而促进鼻咽癌发生发展。 [IL-6促进鼻咽癌发生发展] 促炎因子IL-6是肿瘤相关巨噬细胞(TAMs)分泌的主要促炎因子之一,在炎症促肿瘤发生发展过程中具有很重要的作用,而目前IL-6和肿瘤相关巨噬细胞(TAMs)对鼻咽癌发生发展的影响不是很清楚。因此我们检测分析了鼻咽癌组织中IL-6蛋白表达情况和TAMs浸润程度,IL-6对鼻咽癌细胞生长的影响。采用鼻咽癌组织芯片,免疫组化检测发现鼻咽癌组织中IL-6和CD68(TAMs分子标记物,表达强弱可以反映TAMs在肿瘤组织中浸润程度)均呈强阳性表达,IL-6蛋白表达与TAMs浸润密度正相关,Kaplan-Meier生存分析显示IL-6蛋白表达越高或者TAMs浸润密度越高,鼻咽癌患者预后越差；外源IL-6可促进鼻咽癌细胞增殖、加快细胞周期演进,同时免疫荧光、荧光素酶报告系统及Western blot检测显示IL-6可激活NF-κBp65和p-Stat3蛋白。由此说明鼻咽癌组织中TAMs浸润和IL-6蛋白表达与鼻咽癌发生发展密切相关,IL-6可通过激活NF-κB和Stat3蛋白促进鼻咽癌发生发展。 [LPLUNC1基因抑制鼻咽癌细胞生长] 前期研究显示LPLUNC1基因在鼻咽癌组织中低表达,可能是鼻咽癌潜在的抑瘤基因。应用鼻咽癌组织芯片,免疫组织化学检测我们发现LPLUNC1蛋白在正常鼻咽上皮和腺体中高表达,而在大多数鼻咽癌组织中低表达或阴性表达,LPLUNC1蛋白表达下调与鼻咽癌临床进展分期正相关,Kaplan-Meier生存显示LPLUNC1蛋白阳性表达的鼻咽癌患者临床预后明显好于阴性表达患者,生长曲线绘制、MTT及裸鼠成瘤等实验均显示LPLUNC1基因可明显抑制鼻咽癌细胞生长与增殖；流式细胞和凋亡检测发现LPLUNC1基因可引起鼻咽癌细胞G0/G1期阻滞和诱导凋亡；进一步研究显示,LPLUNC1基因可通过抑制鼻咽癌细胞NF-κB和Stat3信号传导通路,下调CDK4、cyclin D1、Bcl-2基因表达,上调p21、Bax基因表达。随后我们分别使用Jak2和NF-κB特异性抑制剂(AG490、BAY11-7082)分别阻断HNE2/Vector、 HNE2/LPLUNC1细胞中Stat3和NF-κB信号通路,结果发现AG490、BAY11-7082均能明显抑制鼻咽癌细胞增殖,而LPLUNC1基因与各抑制剂之间具有明显的协同作用。上述结果表明LPLUNC1基因可抑制NF-κB和Stat3信号通路的激活,引起G0/G1期阻滞和诱导凋亡,从而抑制鼻咽癌细胞生长。 [LPLUNC1抑制IL-6介导的NF-κB和Stat3信号通路的激活] 我们研究显示IL-6可通过激活NF-κB和Stat3促进鼻咽癌细胞生长；组织芯片免疫组化结果分析发现鼻咽癌组织中LPLUNC1蛋白表达与IL-6蛋白表达和TAMs浸润密度明显负相关,同时应用激光捕获显微切割技术分离获得纯化鼻咽癌和正常鼻咽上皮组织,qRT-PCR检测也证实鼻咽癌组织中LPLUNC1mRNA表达与IL-6mRNA表达明显负相关,提示LPLUNC1可能参与炎症反应,具有抑制IL-6分泌和TAMs浸润的作用。因此,我们探讨了LPLUNC1对IL-6促进鼻咽癌细胞增殖的影响。首先我们确证了LPLUNC1可明显抑制LPS诱导的鼻咽癌细胞和巨噬细胞分泌促炎因子如TNF-α、IL-6、IL-1p和IL-8,参与抗炎反应；随后研究发现,LPLUNC1基因可明显抑制IL-6对鼻咽癌细胞的促增殖和加速细胞周期演进的作用,荧光素酶报告基因检测发现尽管HNE2细胞受到IL-6的刺激,LPLUNC1基因仍可明显抑制NF-κB和Stat3的转录活性,免疫荧光检测显示LPLUNC1基因可明显抑制IL-6诱导的NF-κBp65和Stat3舌化入核,Western blot检测也显示LPLUNC1基因可明显抑制IL-6诱导的NF-κBp65和p-Stat3蛋白表达,NF-κBp65蛋白表达减少的同时伴有IKKP蛋白表达减少和IκBα蛋白表达增加,p-Stat3蛋白表达减少的同时伴有p-Jak2蛋白表达减少,LPLUNC1基因还可通过抑制IL-6介导的NF-κB和Stat3信号通路的激活,下调cyclin D1、Bcl-2基因表达,上调p21、Bax基因表达。因此,LPLUNC1基因可通过抑制IL-6介导的NF-κB和Stat3信号通路的激活,抵抗促炎因子IL-6对鼻咽癌细胞增殖的促进作用。 [LPLUNC1对鼻咽癌5-8F细胞蛋白质表达谱的影响] 蛋白质是生命活动中生物学功能的真正执行者、生命现象的直接体现者,探讨生命活动中尤其是病理过程中发生改变的蛋白质分子具有重大意义。因此,为进一步了解LPLUNC1在鼻咽癌发生发展中的作用,我们采用荧光差异凝胶电泳(2D-DIGE)和质谱分析技术获得了鼻咽癌5-8F细胞过表达LPLUNC1基因前后的差异蛋白质变化。共鉴定出44个差异表达蛋白质,包括上调19个,下调25个。这些差异表达蛋白质按其功能分类主要是(1)分子伴侣蛋白；(2)细胞骨架蛋白；(3)参与细胞新陈代谢的酶类或蛋白；(4)信号转导分子；(5)其他蛋白。结果表明LPLUNC1基因可能通过调控上述蛋白分子表达参与细胞代谢、增殖、转录以及信号转导等生物学过程,从而影响鼻咽癌的生长。随后,我们对上调差异蛋白Prohibitin (PHB)、下调差异蛋白hypoxia up-regulated1precursor (HYOU1)和calreticulin precursor variant (CALR)进行了验证。结果显示,LPLUNC1基因转染5-8F细胞前后上述差异蛋白质表达确实存在差异,与质谱鉴定结果一致；对Prohibitin蛋白进一步研究发现,Prohibitin蛋白在正常鼻咽组织中呈高表达,而在绝大部分鼻咽癌组织中呈低表达,Prohibitin蛋白表达降低与鼻咽癌的临床进展分期和转移相关,Kaplan-Meier生存分析和多因素分析发现Prohibitin蛋白阴性表达与鼻咽癌患者预后差密切相关。提示Prohibitin可能是鼻咽癌的重要分子标志。
[Abstract]:BACKGROUND OF THE STUDY

Chronic inflammation is the seventh major biological characteristic of the tumor , and chronic inflammation plays a very important role in triggering tumor development and promoting tumor development .
Nasopharyngeal carcinoma ( NPC ) is a kind of malignant tumor with obvious regional difference . Nasopharyngeal carcinoma is a kind of malignant tumor with obvious regional difference . The nasopharyngeal epithelial cell has been damaged by various exogenous pathogenic microorganisms such as EBV and toxic chemicals .
LPLUNC1 protein can inhibit the growth and metastasis of nasopharyngeal carcinoma by means of its function of host defense with the aid of its binding domain , and the role of LPLUNC1 gene in the development of nasopharyngeal carcinoma ( NPC ) and its related molecular mechanism are discussed .

Inflammatory factors stimulate the involvement of nasopharyngeal epithelial cells in inflammatory response

The expression of pro - inflammatory cytokines TNF - 伪 , IL - 6 , IL - 1尾 and IL - 8 in nasopharyngeal carcinoma ( NPC ) were measured with low concentration LPS ( 10 ng / ml , similar to the physiological concentration of bacterial lipopolysaccharide , such as bacterial infection ) .

Conclusion IL - 6 promotes the development of nasopharyngeal carcinoma .

IL - 6 and tumor - related macrophages ( TAMs ) have an important role in the development of nasopharyngeal carcinoma .
Exogenous IL - 6 can promote the proliferation of nasopharyngeal carcinoma cells , accelerate the development of cell cycle , and simultaneously immunofluorescence , luciferase reporter system and Western blot analysis show that IL - 6 can activate NF - 魏B and p - Stat3 protein . The expression of TAMs and IL - 6 in nasopharyngeal carcinoma is closely related to the development of nasopharyngeal carcinoma . IL - 6 can promote the development of nasopharyngeal carcinoma by activating NF - 魏B and Stat3 protein .

Inhibitory effect of recombinant LPLUNC1 gene on nasopharyngeal carcinoma cell growth

The low expression of LPLUNC1 gene in nasopharyngeal carcinoma ( NPC ) and the low expression of LPLUNC1 protein in normal nasopharyngeal epithelium and gland were detected by immunohistochemical method . The expression of LPLUNC1 was significantly correlated with the clinical progression of NPC .
LPLUNC1 gene can induce G0 / G1 arrest and induce apoptosis in nasopharyngeal carcinoma cells .
The results showed that AG490 , BAY11 - 7082 could inhibit the proliferation of nasopharyngeal carcinoma cells and inhibit the expression of Stat3 and NF - 魏B in HNE2 / Vector and HNE2 / LPLUNC1 cells .

Activation of IL - 6 - mediated NF - 魏B and Stat3 signaling pathways

Our study shows that IL - 6 can promote the growth of nasopharyngeal carcinoma cells by activating NF - 魏B and Stat3 ;
The expression of LPLUNC1 was negatively correlated with the expression of IL - 6 and the density of IL - 6 in nasopharyngeal carcinoma .
It was found that LPLUNC1 gene could significantly inhibit the expression of NF - 魏B and Stat3 in nasopharyngeal carcinoma cells . The expression of NF - 魏B and Stat3 was significantly inhibited by LPLUNC1 gene .

Effect of LPLUNC1 on protein expression profile of nasopharyngeal carcinoma ( NPC ) 5 - 8F cells

In order to further understand the role of LPLUNC1 in the development of nasopharyngeal carcinoma , we obtained 44 differentially expressed proteins , including up - regulation of 19 and down - regulation of 25 .
( 2 ) cytoskeletal proteins ;
( 3 ) enzymes or proteins involved in cell metabolism ;
( 4 ) signal transduction molecule ;
( 5 ) Other proteins . The results showed that the LPLUNC1 gene could regulate the biological processes of cell metabolism , proliferation , transcription and signal transduction by regulating the expression of the above - mentioned protein molecules , thus affecting the growth of nasopharyngeal carcinoma .
The results showed that the expression of bitin protein in nasopharyngeal carcinoma was significantly lower than that of nasopharyngeal carcinoma ( NPC ) . Kaplan - Meier survival analysis and multivariate analysis showed that the negative expression of bitin protein was closely related to the prognosis of NPC patients .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

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