本文選題：鼻咽癌 + FLOT2��； 參考：《中南大學(xué)》2012年碩士論文

【摘要】：鼻咽癌(nasopharyngeal carcinoma, NPC)是東南亞和我國南方省份高發(fā)的惡性腫瘤。其病因可能涉及EB病毒感染、化學(xué)致癌物作用及基因突變和表觀遺傳學(xué)變異等多個因素。 鼻咽癌細(xì)胞系5-8F和6-10B是從SUNE-1細(xì)胞系中分離出的兩個亞系,雖然遺傳背景相同,但是它們的轉(zhuǎn)移能力差別很大。本課題組利用這一特點(diǎn),通過抑制性消減雜交技術(shù),構(gòu)建了兩株細(xì)胞間差異表達(dá)基因的消減cDNA文庫,從中篩選出了一批在高轉(zhuǎn)移鼻咽癌細(xì)胞系5-8F中表達(dá)上調(diào)的基因,進(jìn)一步挑選出Flotillin-2(FLOT2)基因進(jìn)行后續(xù)研究。 FLOT2蛋白最早是在金魚視網(wǎng)膜神經(jīng)節(jié)細(xì)胞軸突再生過程中發(fā)現(xiàn)的,它具有廣泛的生物學(xué)功能,參與軸突再生、神經(jīng)元分化、胞吞作用、T淋巴細(xì)胞活化、脂筏構(gòu)成、膜蛋白招募及促進(jìn)黑色素瘤發(fā)生和轉(zhuǎn)移等過程。 前期工作中,利用RNA干擾方法降低5-8F細(xì)胞系中FLOT2懸因的表達(dá)水平,我們發(fā)現(xiàn)5-8F細(xì)胞系增殖、侵襲、運(yùn)動、轉(zhuǎn)移能力均下降,而G1期細(xì)胞比例增加。這些結(jié)果提示FLOT2可能與鼻咽癌的轉(zhuǎn)移相關(guān)。 本研究在前期工作的基礎(chǔ)之上,檢測了各種鼻咽癌細(xì)胞系中FLOT2基因mRNA的表達(dá)水平。同時,采用免疫組織化學(xué)方法檢測了38例慢性鼻咽炎患者、45例未轉(zhuǎn)移鼻咽癌患者和87例轉(zhuǎn)移鼻咽癌患者組織切片中FLOT2蛋白的表達(dá)水平。接著抽提了穩(wěn)定干擾FLOT2基因的5-8F細(xì)胞總RNA,進(jìn)行基因表達(dá)譜芯片實(shí)驗(yàn)和生物信息學(xué)分析,并對部分芯片結(jié)果進(jìn)行了初步的驗(yàn)證。最后對FLOT2基因啟動子區(qū)域進(jìn)行生物信息學(xué)預(yù)測,利用Wnt信號通路激活劑BIO刺激5-8F和6-10B細(xì)胞,于不同時間檢測FLO表達(dá)水平和啟動子活性。 RT-PCR結(jié)果顯示,在鼻咽癌細(xì)胞系中,5-8F、HNE-1、HNE-2、 HNE-3、CNE-1、CNE-2、HONE-1、HK-1、C666-1九個細(xì)胞系中表達(dá)較強(qiáng)的FLOT2,而6-10B細(xì)胞中FLOT2mRNA的表達(dá)水平最低,但仍高于NP69正常鼻咽上皮細(xì)胞系。免疫組化結(jié)果顯示,從慢性鼻咽炎組織到未轉(zhuǎn)移鼻咽癌組織,再到轉(zhuǎn)移鼻咽癌組織中,FLOT2的表達(dá)水平依次升高(P0.001)。基因表達(dá)譜芯片和生物信息學(xué)分析發(fā)現(xiàn),FLOT2基因干擾后,5-8F細(xì)胞中p53信號通路活化,TGF-β信號通路被抑制,多個細(xì)胞粘附相關(guān)分子、細(xì)胞周期相關(guān)分子表達(dá)水平發(fā)生了明顯的變化。RT-PCR和/或定量PCR結(jié)果證實(shí),FLOT2表達(dá)下調(diào)后,與鼻咽癌轉(zhuǎn)移負(fù)相關(guān)的E-cadherin以及細(xì)胞周期負(fù)相關(guān)因子p21表達(dá)均明顯上調(diào)。 生物信息學(xué)軟件預(yù)測發(fā)現(xiàn),FLOT2基因轉(zhuǎn)錄起始位點(diǎn)上游2000bp范圍內(nèi)存在多個啟動子和Wnt信號通路轉(zhuǎn)錄因子結(jié)合位點(diǎn)。我們構(gòu)建了包含F(xiàn)LOT2啟動子和Wnt信號通路轉(zhuǎn)錄因子結(jié)合位點(diǎn)的熒光素酶報告基因載體。BIO刺激后48h,6-10B與5-8F細(xì)胞內(nèi)FLOT2基因啟動子區(qū)域活性明顯上調(diào),FLOT2表達(dá)水平也明顯上調(diào)。 綜合上述結(jié)果,我們得出以下結(jié)論：FLOT2在鼻咽癌細(xì)胞系和組織中表達(dá)水平明顯上調(diào),轉(zhuǎn)移的鼻咽癌組織較未轉(zhuǎn)移鼻咽癌組織表達(dá)更高水平的FLOT2;干擾FLOT2基因后,5-8F細(xì)胞內(nèi)相關(guān)信號通路和分子發(fā)生明顯變化,提示FLOT2可能與鼻咽癌的發(fā)生及轉(zhuǎn)移相關(guān),其作用分子機(jī)制可能涉及p53、TGF-β等信號通路及Klf4、 E-cadherin和p21等分子的異常改變。Wnt信號通路通過調(diào)控FLOT2啟動子活性實(shí)現(xiàn)對FLOT2表達(dá)的調(diào)控,進(jìn)而實(shí)現(xiàn)對鼻咽癌轉(zhuǎn)移的調(diào)控。
[Abstract]:Nasopharyngeal carcinoma ( NPC ) is a malignant tumor in South - East Asia and southern provinces of China . Its etiology may involve several factors , such as Epstein - Barr virus infection , chemical carcinogen , gene mutation and epigenetics variation .

Five - 8F and 6 - 10B nasopharyngeal carcinoma cell lines were isolated from the SUNE - 1 cell line , although the genetic background was the same , but their ability to transfer was very different . Using this feature , we constructed a subtractive cDNA library of differential expression genes between two cells , and screened out a batch of genes that were up - regulated in the high - metastatic nasopharyngeal carcinoma cell line 5 - 8F , and further selected the Flotillin - 2 ( FLOT2 ) gene for subsequent research .

FLOT2 protein was found in the process of axonal regeneration of retinal ganglion cells of goldfish . It has extensive biological functions , participates in axonal regeneration , neuronal differentiation , endocytosis , T lymphocyte activation , lipid rafts formation , membrane protein recruitment , and promotes the occurrence and metastasis of melanoma .

In the previous work , RNA interference was used to reduce the level of FLOT2 suspension in the 5 - 8F cell line . We found that the proliferation , invasion , movement and metastasis of 5 - 8F cell line decreased , while the proportion of G1 phase increased . These results suggest that FLOT2 may be related to the metastasis of nasopharyngeal carcinoma .

In this study , the expression level of FLOT2 mRNA in various nasopharyngeal carcinoma cell lines was determined on the basis of the previous work . In the meantime , the expression level of FLOT2 protein in 38 patients with chronic rhinopharyngitis , 45 patients without metastasis of nasopharyngeal carcinoma and 87 patients with metastatic nasopharyngeal carcinoma were detected by immunohistochemical method .

The results of RT - PCR showed that , in nasopharyngeal carcinoma cell lines , the expression level of FLOT2 mRNA was lowest in 5 - 8F , HNE - 1 , HNE - 2 , HNE - 3 , CNE - 1 , CNE - 2 , HONE - 1 , HK - 1 , C666 - 1 , but the expression level of FLOT2 in 6 - 10B cells was significantly higher than that of NP69 .

We constructed a luciferase reporter gene vector containing the transcription factor binding site of the FLOT2 promoter and Wnt signaling pathway . After BIO stimulation , the activity of the promoter region of the FLOT2 gene in the 6 - 10B and 5 - 8F cells was up - regulated , and the expression level of FLOT2 was also significantly up - regulated .

The results showed that the expression level of FLOT2 in nasopharyngeal carcinoma cell lines and tissues was significantly up - regulated . The expression of FLOT2 in nasopharyngeal carcinoma tissues was higher than that of the untreated nasopharyngeal carcinoma . The expression of FLOT2 was correlated with the occurrence and metastasis of nasopharyngeal carcinoma . The mechanism of the action could be related to the abnormal changes of signal pathways such as p53 , TGF - 尾 , Klf4 , E - cadherin and p21 . The Wnt signaling pathway regulates the expression of FLOAT by regulating the activity of the FLOT2 promoter , and then regulates the metastasis of nasopharyngeal carcinoma .

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.63

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