Flotillin-2在鼻咽癌中的表达及作用机制的初步探索

发布时间：2018-05-16 13:47

本文选题：鼻咽癌 + FLOT2　；参考：《中南大学》2012年硕士论文

【摘要】：鼻咽癌(nasopharyngeal carcinoma, NPC)是东南亚和我国南方省份高发的恶性肿瘤。其病因可能涉及EB病毒感染、化学致癌物作用及基因突变和表观遗传学变异等多个因素。鼻咽癌细胞系5-8F和6-10B是从SUNE-1细胞系中分离出的两个亚系,虽然遗传背景相同,但是它们的转移能力差别很大。本课题组利用这一特点,通过抑制性消减杂交技术,构建了两株细胞间差异表达基因的消减cDNA文库,从中筛选出了一批在高转移鼻咽癌细胞系5-8F中表达上调的基因,进一步挑选出Flotillin-2(FLOT2)基因进行后续研究。 FLOT2蛋白最早是在金鱼视网膜神经节细胞轴突再生过程中发现的,它具有广泛的生物学功能,参与轴突再生、神经元分化、胞吞作用、T淋巴细胞活化、脂筏构成、膜蛋白招募及促进黑色素瘤发生和转移等过程。前期工作中,利用RNA干扰方法降低5-8F细胞系中FLOT2悬因的表达水平,我们发现5-8F细胞系增殖、侵袭、运动、转移能力均下降,而G1期细胞比例增加。这些结果提示FLOT2可能与鼻咽癌的转移相关。本研究在前期工作的基础之上,检测了各种鼻咽癌细胞系中FLOT2基因mRNA的表达水平。同时,采用免疫组织化学方法检测了38例慢性鼻咽炎患者、45例未转移鼻咽癌患者和87例转移鼻咽癌患者组织切片中FLOT2蛋白的表达水平。接着抽提了稳定干扰FLOT2基因的5-8F细胞总RNA,进行基因表达谱芯片实验和生物信息学分析,并对部分芯片结果进行了初步的验证。最后对FLOT2基因启动子区域进行生物信息学预测,利用Wnt信号通路激活剂BIO刺激5-8F和6-10B细胞,于不同时间检测FLO表达水平和启动子活性。 RT-PCR结果显示,在鼻咽癌细胞系中,5-8F、HNE-1、HNE-2、 HNE-3、CNE-1、CNE-2、HONE-1、HK-1、C666-1九个细胞系中表达较强的FLOT2,而6-10B细胞中FLOT2mRNA的表达水平最低,但仍高于NP69正常鼻咽上皮细胞系。免疫组化结果显示,从慢性鼻咽炎组织到未转移鼻咽癌组织,再到转移鼻咽癌组织中,FLOT2的表达水平依次升高(P0.001)。基因表达谱芯片和生物信息学分析发现,FLOT2基因干扰后,5-8F细胞中p53信号通路活化,TGF-β信号通路被抑制,多个细胞粘附相关分子、细胞周期相关分子表达水平发生了明显的变化。RT-PCR和/或定量PCR结果证实,FLOT2表达下调后,与鼻咽癌转移负相关的E-cadherin以及细胞周期负相关因子p21表达均明显上调。生物信息学软件预测发现,FLOT2基因转录起始位点上游2000bp范围内存在多个启动子和Wnt信号通路转录因子结合位点。我们构建了包含FLOT2启动子和Wnt信号通路转录因子结合位点的荧光素酶报告基因载体。BIO刺激后48h,6-10B与5-8F细胞内FLOT2基因启动子区域活性明显上调,FLOT2表达水平也明显上调。综合上述结果,我们得出以下结论：FLOT2在鼻咽癌细胞系和组织中表达水平明显上调,转移的鼻咽癌组织较未转移鼻咽癌组织表达更高水平的FLOT2;干扰FLOT2基因后,5-8F细胞内相关信号通路和分子发生明显变化,提示FLOT2可能与鼻咽癌的发生及转移相关,其作用分子机制可能涉及p53、TGF-β等信号通路及Klf4、 E-cadherin和p21等分子的异常改变。Wnt信号通路通过调控FLOT2启动子活性实现对FLOT2表达的调控,进而实现对鼻咽癌转移的调控。
[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 .

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

【参考文献】