EB病毒感染对microRNA-203的表达影响及其相互作用是鼻咽癌发病学的重要机制

发布时间：2018-05-17 02:38

本文选题：EB病毒 + 潜伏膜蛋白1　；参考：《中南大学》2012年博士论文

【摘要】：[背景] EB病毒(Epstein-Barr virus, EBV)是具有高度转化能力的病毒,并与多种恶性肿瘤如伯基特淋巴瘤(BL)和鼻咽癌(NPC)密切相关。但是,EBV在NPC发展中的作用尚未阐明。MicroRNAs (miRNAs)是一类高度保守的非编码小RNA,主要通过与靶基因3’末端非翻译区互补配对,从而抑制靶基因的表达而发挥生物学功能,miRNA可以控制多种生物进程：如细胞增殖,凋亡和分化。越来越多的证据表明miRNA表达异常参与肿瘤的发生。然而,在NPC中EBV能否并如何调控细胞miRNA的表达仍需要阐明。本课题利用前期构建的EBV稳定感染293-EBV细胞系和对照组293细胞系进行miRNA芯片分析。筛选受EBV调控的差异miRNAs,从miRNA的角度研究EBV促进NPC发生发展的机制。 [miR-203在EBV感染的上皮细胞、淋巴细胞和低分化鼻咽癌组织中表达下调] 对293及293-EBV细胞进行miRNA芯片检测和分析,筛选差异表达超过2.5倍的miRNAs,其中3个表达上调,15个表达下调。miR-203是上皮细胞中特异表达的一个miRNA,并在多种上皮肿瘤中表达下调,具有抑瘤基因的功能,因此,本课题选择miR-203作为下一步研究对象。芯片结果显示miR-203在293-EBV细胞中下调约3.7倍。qRT-PCR证实miR-203表达水平和芯片结果保持一致；在EBV感染的上皮细胞、淋巴细胞中miR-203明显下调；然而在EBV丢失上皮细胞中miR-203的表达水平恢复明显；证实miR-203在低分化NPC组织较正常鼻咽组织中的表达水平下调4.25倍。 [miR-203调控新靶基因E2F3、CCNG1参与抑制上皮细胞的生长和恶性转化能力] 通过miRBase软件分析差异表达miRNA分子的基本信息,在线miRNA靶基因预测软件targetscan和miRanda对下游靶基因进行分析,并利用荧光素报告载体活性检测实验证实E2F3和CCNG1是miR-203的直接调控的新靶基因。RT-PCR和Westernblot结果显示miR-203可以在mRNA和蛋白质水平调控靶基因的表达。 MTT和流式细胞结果表明miR-203具有抑制上皮细胞生长能力,能抑制细胞从G0/G1期向S期转换的进程；miR-203明显抑制裸鼠移植瘤肿瘤细胞的生长。免疫组化和原为杂交结果显示miR-203过表达的裸鼠瘤组织中靶基因E2F3和CCNG1表达下调。在上皮细胞中过表达靶基因E2F3和CCNG1能够逆转miR-203对上皮细胞生物学功能的影响。 [LMP1是EBV下调miR-203的关键病毒蛋白,并且通过激活JNK和NF-κB信号通路调控miR-203的表达] 潜伏膜蛋白LMP1和LMP2是EBV编码的重要癌蛋白。qRT-PCR结果证实EBV通过LMP1参与调控Pri-miR-203和Mature-miR-203表达下调而LMP2却无此功能；在EBV和LMP1稳定转染的上皮细胞中沉默LMP1表达后,miR-203的表达量恢复明显。免疫组化和原位杂交结果显示miR-203在NPC旁较癌组织中表达高；LMP1表达阴性NPC组织较阳性癌组织的miR-203表达明显增高。LMP1发挥生物学功能的主要区域是C端的CTAR1、CTAR2和CTAR3结构域。Western-blot和qRT-PCR结果显示LMP1蛋白CTAR1和CTAR2结构域通过激活JNK和NF-κB信号通路调控miR-203的表达；而p38信号通路对miR-203的表达没有明显影响。同时,qRT-PCR结果证实LMP1蛋白CTAR3结构域不能下调miR-203的表达。 [miR-203与鼻咽癌的侵袭转移及患者血清中EBV抗体VCA-IgA的表达水平密切相关] 研究显示miR-203在EBV感染的NPC组织中低表达,miR-203可能是NPC潜在的抑瘤基因。本研究应用NPC组织芯片,原位杂交结果显示NPC组织中miR-203表达水平与NPC淋巴结转移和患者血清EBV抗体VCA-IgA表达量紧密相关。NPC组织中miR-203表达越低,淋巴结转移率越高,血清EBV抗体VCA-IgA表达水平越高。
[Abstract]:Background background

Epstein - Barr virus ( EBV ) is a highly transformed virus and is closely associated with a variety of malignancies such as Burkitt ' s lymphoma ( BL ) and nasopharyngeal carcinoma ( NPC ) . However , the role of EBV in the development of NPC has not yet been elucidated . However , the role of EBV in the development of NPC has not yet been clarified . MicroRNAs ( microRNAs ) are highly conserved non - coding small RNAs that can control various biological processes , such as cell proliferation , apoptosis and differentiation .

In this study , miRNA chip analysis was carried out on 293 - EBV and 293 cell lines in the control group , and the mechanism of EBV for the development of NPC was studied from the angle of miRNA by screening the differentially expressed miRNA regulated by EBV .

Down - regulation of miR - 203 in EBV - infected epithelial cells , lymphocytes and low - differentiated nasopharyngeal carcinoma tissues

The results showed that miR - 203 was down - regulated in 293 - EBV cells by about 3.7 times , and the results showed that miR - 203 was down - regulated by about 3.7 times in 293 - EBV .
miR - 203 was down - regulated in lymphocytes of EBV - infected epithelial cells .
However , the expression level of miR - 203 in EBV - lost epithelial cells was restored .
It was confirmed that miR - 203 was down - regulated by 4.25 times in lower - differentiated NPC tissues than in normal nasopharyngeal tissues .

miR - 203 regulates the expression of E2F3 and CCNG1 in inhibiting the growth and malignant transformation ability of epithelial cells .

Through the miRBase software analysis , the basic information of miRNA molecules is analyzed , the target gene of the on - line miRNA is predicted by targetscan and miRanda , and the downstream target gene is analyzed by the miRNA target gene prediction software targetscan and miRanda , and a novel target gene which is directly regulated by miR - 203 is confirmed by using a fluorescein reporter vector activity detection experiment . The RT - PCR and Western blot results show that miR - 203 can regulate the expression of the target gene at mRNA and protein levels .

MTT and flow cytometry showed that miR - 203 had the ability to inhibit the growth of epithelial cells and inhibit the transition of cells from G0 / G1 phase to S phase ;
miR - 203 significantly inhibited the growth of tumor cells in nude mice . The expression of E2F3 and CCNG1 in nude mice with overexpression of miR - 203 was downregulated by immunohistochemistry and in situ hybridization . The overexpression of E2F3 and CCNG1 in epithelial cells could reverse the effects of miR - 203 on the biological function of epithelial cells .

LMP1 is a key viral protein down - regulation of miR - 203 by EBV , and the expression of miR - 203 is regulated by activating the NF - 魏B signal pathway .

latent membrane proteins LMP1 and LMP2 are important cancer proteins encoded by EBV . qRT - PCR results demonstrate that EBV is regulated by LMP1 in regulating the expression of Pri - miR - 203 and miR - miR - 203 , whereas LMP2 does not have this function ;
The expression of miR - 203 was restored after the expression of LMP1 was silenced in the stably transfected epithelial cells of EBV and LMP1 . The results of immunohistochemistry and in situ hybridization showed that miR - 203 was highly expressed in the adjacent tissues of NPC ;
In LMP1 , the expression of miR - 203 was significantly increased in the negative NPC tissues . The main regions of LMP1 play a biological function : CTAR1 , CTAR2 and CTAR3 domains at the C - terminal . Western - blot and qRT - PCR showed that the LMP1 protein CTAR1 and CTAR2 domains regulate the expression of miR - 203 by activating the MAPK and NF - 魏B signaling pathways .
At the same time , the results of qRT - PCR confirmed that the LMP1 protein CTAR3 domain could not downregulate the expression of miR - 203 .

Conclusion miR - 203 is closely related to the invasion and metastasis of nasopharyngeal carcinoma ( NPC ) and the level of EBV - IgA in patients with nasopharyngeal carcinoma ( NPC ) .

The expression level of miR - 203 in NPC tissue was closely related to NPC ' s lymph node metastasis and the expression of EBV antibody of EBV . The higher the expression of miR - 203 in NPC tissue , the higher the expression level of miR - 203 in NPC tissue , the higher the expression level of EBV antibody in serum .
【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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