BLCAP A-I RNA编辑在宫颈癌-癌旁组织中的差异以及编辑对BLCAP功能的影响

发布时间：2018-03-12 19:35

本文选题：BLCAP基因　切入点：A-IRNA编辑　出处：《武汉大学》2017年博士论文　论文类型：学位论文

【摘要】：根据世界卫生组织(WTO)对2012年全球癌症患者的统计显示,宫颈癌的发生在女性癌症患者中排行第四,其发病率和死亡率仅次于乳腺癌、结直肠癌和肺癌;在发展中国家,宫颈癌在女性癌症患者中的发病率和死亡率更是排行第二和第三。虽然人乳头瘤病毒(high-risk human papillomavirus,HPV)作为宫颈癌的主要的致病因素这个观点已经得到很好的阐述,且HPV疫苗的使用也使其发病率大大降低,但对于宫颈癌这个多步骤多因素的发展过程,探究其致病机制仍任重道远。膀胱癌相关蛋白(Bladder cancer associated protein,BLCAP)基因是本实验室利用基因表达芯片筛选出的一个在宫颈癌组织中表达下降最为明显的基因。BLCAP于2002年在研究侵袭性膀胱癌时被发现,由两个内含子和一个外显子组成,编码一个大小为10kDa的蛋白。课题组先前的研究显示BLCAP在宫颈癌组织中表达明显下调,且通过克隆形成、生长计数以及裸鼠成瘤实验,发现BLCAP可能是一个潜在的宫颈癌抑癌基因。除此之外,有研究报道BLCAP也是一个A-IRNA编辑事件的底物,受到ADAR编辑酶的催化调控。RNA编辑(RNA editing)是指在mRNA水平上改变遗传信息的过程。虽然BLCAP及其产物BLCAP的功能已经得到初步研究,但是BLCAP如何发挥抑癌作用以及A-I RNA编辑对其编辑底物BLCAP功能的影响依旧需要更多的探索。在本研究中,我们针对35对宫颈癌-癌旁组织的BLCAP进行测序,希望了解BLCAP在宫颈癌中的A-I RNA编辑情况。通过使用Illumina公司Miseq PE300测序平台对35对宫颈癌及癌旁组织标本中BLCAP的编码区进行外显子测序,我们发现BLCAP编码区第五位、十四位、四十四位A碱基表现出高编辑状态,且编辑水平在癌和癌旁组织中存在显著的统计学差异。基于直接测序的结果,我们将高通量数据库按照三个位点发生编辑现象的八种情况进行分类,结果显示这三个位点间的编辑情况常常关联发生。另一方面,通过将BLCAP的编辑水平与A-IRNA编辑的两种催化酶ADAR1和ADAR2进行关联性分析,并结合HeLa细胞上干扰ADAR1、ADAR2分析BLCAP编辑水平的实验结果,发现ADAR1主要介导了BLCAP A-I RNA编辑的催化。随后,我们用信息学工具ELM(Eukaryotic Linear Motif)软件对BLCAP进行生物信息学预测,发现两个编辑频率最高的位点均在BLCAP的YXXQ基序中,并且YXXQ基序可能与STAT3的SH2结构域相互作用。信号转导与转录活化因子 3(Signal transducer and activator of transcription 3,STAT3)是一种转录因子,参与调节细胞内一系列的生理活动。在经典的STAT3活化通路中,白细胞介素家族-6作为主要刺激物,激活Janus激酶(Janus kinase,JAK);JAK激活后发生聚集,继续活化STAT3使STAT3羧基端的酪氨酸残基磷酸化;磷酸化后的STAT3转移到细胞核内与其靶基因的特异性启动子结合,从而调控其靶基因的表达。STAT3的主要目标基因包括Bcl-2家族蛋白、Cyclin周期蛋白、MMP家族蛋白,这些蛋白参与抗凋亡、促增殖、诱导血管生成以及逃避抗肿瘤免疫。STAT3的持续性激活参与很多肿瘤的发生和发展,因此STAT3被视为一种有前景的肿瘤治疗靶基因。在宫颈癌中,STAT3的磷酸化水平持续性升高并且高活化状态的STAT3可以预测不良预后,表明STAT3在宫颈癌发生中发挥重要作用。根据ELM的预测结果,我们研究了 BLCAP与STAT3之间的相互关系。首先我们构建了 STAT3-HA真核表达质粒,在293T细胞中同时过量表达BLCAP与STAT3后进行免疫共沉淀(Co-Immunoprecipitation,Co-IP)实验,结果显示外源性BLAP能和外源性STAT3相互作用;在293T细胞和HeLa细胞中分别过量表达BLCAP后进行Co-IP实验,结果显示外源性BLAP能和内源性STAT3相互作用。随后我们探究了 BLCAP对STAT3的活化有无影响。在宫颈癌细胞系HeLa和C33A细胞上分别过量表达BLCAP或RNA干扰BLCAP,结果均提示BLCAP能抑制STAT3的磷酸化水平。同样的,我们在HeLa细胞上过量表达或RNA干扰BLCAP,对STAT3下游基因进行检测,结果显示BLCAP也能抑制STAT3下游基因。根据以上结果,我们证明了在宫颈癌细胞系中BLCAP能抑制STAT3信号通路的活化。由于A-IRNA编辑替换了 BLCAP YXXQ基序中的两个重要氨基酸,我们模拟A-I RNA编辑的情况构建了两个BLCAP突变体(CCLQ-FLAG和CCLR-FLAG)进一步探索A-I RNA编辑在BLCAP抑制STAT3磷酸化的过程中的作用。免疫共沉淀实验结果显示,突变体CCLQ-FLAG与STAT3的相互作用弱于野生型(BLCAP-FLAG),突变体CCLR-FLAG对STAT3的相互作用弱于CCLQ-FLAG。在HeLa和C33A细胞上过量表达野生型和突变体,突变体CCLR-FLAG和CCLQ-FLAG对STAT3的磷酸化作用增强;对STAT3下游基因进行检测,我们也得到相同的结果。以上结果说明,A-IRNA编辑事件通过编辑BLCAP YXXQ的两个重要位点,使BLCAP丧失了对STAT3信号通路的抑制作用。本研究中为BLCAP发挥其抑癌功能提供了新的机制,同时证明了 A-IRNA编辑事件通过改变BLCAP的重要编辑位点导致蛋白质功能的改变,从而对宫颈癌的发生发展中起到重要作用。
[Abstract]:According to the WHO (WTO) of statistics show that 2012 global cancer patients, the incidence of cervical cancer is ranked fourth in the women's cancer, the morbidity and mortality after breast cancer, colorectal cancer and lung cancer; in developing countries, cervical cancer incidence and mortality in female cancer patients in the ranks second and third. Although human papillomavirus (high-risk human, papillomavirus, HPV) as the main point of the pathogenic factors of cervical cancer has been well described, and the use of HPV vaccine also makes its incidence rate is greatly reduced, but for the development of multi - factors of cervical cancer in this step, to explore the pathogenesis of bladder cancer is still a long way to go. Related protein (Bladder cancer associated protein, BLCAP) gene in our laboratory using gene chip screening out of an expression in cervical cancer tissues decreased most The.BLCAP gene was found obviously in invasive bladder cancer research in 2002, consists of two introns and exons, encoding a 10kDa protein. Previous study showed the expression of BLCAP in cervical cancer tissues was significantly reduced, and the clone formation, growth and counting of nude mice in experiment, found that BLCAP may be a potential tumor suppressor gene in cervical cancer. In addition, studies have reported that BLCAP is a A-IRNA editing substrate, by ADAR editing enzyme regulation (RNA editing).RNA editing is a process to change the genetic information at the level of mRNA. Although BLCAP and BLCAP products the function has been preliminarily studied, but the BLCAP how to play the role of tumor suppressor A-I and RNA editing effect on the editing function of BLCAP substrate still need more exploration. In this study, we focused on 35 of cervical cancer - cancer Tissue adjacent to BLCAP sequencing, hope to know BLCAP A-I in cervical cancer. In 35 RNA editing of BLCAP cervical cancer and paracancerous tissues in the encoding region by using Illumina Miseq PE300 sequencing platform exon sequencing, we found that fifth BLCAP encoding region, fourteen bit, forty-four bit A base show high editing, significant differences exist and editing level in cancer and cancer adjacent tissues. Direct sequencing based on the results, we will classify the high-throughput database in accordance with the eight cases of three loci editing phenomenon, results show that the editing between the three loci are often associated with. On the other hand by two, the catalytic enzyme ADAR1 and ADAR2 editing and A-IRNA BLCAP editorial for association analysis, combined with the interference of ADAR1 on HeLa cells, ADAR2 BLCAP analysis of the experimental results of the editing level, hair ADAR1 is mainly mediated by A-I catalyzed BLCAP RNA editing. Then, we use bioinformatics tool ELM (Eukaryotic Linear Motif) software for bioinformatics prediction of BLCAP, found that two of the highest frequency of the editing sites are in the YXXQ motif in the BLCAP and YXXQ motif and SH2 domain may be STAT3 each other. Signal transducer and activator of transcription 3 (Signal transducer and activator of transcription 3, STAT3) is a transcription factor that is involved in the regulation of a series of physiological activity in the cell. In the classic STAT3 activation pathway, interleukin -6 family as the main stimuli, activation of Janus kinase (Janus, kinase, JAK JAK); after activation, aggregation, activation of STAT3 to make the phosphorylation of tyrosine residues of the C-terminus of STAT3; phosphorylation of STAT3 after transfer to specific nucleus and its target gene promoter binding, the regulation of its target gene The main target of gene expression of.STAT3 family proteins including Bcl-2, Cyclin, cyclin, MMP family proteins, these proteins involved in anti apoptosis, proliferation, angiogenesis and persistent escape antitumor immune activation of.STAT3 is involved in the development and progression of many cancers, because this STAT3 is regarded as a promising target for cancer therapy gene in cervical cancer, the phosphorylation level of STAT3 increased continuously and the high activation of STAT3 can predict the prognosis, suggested that STAT3 play an important role in the development of cervical cancer. According to the prediction results of ELM, we study the relationship between BLCAP and STAT3. First, we constructed STAT3-HA eukaryotic expression plasmid in 293T cells and overexpression of BLCAP and STAT3 after immunoprecipitation (Co-Immunoprecipitation, Co-IP) the experimental results showed that exogenous BLAP and exogenous STAT3 interaction; In 293T and HeLa cells were overexpressed after BLCAP Co-IP experiment, results showed that exogenous BLAP and endogenous STAT3 interaction. Then we explored the activation of BLCAP on STAT3 has no effect. In cervical cancer cell lines HeLa and C33A cells respectively, overexpression of BLCAP or RNA interference BLCAP, the results showed BLCAP can inhibit the phosphorylation of STAT3. Also, we overexpressed or RNA interference BLCAP in HeLa cells, to detect the STAT3 downstream genes, the results show that BLCAP can inhibit the downstream genes of STAT3. According to the above results, we prove that the activation in the cervical cancer cell lines BLCAP can inhibit STAT3 signaling pathway by A-IRNA. Edit replace two important amino acid BLCAP YXXQ motif, we simulate the A-I RNA editor of the constructed two BLCAP mutants (CCLQ-FLAG and CCLR-FLAG) to further explore the A-I RNA series Series of inhibition of STAT3 phosphorylation in the BLCAP process. Co immunoprecipitation results show that interaction between mutant CCLQ-FLAG and STAT3 is weaker than the wild type (BLCAP-FLAG), the interaction of mutant CCLR-FLAG on STAT3 in HeLa is weaker than that of CCLQ-FLAG. and C33A cells on the excess expression of wild type and mutant, enhanced phosphorylation mutants CCLR-FLAG and CCLQ-FLAG on STAT3; to detect the STAT3 gene, we get the same results. These results suggest that A-IRNA editing events through two important site editor BLCAP YXXQ, the BLCAP lost the inhibition of the STAT3 signal pathway. In this study, BLCAP provides a new mechanism to play its suppression cancer also proved an important function of editing sites A-IRNA editing by changing the BLCAP lead to altered protein function, so as to the occurrence and development of cervical cancer plays an important role Use.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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