本文選題：BLCAP基因　切入點：A-IRNA編輯　出處：《武漢大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：根據(jù)世界衛(wèi)生組織(WTO)對2012年全球癌癥患者的統(tǒng)計顯示,宮頸癌的發(fā)生在女性癌癥患者中排行第四,其發(fā)病率和死亡率僅次于乳腺癌、結(jié)直腸癌和肺癌;在發(fā)展中國家,宮頸癌在女性癌癥患者中的發(fā)病率和死亡率更是排行第二和第三。雖然人乳頭瘤病毒(high-risk human papillomavirus,HPV)作為宮頸癌的主要的致病因素這個觀點已經(jīng)得到很好的闡述,且HPV疫苗的使用也使其發(fā)病率大大降低,但對于宮頸癌這個多步驟多因素的發(fā)展過程,探究其致病機制仍任重道遠。膀胱癌相關(guān)蛋白(Bladder cancer associated protein,BLCAP)基因是本實驗室利用基因表達芯片篩選出的一個在宮頸癌組織中表達下降最為明顯的基因。BLCAP于2002年在研究侵襲性膀胱癌時被發(fā)現(xiàn),由兩個內(nèi)含子和一個外顯子組成,編碼一個大小為10kDa的蛋白。課題組先前的研究顯示BLCAP在宮頸癌組織中表達明顯下調(diào),且通過克隆形成、生長計數(shù)以及裸鼠成瘤實驗,發(fā)現(xiàn)BLCAP可能是一個潛在的宮頸癌抑癌基因。除此之外,有研究報道BLCAP也是一個A-IRNA編輯事件的底物,受到ADAR編輯酶的催化調(diào)控。RNA編輯(RNA editing)是指在mRNA水平上改變遺傳信息的過程。雖然BLCAP及其產(chǎn)物BLCAP的功能已經(jīng)得到初步研究,但是BLCAP如何發(fā)揮抑癌作用以及A-I RNA編輯對其編輯底物BLCAP功能的影響依舊需要更多的探索。在本研究中,我們針對35對宮頸癌-癌旁組織的BLCAP進行測序,希望了解BLCAP在宮頸癌中的A-I RNA編輯情況。通過使用Illumina公司Miseq PE300測序平臺對35對宮頸癌及癌旁組織標(biāo)本中BLCAP的編碼區(qū)進行外顯子測序,我們發(fā)現(xiàn)BLCAP編碼區(qū)第五位、十四位、四十四位A堿基表現(xiàn)出高編輯狀態(tài),且編輯水平在癌和癌旁組織中存在顯著的統(tǒng)計學(xué)差異。基于直接測序的結(jié)果,我們將高通量數(shù)據(jù)庫按照三個位點發(fā)生編輯現(xiàn)象的八種情況進行分類,結(jié)果顯示這三個位點間的編輯情況常常關(guān)聯(lián)發(fā)生。另一方面,通過將BLCAP的編輯水平與A-IRNA編輯的兩種催化酶ADAR1和ADAR2進行關(guān)聯(lián)性分析,并結(jié)合HeLa細胞上干擾ADAR1、ADAR2分析BLCAP編輯水平的實驗結(jié)果,發(fā)現(xiàn)ADAR1主要介導(dǎo)了BLCAP A-I RNA編輯的催化。隨后,我們用信息學(xué)工具ELM(Eukaryotic Linear Motif)軟件對BLCAP進行生物信息學(xué)預(yù)測,發(fā)現(xiàn)兩個編輯頻率最高的位點均在BLCAP的YXXQ基序中,并且YXXQ基序可能與STAT3的SH2結(jié)構(gòu)域相互作用。信號轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄活化因子 3(Signal transducer and activator of transcription 3,STAT3)是一種轉(zhuǎn)錄因子,參與調(diào)節(jié)細胞內(nèi)一系列的生理活動。在經(jīng)典的STAT3活化通路中,白細胞介素家族-6作為主要刺激物,激活Janus激酶(Janus kinase,JAK);JAK激活后發(fā)生聚集,繼續(xù)活化STAT3使STAT3羧基端的酪氨酸殘基磷酸化;磷酸化后的STAT3轉(zhuǎn)移到細胞核內(nèi)與其靶基因的特異性啟動子結(jié)合,從而調(diào)控其靶基因的表達。STAT3的主要目標(biāo)基因包括Bcl-2家族蛋白、Cyclin周期蛋白、MMP家族蛋白,這些蛋白參與抗凋亡、促增殖、誘導(dǎo)血管生成以及逃避抗腫瘤免疫。STAT3的持續(xù)性激活參與很多腫瘤的發(fā)生和發(fā)展,因此STAT3被視為一種有前景的腫瘤治療靶基因。在宮頸癌中,STAT3的磷酸化水平持續(xù)性升高并且高活化狀態(tài)的STAT3可以預(yù)測不良預(yù)后,表明STAT3在宮頸癌發(fā)生中發(fā)揮重要作用。根據(jù)ELM的預(yù)測結(jié)果,我們研究了 BLCAP與STAT3之間的相互關(guān)系。首先我們構(gòu)建了 STAT3-HA真核表達質(zhì)粒,在293T細胞中同時過量表達BLCAP與STAT3后進行免疫共沉淀(Co-Immunoprecipitation,Co-IP)實驗,結(jié)果顯示外源性BLAP能和外源性STAT3相互作用;在293T細胞和HeLa細胞中分別過量表達BLCAP后進行Co-IP實驗,結(jié)果顯示外源性BLAP能和內(nèi)源性STAT3相互作用。隨后我們探究了 BLCAP對STAT3的活化有無影響。在宮頸癌細胞系HeLa和C33A細胞上分別過量表達BLCAP或RNA干擾BLCAP,結(jié)果均提示BLCAP能抑制STAT3的磷酸化水平。同樣的,我們在HeLa細胞上過量表達或RNA干擾BLCAP,對STAT3下游基因進行檢測,結(jié)果顯示BLCAP也能抑制STAT3下游基因。根據(jù)以上結(jié)果,我們證明了在宮頸癌細胞系中BLCAP能抑制STAT3信號通路的活化。由于A-IRNA編輯替換了 BLCAP YXXQ基序中的兩個重要氨基酸,我們模擬A-I RNA編輯的情況構(gòu)建了兩個BLCAP突變體(CCLQ-FLAG和CCLR-FLAG)進一步探索A-I RNA編輯在BLCAP抑制STAT3磷酸化的過程中的作用。免疫共沉淀實驗結(jié)果顯示,突變體CCLQ-FLAG與STAT3的相互作用弱于野生型(BLCAP-FLAG),突變體CCLR-FLAG對STAT3的相互作用弱于CCLQ-FLAG。在HeLa和C33A細胞上過量表達野生型和突變體,突變體CCLR-FLAG和CCLQ-FLAG對STAT3的磷酸化作用增強;對STAT3下游基因進行檢測,我們也得到相同的結(jié)果。以上結(jié)果說明,A-IRNA編輯事件通過編輯BLCAP YXXQ的兩個重要位點,使BLCAP喪失了對STAT3信號通路的抑制作用。本研究中為BLCAP發(fā)揮其抑癌功能提供了新的機制,同時證明了 A-IRNA編輯事件通過改變BLCAP的重要編輯位點導(dǎo)致蛋白質(zhì)功能的改變,從而對宮頸癌的發(fā)生發(fā)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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.33

【參考文獻】

相關(guān)期刊論文 前1條

1 Qian-Rong Qi;Zeng-Ming Yang;;Regulation and function of signal transducer and activator of transcription 3[J];World Journal of Biological Chemistry;2014年02期

，

本文編號：1603002