【作者】 李子坚；

【导师】 曹亚； 孙仑泉；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 miRNA(microRNA)是近年来发现的一类长约22nt的非编码小RNA分子。在胞浆内与特定蛋白形成微核酸蛋白复合体(miRNPs,micro-ribonucleouproteins),通过对靶基因mRNA的序列特异性裂解、抑制翻译的起始或延长、降解多聚腺苷酸尾或者介导肽链水解等机制在翻译水平抑制其靶基因的表达。但是,人们对miRNA在形成蛋白复合体并靶向调节基因的过程中的具体机制,仍然知之甚少。miRNAs广泛存在于从病毒到高等多细胞生物,目前采用生物信息学预测,在低等多细胞动物和植物中存在一两百条miRNAs,而在人类可能存在至少一千条。对miRNAs功能的研究提示几乎参与所有细胞内进程的调节,在包括肿瘤在内的人类所有病理过程中都能观察到其表达的异常。这一发现与生物信息学所预测的,哺乳动物中约30%蛋白编码基因受到miRNAs的调节相一致。病毒在感染宿主细胞后,需要调节自身和宿主两方面的基因表达,使其自身存在得以维持和复制,潜伏感染以维持自身存在,部分进入复制周期以使自身繁殖扩增,两者之间的平衡,避免了自身过多复制引起宿主细胞的死亡。这种平衡对病毒来说很重要。多数病毒也能编码miRNAs并广泛参与病毒与宿主的相互作用。EB病毒是人类疱疹病毒,与包括鼻咽癌在内的多种人类疾病密切相关。该病毒目前发现编码至少32条miRNAs,据其在基因组中的位置分为两组:BHRF1组和BART组。其中BHRF1组位于病毒复制早期基因BHRF1内,BART组则位于病毒潜伏期转录产物BART内。BART组miRNA已证实可以抑制病毒复制,维持其较低的复制水平。BHRF1组在病毒裂解复制水平增高时表达增加,提示其与病毒裂解复制的调控相关。但是,生物信息学分析并没有发现BHRF1组miRNA在病毒裂解复制期基因中存在作用位点,只有ebv-miR-BHRF1-1在人p53mRNA 3’-非编码区(3’-UTR,3’-Untranslated regions)存在两个作用靶位点。P53是重要的抑癌基因,在多种肿瘤中存在突变。但是,在鼻咽癌中p53基因的突变频率很低(＜10%),并出现了p53蛋白积聚现象。作为调节细胞周期进程和细胞凋亡的重要基因,多数病毒在宿主细胞内进行自身复制时,需要通过不同的途径来抑制p53的功能,来使宿主细胞内环境处于类S期,并抑制细胞的凋亡,为病毒自身复制提供原料和赢得时间。而且研究发现,EB病毒裂解复制立刻早期基因蛋白Zta,作为病毒裂解复制的开关基因,在病毒进入裂解复制周期时可以和p53结合,抑制其转录激活功能。说明EB病毒在复制过程中同样需要抑制p53的功能以为其自身复制提供有利条件。那么野生型p53蛋白在鼻咽癌中的积聚,可能在鼻咽癌中EB病毒低水平复制的维持中具有重要意义。由此,我们推测:EBV编码的ebv-miR-BHRF1-1可能通过调节宿主细胞相关基因p53的表达,进而调节p53与Zta相互作用而参与调节EBV潜伏与裂解两种状态的转换。我们采用TPA诱导SUNE1内病毒裂解复制水平增高后,采用RT-PCR方法证实在病毒裂解复制水平增高后,ebv-miR-BHRF1-1的表达也随之上调,而p53蛋白水平和下游基因mdm2水平随之下调,同时细胞周期检测证实S期细胞增加而凋亡细胞减少,ebv-miR-BHRF1-1的上调与p53的下调及功能受抑有利于病毒复制,且两者之间存在联系。然后,我们采用反义RNA特异性抑制ebv-miR-BRHRF1-1之后,再以TPA处理SUNE1,发现病毒裂解复制早期蛋白Zta和Ea-D上调幅度减小,说明裂解复制水平提高的幅度减小,而p53蛋白水平和mdm2水平的下调也减弱,mdm2水平甚至得到逆转,说明p53功能的受抑制程度也得到了逆转。接下来,我们构建ebv-miR-BHRF1-1的过表达载体,转染SUNE1细胞,观察GFP蛋白表达证实转染效率,以RT-PCR检测证实成熟ebv-miR-BHRF1-1得到表达,再检测病毒复制早期蛋白Zta和Ea-D水平以及p53蛋白和mdm2蛋白水平,比较对病毒复制水平的影响和对p53的抑制作用。发现在过表达ebv-miR-BHRF1-1后,病毒裂解复制水平提高而p53蛋白水平和功能受到了抑制。以上研究结果表明,EB病毒编码的miR-BHRF1-1通过调节宿主细胞相关基因p53的表达参与病毒裂解复制的调节,初步阐明EBV编码的miR-BHRF1-1参与调节EBV两种感染状态的平衡以及维持EBV的持续存在中的作用机制。为EBV相关肿瘤的发生机制提供新的理论依据,并为以EBV编码的miRNAs为切入点治疗EBV相关肿瘤的实验研究提供新的理论和实验依据。更多还原

【Abstract】 miRNAs(microRNAs)constitute a large family of approximately 22-nucleotide-long non-coding small RNAs that have been found in recent years,miRNAs have been known as key post-transcriptional regulators of gene expression through translational repression or mRNA degradation in metazoans and plants.However,the mechanistic details of the functions of miRNAs in protein synthesis repression are still poorly understood,miRNAs have been found to located from virus to multicellular organisms.About 100-200 miRNAs are expressed in lower metazoans and plants,but at least 1000 are predicted to operate in humans,miRNA are predicated to control the activity of approximately 30%of all protein-coding genes,which have been shown to participate in the regulation of almost all cellular process investigated so far and the changes in their expression are observed in human pathologies,including cancer.After infecting the host cell,virus regulate its and the host cell’s gene expression to maintain its existence and get moderate replication,in witch,latent infectious state maintains the infection and replication results in diffusion.It is very important that the balance between the two states can avoid the host cell death caused by excessive reproduce.Most of viruses have been found to encode miRNAs that widely participate in the host-virus interaction.Epstein-Barr virus,which belongs to the human herpes virus,has high correlation with many human diseases including nasopharyngeal carcinoma.Epstein-Barr virus encodes at least 32 miRNAs within two clusters depending on their gene location in the virus’ genome:the BHRF1-cluster and the BART-cluster.The BHRF1-cluster miRNAs located in the BHRF1,one of the lytic early genes,while the BART-cluster miRNAs is included in the transcripts named BART,which is a product of latent infection.It has been proved that the BART-cluster miRNAs can suppress virus reproduction to maintain the low reproduction of virus during latent infection.The high level of BHRF1-cluster miRNAs in cell lines with high level virus reproduction indicates that the BHRF1-cluster miRNAs may correlate to the regulation of lytic cycle.But bioinformatics predict that there are no potential target sites of the BHRF1-cluster miRNAs on the EBV encoded mRNAs.Interestingly,two target sites of miR-BHR1-1 lies in the 3’-UTR (3’-Untranslated regions)of p53 mRNA.P53 is an important tumor suppress gene which mutate in many neoplasm.But p53 mutation is infrequently observed in NPC biopsies. Meanwhile,accumulation of wild-type p53 is observed.During their reproduction many virus need to suppress the function of p53 via multiple mechanisms to provide an s-phase-like host cell environment and to prevent cell apoptosis to get enough time for their reproduction. Present investigation tells us that Zta,the product of EBV lytic immediate early gene BZLF1,can bind to p53 and suppress its transcript active function.It indicates that EBV needs to suppress the p53 function to create an advantageous environment for virus reproduction.So,the wild-type p53 with high level in NPC biopsies may have significance in the maintenance of virus latent infection in NPC.So we assume that the EBV encoded miR-BHRF1-1 may contribute to the induction of lytic cycle of EBV through inhibits the translation of P53 in NPC.First,our study showed that after being induced by TPA into lytic cycle the miR-BHRF1-1 increased while the p53 and its downstream gene production mdm2 decreased.Moreover,the percent of S-phase cell enhanced while apoptosis declined.These phenomena indicate that there may be relations between the up-regulation of miR-BHRF1-1 and the down-regulation of p53,and this relation may be involved in the lytic cycle of EBV in NPC.Second,after being transfected with anti-sense RNA targeted to miR-BHRF1-1,the inductive effect of TPA on EBV to lytic cycle in SUNE1 was retarded.At the same time,the suppression of the p53 and mdm2 after inducing EBV entry lytic cycle was decreased;what’s more, the suppression of mdm2 even reversed. Then,we constructed the expression plasmid of miR-BHRF1-1 and transiently transfected SUNE1 for 48 hours.After normalizing the transfective efficiency by observing the GFP expression under fluoroscopy,and we proved the expression of mature miR-BHRF1-1 by RT-PCR.Successively,we detected the Zta and Ea-D and proved that the reproduction of EBV was enhanced.Then we detected the p53 and mdm2 simultaneously and proved that the p53 was suppressed.The above data indicate that EBV encoded miR-BHRF1-1 are involved in the switch between different infective states of EBV to maintain the existence of virus by inhibiting the p53.And this discovery proves a new clue for pathogenesis of EBV-related neoplasm and provides new experimental base for research of using EBV encoded miRNA for the treatment of EBV related neoplasm.更多还原