| Nasopharyngeal carcinoma(NPC)is one of the commonest malignant tumors in Southeast Asia and southern China that origins from mucous membrane of nasopharynx,with an incidence of 25-50 per 100,000 populations.The pathological mechanisms responsible for the initiation and progression of NPC have been attributed to EBV infection,genetical predisposition and exposure to certain chemical carcinogens.Despite the regeneration and improvement of the treatment for NPC,the 5-year survival of patients does not improve very much,one of the reasons depend on its early metastasis.Once the cervical lymph node metastasis occurs,the 5-year survival tends to less than 50%,if long-distance metastasis,it left only 20%.Therefore,identify the key molecular,elucidate the molecular mechanism,and adopt effective treatment to control the metastasis are critical for the successful therapy of NPC.Epstein-Barr virus(EBV),as a ubiquitous herpesvirus that can be detected in more than 95%of the undifferentiated and non-keratinizing NPC.Therefore,the mechanism of EBV in the development of NPC has not only been the key point,but also been the breakpoint for early diagnosis,new medical treatment and prognostic.Previously researches have reported the relationship between EBV and NPC in multiple levels,however,there is not enough evidence to reveal its role in the progression of NPC.To date numerous studies have focused on exploring the functions of viral latent proteins,such as LMP1 and LMP2A,for their tumorigenesis and cancer progression properties.Whereas,a large sample meta-analysis study reported that the detected percentage of LMP1 and LMP2A in NPC just was more than 90%.What’s more,among the LMP1 positive patients only 66.75%of them encountered metastasis,and the metastasis percentage were still 46.98%in LMP1 negative ones,suggesting that the pathogenesis and progression of these tumors likely depends upon factors beyond the expression of viral proteins.It is well known that miRNAs are one of the important regulatory factors participate in different progessions,including cell differentiation and apoptosis,tissue development,and disease(including malignant tumors),in post-transcription or translation level.It is has been found that the virus-encoded miRNAs hold extremely similar biological processing and induced silencing complex functions as human miRNAs,and play important roles in the process of viral escape and latent infection by regulating gene expression of their own or their host.Epstein-Barr Virus(EBV)is the first virus found to be able to encode miRNAs.In 2004,Pfeffer et al firstly isolated small noncoding RNAs from EBV,they belonged to the human herpesvirus family,these miRNAs had been studied and revealed that they played roles in the process of host infection,and were named as EBV-miR-BHRF1-1,EBV-miR-BHRFl-2,EBV-miR-BHRFl-3,EBV-miR-BART1 and EBV-miR-BART2,these were the earliest discovery of virus-encoded miRNAs.In the following year,Pfeffer et al identified 33 virus-encoded miRNAs from the 29 kinds of viruses with computer analysis and experimental research.The findings of Pfeffer et al led the viral miRNA studies into a new research focus,opened a new research branch provided a new direction and new content for viral miRNAs regulating host and viral gene expression,and promoted an important step in the interaction model between virus and host cells.To date,most of the identified virus-encoded miRNAs belong to the tumorigenic virus,therefore,it can be speculated that there is an extremely relationship between viral miRNAs and tumor genesis and development.The neodoxy of virus tumorgenic thinks that the sequences of the viral miRNAs are directed to the tumor suppressor genes of host cells.For example,EBV-eneoded miR-BHRF1 bears the potential sites combined with the tumor suppressor P53 and the binding sites of apoptosis regulating factor Bcl-2 mRNA in the 3’ end of non-coding region involved in the regulation of host cell apoptosis and proliferation,which may lead to disorders of host tumor suppressor gene loss of function and immune surveillance function,eventually causing the development of tumors.Another report prompted that EBV-miR-BART5 could combine with PUMA gene of host cells and inhibit cell apoptosis.In the aspect of self regulation,it is predicted that EBV-miR-BART2,which is from the antisense strand of transcription of DNA polymerase(BALF5),contains a fully complementary binding sites of BALF5 3’UTR,may guide BALF5 mRNA degradation and BALF5 expression,leading to a latent infection in host.Previously studies showed that the 3’UTR sequences of the degradation product(3.7 kb)processed from a full-length 5.0kb BALF5 mRNA were consistent with the cleavage sites(predicted by Pfeffer et al)guided by miR-BART2.Furthermore,miR-BART2 can bind with the 3 ’UTR of BMLF1(EB2)mRNA,miR-BART1 can bind with the 3’ UTR of BBLF4 and LMP2A mRNA,so as to achieve the purpose of the regulation of viral gene expression;in the immune regulation,EBV-miR-BHRF1-3 can bind with CXCL-11(an interferon-induced T cell chemokine)to evade immune surveillance.However,many of the related studies are still localized on the analysis the profiling of EBV miRNA in NPC,or the prediction of partial target genes and its preliminary studies.The role of EBV encoding miRNAs in the development of NPC,especially the biological role and regulatory mechanism in the process of invasion and metastasis are new topics need to be explored.1.miRNA profiling analysis and real time qPCR validation of miRNAs in human NPC and chronic nasopharyngitis specimensmiRNA microarray can be used to identify the differentially expressed miRNAs,and bring new hopes to the early diagnosis and treatment of tumor.To better understand the roles of miRNA in the process of malignant progression and metastasis in NPC and identify the marker suppressing the early metastasis of NPC,we analysised miRNA expression profiling in 20 poor or undifferentiated NPC and 20 CNP specimens using a miRNA microarray platform.As a result,we identified 70 miRNAs whose expression levels were significantly altered in NPC samples((universal test:P<0.005,fold-changes>1.5;false discovery rate:<0.05;).Among the significantly altered miRNAs,46 miRNAs were significantly downregulated and 24 miRNAs were significantly upregulated.Notably,EBV-miR-BARTs miRNAs held most of the up-regulated ones in NPC,taking up 62.5%(15/24)of upmodulated miRNAs.What’s more,some of the EBV-miR-BARTs showed extremely higher expression in NPC than in CNP.The fold changes of EBV-miR-BART1 EBV-miR-BART7,EBV-miR-BART3,EBV-miR-BART10,EBV-miR-BART8,EBV-miR-BART9 and EBV-miR-BART5 were more than 7.8-fold increased,of which EBV-miR-BART1 was ranked No.1 in NPC compared with CNP with 19.27 fold-increase.Due to the results of the expression profile of the chip may be false positive or false negative,we need to verify the microarray analysis of the data with other methods.To validate the microarray data,we estimated the relative expression levels of selected EBV-encoded BART-miRNAs and human miRNAs in the same NPC and CNP samples used for miRNA microarray with Taqman RT-qPCR.Consistently,EBV-encoded BART-miRNAs and human miRNAs showed significant difference in NPC and in CNP(Independent t-test,P<0.05).The similar tendency of these miRNAs between the RT-qPCR and our miRNA microarray analysis confirmed that the result of miRNA microarray is credible,and can be used for the following studies.2.The roles of the differentially expressed EBV-miR-BART1 in the tumorigenesis and development in NPCWe identified a batch of up-regulated miRNAs,correlated with the tumorigenesis and development of NPC,using a miRNA platform containing miRNAs from multiple species.EBV-miR-BART1 is one of highly expressed EBV BART miRNAs in NPC and its functional effects on NPC progression is far less clear,we selected this viral miRNA for further investigation.EBV-miR-BART1 is an EBV-encoding miRNA encoded by EBV BART gene,and we identified that it is upregulated in NPC tissues with miRNA microarray.In current study,an additional 27 NPC with TNM stage and 12 CNP samples dignosised by pathologist were used to validate that the expression levels of EBV-miR-BART1-5p and EB V-miR-BART1-3p were significantly upregulated(t=-3.749,P<0.001;t=-4.094,P<0.001,respectively)in NPC with Taqman RT-qPCR techniques.Statistic analysis indicated that there was a significantly positive correlation of EBV-miR-BART1 with N stage was observed in NPC patients(P<0.05),as well as clinical stages.To further investigate EB V-miR-BART1’s role in the process of metastasis and invasion in NPC.Applying of miRNA lentivirus and miRNA inhibitors strategies.We infected EBV negative NPC cell lines(CNE1,5-8F)with pre-EB V-miR-BART1 expression lentivirus and then we transfected miRNA inhibitors,which were specific for the two mature sequences separately,into EBV-miR-BART stably expressed CNE1-BART1 and 5-8F-BART1 cells,to analysis the effects of EBV-miR-BART1 on NPC cell lines.The colony formation assay was used to analysis colony-fomation ability of NPC cell lines with EBV-miR-BART1 overexpression.The results showed that,the colony formation efficiency was increased 31.2%(t=-5.391,P<0.001)in CNE1-BART1/MOCK and 39.5%(t=-13.328,P<0.001)in 5-8F-BART1/MOCK cells respectively.As domenstrated in FACS cell cycle analysis,EBV-miR-BART1 overexpression could promote NPC cells to proliferation phase and improved cell proliferation ability in vitro.We also performed tumor growth assay in nude mice.The results showed that EBV-miR-BART1 overexpression could improve NPC cell proliferation abilities in vivo(P<0.05).Transwell and Boyden assays were used to detect effect of EBV-miR-BART1 overexpression in NPC cell migration,the results domenstrated that the migration abilities were significantly upregulated by 2.87-fold(t=-7.113,P<0.01)in CNE1-BART 1/MOCK and 1.76-fold(t=-5.513,P<0.001)in 5-8F-BART1/MOCK cells,and the invasion abilities were significantly upregulated by 1.85-fold(t=-6.226,P<0.001)in CNE1-BART 1/MOCK and 1.69-fold(t=-7.003,P<0.001)in 5-8F-BART1/MOCK cells.Wound healing results,being consistent with migration assays,showed that EBV-miR-BART1 overexpression could strengthen the migration abilities of NPC cells.The results of xenograft NPC cells into the liver’s envelope of nude mice revealed that EBV-miR-BART1 overexpression could enhance lymph metastasis abilities of NPC cells.In addition,miRNA inhibitors were used to inhibit EBV-miR-BART1 expression in EBV-miR-BART1-stably-expressed cells,and following that we detected the migration abilities of NPG cells.Correspondingly,the results indicated that cell migration abilities were significantly downregulated(P<0.05).Moreover,the homologus inhibitors of EBV-miR-BART1 two mature sequence could inhibit cell invasion and metastasis in varying degress respectively.Meanwhile,a synergetic restriction of cell migration obviously appeared in co-treatment with two miRNA-inhibitors(P<0.05).Collectively,these data supported that EBV-miR-BART1 involved in the development of nasopharyngeal carcinoma,its expression imbalance can promote the proliferation,invasion and metastasis of nasopharyngeal carcinoma cells in vitro,also EBV-miR-BART1 upregulated the tumor formation ability and lymph node metastasis of nasopharyngeal carcinoma cells in vivo.EBV-miR-BART1 acts as a candidate oncogene in NPC.3.Figure out the host target of EBV-miR-BART1In current study,we selected EBV-miR-BART1 overexpression NPC cells(CNE-BART1)and its corresponding control cells(CNE1-MOCK)as suitable cell model,applied high-throughput deep sequencing technique,combining biological informatics analysis and literature retrieval to screen out the host target genes of EBV-miR-BART1.Real-time quantitative PCR was used to verify relative expression of the candidate target gene in EBV-miR-BARTl overexpression and its corresponding control cells,luciferase reporter assay was utilized to detect the effect of candidate target gene on luciferase abilities,immunocytochemistry technique was applied to identify the expression of target gene in EBV-miR-BART1 overexpression and its corresponding cells.Expressions of target gene in NPC and CNP tissues were measured by RT-qPCR.Appling RNA deep-sequencing high-throughput techniques,comparing the sequencing result of CNE1-BART1 group and CNE-MOCK group cells,we totally acquired 1550 altered genes,expressed more than 2-fold change,correlated with the effect of EBV-miR-BARTl.Among them,there were 555 and 995 genes positively and negatively correlated with EBV-miR-BART1 overexression respectively.The GOfact and KEGG pathway analysis indicated that they participated in regulation of diverse pathways,including Focal adhesion,cell cycle,cell adhesion,P53 signaling pathway,pathway in cancer,MAPK,ECM receptor,ErbB,Wnt and TGF-beta signaling pathways etc.Since these pathways involved in multiple steps during the development of tumor development.we speculated that EBV-miR-BART1 overexpressed in NPC cells could regulate gene expression with direct or indirect manners and then induced alteration of biological characteristics of NPC cells.Some mainly invasion and metastasis related genes are included,such as PTEN,N-RAS,CHUK,BAX,TNFRSF10D,CDK1,CYCS,ROCK1,MGST2 and PPP1C etc.Pathways associated with invasion and metastasis included pathway in cancer,p53 pathway,cell adhesion signaling pathway,Wnt signaling pathway and VEGF signaling pathway etc.We next acquired PTEN(phosphatase and tensin homolog),as a possible target gene of EBV-miR-BART 1,via literature retrival and RNAhybrid bioinformatics prediction.In addition,bioinformatics prediction showed that both of EBV-miR-BARTl mature sequences could combined with PTEN 3’UTR,suggesting that PTEN was a candidate target gene of EBV-miR-BART1.To validate this hypothesis,we constructed the recombinant plasmid pEZX-wt PTEN 3’UTR and two mutant vectors corresponding to the separated binding sites of EBV-miR-BART1-5p and EBV-miR-BARET1-3p,named as pEZX-mutl PTEN 3’UTR.pEZX-mut2 PTEN 3’UTR respectively,and performed dula luciferase reporter assay.The results showed a significant decrease of luciferase activity when EBV-miR-BART1-5p,1-3p and pEZX-wt PTEN 3’UTR vector were cotransfected in 293T cells(F=233.209,P<0.001),suggesting EBV-miR-BART1 could directly target the 3’UTR of PTEN.Next,we examined the expression of PTEN in EBV-miR-BART 1 overexpression cells and their corresponding control cells with immunocytochemistry.The results indicated that PTEN was downregulated in EB V-miR-BART 1 overexpression cells,compared to their control ones.Moreover,we detected the relative expression level of PTEN in NPC and CNP tissues.Compare to the expression in CNP tissues,the mean level of PTEN was downregulated 7.09-fold(t=4.750,P<0.001),what’s more,correlation analysis indicated that mRNA level of PTEN was negatively correlated with both EB V-miR-BART l-5p and 1-3p(t=-0.493,P<0.01;t=-0.512,P<0.01,respectively).Taken together,PTEN was a target gene of EB V-miR-BART 1 in NPC.To elucidate whether the metastasis and invasion effect of EBV-miR-BART1 was mediated by repression of PTEN in NPC cells,we performed gain-of-function studies.Transwell and Boyden assays showed that ectopic expression of PTEN significantly decreased EBV-miR-BARTl-overexpression induced cell migration and invasion increase(P<0.05).These effects were similar with the effects induced by EBV-miR-BARTl s inhibitors.Collectively,these results suggested that the migration and invasion increase effect of EBV-miR-BART1 was mediated by repression PTEN in NPC cells.4.The molecular mechanisms of metastasis and invasion in NPC induced by EBV-miR-BART1PTEN(Phosphatase and tensin homolog),reported for the first time since 1997,is the first ever found tumor suppressor gene which possessed a dual specificity phosphatase activity.It is another widely known tumor suppressor gene closely related to tumor development following P53.PTEN protein acts important roles in cell growth,apoptosis,adhesion,migration,invasion,it is also a prognosis evaluation index for many tumors.To investigate the exact regulatory mechanisms of PTEN’ down-modulation regulated by EBV-miR-BART1 overexpression,We applied western blotting and immunofluorenscence techniques to identify the alteration of molecular in related signaling pathways.Western blotting indicated that EBV-miR-BART1 overexpression could down-regulate PTEN protein level,up-modulate phosphorylation levels of its downstream molecular,including FAK,Shc,ERK and AKT etc.And then boost up cell ability,promote migration of NPC cells.Based one these data,we stained F-actin with FITC-phalloidin in EBV-miR-BART1 overexpression cells and their corresponding controls.Confocal microscopy were used to observe the shape of intracellular cytoskeletal proteins.Confocal microscopy displayed a higher densityand better organization of F-actin filaments in EBV-miR-BART1-stably-expressed cells,while in the corresponding control cells microtubules structures were scarce,disarranged.This phenomenon prompted that EBV-miR-BART1 could regulate PTEN and its downstream signals,proceeding rearrangement of F-actin to remodel cellular movement potential,finally leading tumor metastasis.Conlusion:1.Using miRNA expression profiling analysis we identified 70 dysregulated miRNAs between NPC and CNP tissues,and there were 46 upregulated and 24 downregulated miRNAs.Moreover,among the upregulated miRNAs,the EBV-miR-BARTs took up 62.5%(15/24),prompted that upregulation of EBV-miR-BARTs might be important biomarkers of the development and progression of nasopharyngeal carcinoma..2.EBV-miR-BART1 promoted ability of cell proliferationand clony formation,and also increased potential of cell migration and invasion.EBV-miR-BART1 was related with lymph node metastasis,clinical stage of patients with NPC.3.In this study,tumor suppressor gene PTEN was identified as a target gene of EBV-miR-BART1.Low expression of PTEN could promote lymph node metastasis and clinical progress in NPC,ectopic expression of PTEN could eliminate migration abilities induced by EB V-miR-BARTl overexpression.4.Downregulation of PTEN could upregulate the phosphorylation of downstream genes:FAK、Shc、ERK and AKT,then led to actin rearrangement of cytoskeleton and epithelial-meschymal like transition and jointly contributed to NPC metastasis. |
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