| BackgroundNasopharyngeal carcinoma(NPC)is a malignancy derived from nasopharyngeal epithelium and is highly prevalent in Southern China and Southeast Asia.Nasopharyngeal carcinoma,an occult disease with a higher degree of malignancy,can frequently lead to occult and typical cervical lymph node metastasis in its early stage.Its occurrence is a complex process accumulating with multiple stages,multiple ways,multiple mechanisms and various genetic variations.At present,the major risk factors for NPC are EB virus infection,genetic susceptibility and environmental factors.Although radiotherapy can effectively control early stage NPC,the average 5-year survival rate after therapy is less than 30-40%in the patients with advanced NPC.Most seriously,due to no obvious symptoms at early stage,more than 60%of clinical NPC patients are easily overlooked and reach advanced stages.Thus,it is necessary to discover novel therapeutic targets and interventions for effectively treating NPC.MicroRNAs(miRNAs)are endogenous non-coding RNAs that suppress gene expression post-transcriptionally by targeting the 3'UTRs of specific mRNAs.They have emerged as important regulators of physiology and disease,and presented the important therapeutic potential.The aberrant expression of many miRNAs has been linked to human diseases including cancers.Recently,aberrant expression of some miRNAs,such as miR-184,miR-34b,miR-92a,miR-93,miR-27a,miR-101,miR-497,and miR-663,have been linked to various cancers including NPC,relating to multiple cellular responses such as cell proliferation,cell-cycle progression and tumorigenesis.To date,multiple cellular miRNAs,such as miR-148a,miR-155,miR-377,miR-92a,miR-200c,miR-23a,miR-130b,miR-30a,miR-149 and miR-93 have been reported to modulate the epithelial-mesenchymal transition(EMT)and metastasis in various cancers,but only three of them are implicated in the EMT in NPC.According to the latest literatures,many miRNAs have been related to sternness and drug resistance in various cancers.They are miR-22,miR-135a,miR-29c,miR-124,miR-142,miR-499,miR-133,miR-103/107 family,miR-24,miR-200a and so on,of which miR-200a is uniquely reported to promote stemness in nasopharyngeal carcinoma cells.Therefore,it is necessary to explore the effect and mechanism of miRNAs in NPC so as to provide promising bio-therapeutic targets.Epstein-Barr virus(EBV)infection is closely related to nasopharyngeal carcinoma.EBV DNA is detectable in most of patients with non-keratinizing NPC and in almost all of undifferentiated NPC.EBV,as a major etiological factor for NPC,encodes 25 viral miRNA precursors including 3 BHRF1 pre-miRNAs and 22 BART pre-miRNAs.Investigations gradually disclose that this virus can actively deploy its BART miRNAs to flexibly manipulate various viral and host cellular functions.Several EBV-encoded BART miRNAs have been associated with viral latency,immune escape,cell survival,cell proliferation and apoptosis.For example,EBV-miR-BART 1 influences multiple metabolism-associated genes in NPC.EBV-miR-BART2 suppresses viral DNA polymerase BALF5 to reduce EBV lytic replication.Both EB V-miR-B ART5-5p and miR-B ART 19-5p inhibit viral oncoprotein LMP1 expression,while EBV-miR-BART22 decreases LMP2A.Moreover,EBV-miR-BART15-3p suppresses an apoptosis inhibitor BRUCE to induce apoptosis and miR-BART3*targets a tumor suppressor DICE1 to promote cellular growth and transformation in NPC.These findings open up a promising therapeutic option and encourage more studies to develop novel miRNA-based therapies for NPC.As a member of BART-miRNA family,EBV-miR-BART7-3p is emerging as one of the most important regulatory factors in NPC.Other two investigations reported the high EBV-miR-BART7-3p expression in NPC tissue/plasma samples and its correlation with NPC cell proliferation in vitro,suggesting its involvement in NPC tumorigenesis though the underlying molecular mechanism still remains unclear.In the present study,we firstly screened out the most differentially expressed EBV-encoded miRNAs between NPC tissue samples and non-cancerous nasopharyngeal tissue(NP)tissue samples by using in-house miRNA array detection.We further observed that the high expression of EBV-miR-BART7-3p is positively related to the degree tumor size(T stage),the degree of spread to regional lymph nodes(N stage)and clinical stage of NPC by using real-time PCR(qPCR)assay.These results reveal that EBV-miR-BART7-3p may play a role in the development of NPC.Secondly,we established NPC cell lines stably overexpressing this EBV miRNA via lentivirus infection.We observed the change in the proliferation,migration,invasion and stem-like ability of NPC cell lines in the presence of upregulated EBV-miR-BART7-3p.Finally,according to the literature search and computational prediction identifying human host target genes of EBV-miR-BART7-3p and related signaling pathways,we investigated the molecular mechanism underlying its biological effects.Accordingly,based on the above researches,we carried out a therapeutic experiment by inhibiting EBV-miR-BART7-3p,providing a theoretical basis for new strategies of NPC treatment.Research Objectives1.To explore the biological functions of EBV-miR-BART7-3p in NPC,and provide the theoretical basis for molecular mechanism and therapeutic target in NPC.2.To identify EBV-miR-BART7-3p target gene in human host.3.To investigate the molecular mechanism of EBV-miR-BART7-3p targeting host genes and the downstream signaling pathways.4.Using EBV-miR-BART7-3p as a therapeutic target for in vivo and in vitro experiment,to identify whether EBV-miR-BART7-3p can be a new target for the treatment of patients with NPC.Methods and resultsPart ? EBV-miR-BART7-3p promotes the EMT and metastasis of NPC cells by suppressing the tumor suppressor PTENMethods1.Applying miRNA microarray technology to detect the differentially expressed viral miRNAs with the highest fold-change between NPC and NP,and then verifying results by qPCR.2.Establishing NPC cell lines stably overexpressing EBV-miR-BART7-3p(CNE1-BART7-3p and 5-8F-BART7-3p)by the construction and infection of lentiviral vector.3.Conducting a series of in vitro(transwell and boyden chamber)and in vivo(liver capsule injection of mice)experiments to evaluate the effect of EBV-miR-BART7-3p on NPC invasion and metastasis.4.Using morphological observations,western blot assay and immuno-histochemistry(IHC)to explore the effect of EBV-miR-BART7-3p on the EMT in NPC cells.5.Predicting the host target genes of EBV-miR-BART7-3p by using literature retrieval and bioinformatics,further confirming target genes by performing luciferase reporter assay,western blot and IHC,and evaluating the correlation between EBV-miR-BART7-3p and target gene by using qPCR assay.6.Using western blot,IHC and immunofluorescence(IFC)to determine the downstream signaling pathway of EBV-miR-BART7-3p-regulated target gene,and then detecting the alterations in phenotype,function and underlying mechanism after transfecting siRNA or target gene expressing(not including 3'UTR)vector.7.Silencing the endogenous EBV-miR-BART7-3p in the EBV positive NPC cells and detecting the alteration in function and mechanismResults1.We conducted a miRNA expression profiling analysis in 16 NPC tissue samples and 20 non-cancerous nasopharyngeal tissue(NP)samples and found EBV-miR-BART7-3p was highly expressed in NPC samples relative to NP samples.Furthermore,in additional 42 NPC patients,we observed a positive correlation of EBV-miR-BART7-3p expression with the degree of spread to regional lymph nodes(N stage)and clinical stage of NPC2.In order to explore the role of EBV-miR-BART7-3p in nasopharyngeal carcinoma,using lentiviral particles carrying EBV-miR-BART7-3p precursor,we generated two EBV-negative NPC cell lines stably expressing EBV-miR-BART7-3p(CNE1-BART7-3p and 5-8F-BART7-3p cells)and the corresponding control cells(CNE1-NC and 5-8F-NC cells).The expression levels of EBV-miR-BART7-3p in these two stable cells were within a similar physiological range to pooled NPC tissue samples by qPCR assay.3.In vitro migration assays revealed that the exogenic expression of EBV-miR-BART7-3p significantly enhanced the migration of these two cell lines,consistent with the results of the invasion assays using a matrigel invasion chamber system.Conversely,silencing of EBV-miR-BART7-3p attenuated the migration and invasion of stably NPC cells.5-8F-BART7-3p and its control cells(5-8F-NC)were transplanted under the liver capsule of mice.The average number of metastatic lymph nodes and intrahepatic metastasis was significantly bigger in 5-8F-BART7-3p cell-injected mice4.We observed that EBV-miR-BART7-3p led to the deceased expression of epithelial marker(E-cadherin)and increased expression of mesenchymal markers(vimentin and fibronectin)in two NPC cell lines along with the EMT-like morphological changes identified by relatively losing cell-to-cell adhesions and acquiring spindle-like phenotype.Using immunohistochemistry assay to detect the expression of EMT markers,we observed the loss of E-cadherin and the increased expression of vimentin,FOXM1 and Fibronectin in EBV-miR-BART7-3p-induced tumor tissues relative to control tumor tissues.5.After using bioinformatics and literature retrieval to determine target gene,luciferase reporter assays identified EBV-miR-BART7-3p directly targeted human PTEN gene.Western blot and qPCR showed that the expression level of PTEN was down-regulated in EBV-miR-BART7-3p-stably expressing NPC cells;Furthermore,compared with NP tissue,the expression of PTEN was down-regulated in NPC tissues,and negatively correlated with EBV-miR-BART7-3p expression(r=-0.7229,p=0.0125).6.Western blot showed that EBV-miR-BART7-3p suppressed PTEN,thereby activating PI3K/Akt/GSK-3? signaling pathway.IHC and IFC assays displayed that EBV-miR-BART7-3p promoted snail and ?-catenin into the nucleus and enhanced the metastasis and invasion of NPC cells.After transfecting siRNA of PTEN into NPC cells,the ability of metastasis and invasion were increased and signaling pathway was also modulated accordingly.After transfecting PTEN vector(not including 3'UTR)into CNE1-BART7-3p and 5-8F-BART7-3p cells,the ability of metastasis and invasion were enhanced and PI3K/Akt/GSK-3?signaling pathway was down-regulated.7.After silencing of EBV-miR-BART7-3p expression in HONE1-EBV cells,transwell and boyden chamber experiment showed that metastasis and invasion of NPC cells were decreased and PI3K/Akt/GSK-3? signaling pathway was suppressed.After transfection of PTEN vector(not including 3'UTR),the metastasis and invasion of HONE1-EBV cells were decreased.Part ? EBV-miR-BART7-3p enhances the growth and proliferation of NPC cells via stimulating transcription factors c-Myc and c-JunMethods1.Using MTT,plate clone formation assay,cell cycle assay and in vivo experiment to detect the impact of EBV-miR-BART7-3p on the growth and proliferation in NPC cells.2.Using western blot and qPCR assay to verify the downstream pathway of target gene which was regulated by EBV-miR-BART7-3p,and detecting the alteration in function and mechanism of NPC cells transfected with siRNA silencing the expression of target gene.Results1.MTT and colony formation assays showed that this viral miRNA significantly promoted cell growth,proliferation and colony formation.Flow cytometric evaluation displayed that these two stable cell lines had an increased proportion of G2 phase and a decreased proportion of G1 phase relative to the control cell lines.Contrarily,after the transfection with EBV-miR-BART7-3p inhibitor(anti-miR),these two stable cell lines presented an obviously reduced cell growth and colony formation as well as an increased G1 phase and fewer cells in G2 phase.2.The mice injected with 5-8F-BART7-3p cells appeared to carry larger tumor burdens and display relatively higher expression levels of Ki67 and PCNA in tumor tissues relative to the controls.3.Western blot assay indicated that EBV-miR-BART7-3p stimulated PI3K/Akt/c-myc and c-Jun through suppressing PTEN in NPC cells.The levels of cyclins and cyclin-dependent kinases were generally increased more than 2-fold upon EBV-miR-BART7-3p overexpression,whereas the levels of cell cycle inhibitor were decreased at least 2-fold by qPCR assay.The opposite results appeared in 5-8F-BART7-3p and CNE1-BART7-3p cells after treating cells with anti-BART7-3p.4.Si-PTEN-transfected CNE1 and 5-8F-cells presented a higher ability to grow and proliferate relative to the control cells.Similarly,flow cytometry analysis revealed a decreased percentage of G1 phase cells and increased percentage of G2 phase cells in two NPC cell lines after introducing si-PTEN.Western blotting assay confirmed a reduced PTEN protein expression,followed by an obviously induced expression of p-Akt,c-Myc,c-Jun,CCND1 and CCNE1.Part ? EBV-miR-BART7-3p promotes the stem-like phenotype and reduces the sensitivity of chemotherapeutic drugs in NPC cells by suppressing Smad7Methods1.Using lentiviral infection to construct stable NPC cells(CNE2-BART7-3p and 5-8F-BART7-3p),and then detecting the transfection efficiency by qPCR assay using the NPC tissue as the positive control.2.Using in vitro(culturing tumor spheres,sorting side population cells)and in vivo(tumor formation rate of nude mice)experiments as well as western blot and qPCR assays to evaluate the effect of EBV-miR-BART7-3p on sternness in NPC cells.3.Using clinical first-line chemotherapy drugs(cisplatin,fluorouracil and paclitaxel)in NPC cell lines to compare the drug sensitivity of CNE2-BART7-3p and 5-8F-BART7-3p cells with control cells.Using an intraperitoneal injection of chemotherapeutic drugs to treat nude mice with subcutaneous tumors,and then evaluating therapy effect.4.Preliminarily predicting the other host target genes of EBV-miR-BART7-3p by literature retrieval and bioinformatics approach,and further confirming target genes by lucifearse reporter assay and western blot.5.Applying western blot,IHC,IFC assays to determine Smad7-related signaling pathway regulated by EBV-miR-BART7-3p,and further validating results in EBV positive NPC cells.After transfection with siRNA or target gene(not including 3'UTR)expression vector,detecting associated phenotypes,functions and related mechanism.Results1.Using lentiviral particles carrying EBV-miR-BART7-3p precursor,we generated two NPC cell lines stably expressing EBV-miR-BART7-3p(CNE2-BART7-3p and 5-8F-BART7-3p cells).The expression levels of EBV-miR-BART7-3p in these two stable cells were within a similar physiological range to pooled NPC tissue samples.2.Compared with the corresponding control group,it was in stably NPC cells that the number/size of tumor spheres and the number of side population cells were increased.After transfecting EBV-miR-BART7-3p inhibitor into HONE1-EBV and HK1-EBV cells,the number of side population cells decreased.Western blot and qPCR showed that "stemness"-associated markers(ABCG2,Oct4,Nanog and Sox-2)were up-regulated.In vivo experiment displayed an increase in the tumor formation rate for BART7-3p staly expressing cells after the different amounts of NPC cells were injected into the nude mice subcutaneously.3.Drug sensitivity test showed that stably NPC cells grew faster and less died than the control group.In vivo experiment also displayed that,after the intraperitoneal injection of cisplatin in nude mice with BART7-3p cells-derived tumors,the tumor grew more rapidly with a larger volume compared with the control group.4.Using bioinformatics and luciferase reporter assay,we found another target gene Smad7 regulated by EBV-miR-BART7-3p.Western blot and qPCR validated that Smad7 expression level was down-regulated in stably NPC cells.Further qPCR assay showed that Smad7 expression was down-regulated in NPC tissues compared with the NP tissue,and negatively correlated with EBV-miR-BART7-3p expression(r=-0.7229,p<0.001).5.Western blot showed EBV-miR-BART7-3p targeted Smad7 and activated TGF-?signaling pathway.IFC supported that EBV-miR-BART7-3p promoted p-Smad2 and p-smad3 into the nucleus and enhanced the stem-like phenotypes of NPC cells.6.After transfecting siRNA into NPC cells,the ability to form tumor sphere was increased and western blot showed an activated signaling pathway.After transfecting Smad7 expression vector(not including 3'UTR)into CNE2-BART7-3p and 5-8F-BART7-3p cells,the ability to form tumor sphere was enhanced as well and TGF-? signaling pathway was downregulated.Part ? Gold-nano-particles(AuNPs)carrying anti-EBV-miR-BART7-3p inhibit the growth of EBV-positive NPCMethods1.Using a layer-by-layer method of nanotechnology to construct EBV-miR-BART7-3p antisense nucleotide(nano-anti-miR,carrying GFP),and then employing transmission electron microscopy and dynamic light scattering to observe its morphology,stability and potential2.Using confocal microscopy to observe the transfection efficiency,and qPCR-verifying the results3.Transfecting Gold-nano particles carrying EBV-miR-BART7-3p antisense nucleotide into positive-EBV NPC cells,and using a series of in vitro experiments(such as MTT and western,blot)to evaluate the alteration in cell growth and proliferation.4.Injecting Nano-anti-miR into the solid tumor of each mouse model,and then observing tumor growth.Results1.We fabricated a nano-carrier(gold-PEI)using a layer-by-layer method for delivering anti-EBV-miR-BART7-3p into HONE1-EBV cells.The size of Gold-PEI nano-carrier was about 20-30 nm as measured by dynamic lighting scatter(DLS).The zeta potential of gold-PEI was positive charge(about 20 mV).The nano-anti-miR displayed an approximately spherical shape with good dispersion and its size was similar to that of gold-PEI observed by TEM.2.There were much more visible green fluorescence particles in HONE-EBV cells transfected with nano-anti-miR by confocal microscopy observation.The inhibition efficiency of nano-anti-miR was evaluated by qPCR,revealing that nano-anti-miR effectively inhibited EBV-miR-BART-3p expression level in HONE-EBV cells.3.After transfection of nano-anti-miR in HONE1-EBV cells,MTT experiments showed that the cell growth rate were decreased(P<0.001)and PI3K/Akt signaling pathway was downregulated.4.The nano-anti-miR was further injected into the solid tumor originating from HONE1-EBV cells of each mouse per three days.We observed that nano-anti-miR obviously inhibited tumor growth compared with NC control or nano-carrier groups.Western blot assay confirmed that PTEN expression was decreased in tumor tissues,whereas p-Akt,c-Myc and CCND1 were increased upon the treatment of nano-anti.Conclusions1.EBV-miR-BART7-3p is expressed and positively correlated with TMN stage and clinical stage of patients in NPC.2.EBV-miR-BART7-3p promotes the metastasis,invasion and EMT of NPC cells by targeting PTEN and regulating its downstream PI3K/Akt/GSK-3? signaling pathway.3.EBV-miR-BART7-3p promotes the growth,proliferation and tumorigenesis of NPC cells by increasing the expression of transcription factor c-Myc and c-Jun.4.EBV-miR-BART7-3p promotes the stem-like phenotypes and reduces the sensitivity to chemotherapeutic drugs of NPC cells by regulating Smad7 and its downstream TGFP signaling pathway.5.Gold nano-particles(AuNPs)carrying anti-EBV-miR-BART7-3p can inhibit the growth of EBV-positive NPC in vitro and in vivo. |
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