| Background and objectiveNasopharyngeal carcinoma （NPC） is the most common cancer arising from the mucosal epithelium of the nasopharynx, and is extremely common in southern regions of China, particularly in Guangdong, Guangxi, Hunan, Fujian. NPC frequently metastasizes to regional lymph nodes. The etiology of NPC seems to follow a multi-step process, in which Epstein-Barr virus infection, dietary, smoking, genetic factors and environmental carcinogens seem to play important roles. Typical conventional treatments of NPC include radiotherapy, surgery and chemotherapy. Recently, molecular targeted therapy has become the hotspot and focus of comprehensive treatment of NPC.As was shown by Weinberg in2011, The hallmarks of cancer comprise ten biological capabilities acquired during the multistep development of human tumors including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, genome instability, inflammation, reprogramming of energy metabolism and evading immune destruction. There are many kinds of cells in "tumor microenvironment", such as tumor cells, inflammatory cells, cancer stem cells （CSCs）. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis, moreover, they can resistant to chemotherapy and radiotherapy. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival cancer patients.Metastasis is a basic biological characteristics of malignant tumor, and the primary factors affecting patient survival, which has become a hot and difficult field of cancer research. The most prominent feature of tumor metastasis is the tissue specificity, which is different tumor can form metastases in the same or different parts of the human body. Epithelial-mesenchymal transition or transformation （EMT） is a process characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell motility. EMT is an important contributor to the invasion and metastasis of epithelial-derived cancers.miRNAs are small non-coding RNA molecules, and important regulatory molecules in animals and plants. Each miRNA may have a plurality of target genes, while several miRNAs can also target the same gene. Recent studies show that some miRNAs regulate cell proliferation and apoptosis processes in cancer. In addition, some miRNAs may function as oncogenes or tumor suppressors. miRNA expression profiles may become useful biomarkers for cancer diagnostics. Moreover, miRNA therapy could be a powerful tool for cancer prevention and therapeutics. Therefore, aberrant expression of miRNAs has been implicated in numerous disease states, and miRNA-based therapies are under investigation.miR-9, initially demonstrated to function in neurogenesis, has been confirmed to be implicated in cancer. miR-9is under-expressed in many types of cancers, including breast cancer, hepatocellular carcinomas, gastric cancer, ovarian cancer, colon cancer, melanoma, clear cell renal cell carcinoma and medulloblastoma, all of which is indicative of a tumor suppressor potential, whereas miR-9is over-expressed in brain cancer and Hodgkin’s lymphoma, suggesting oncomir activity for miR-9in these two cancers. Some evidence indicates that the functions of miR-9in cancer cells are recently implicated in regulating proliferation, EMT, metastasis, apoptosis and tumor angiogenesis, etc. However, the contribution of miR-9which is commonly down-regulated in NPC to the pathogenesis of NPC remains to be characterized.MethodsPart I miR-9inhibits cell growth and tumorigenesis through repressing CCNG1in NPC1Establishment of miR-9stably over-expressed NPC cells:Particles expressing vector along with packaging plasmids psPAX2and pMD2.G were transfected into HEK293T cells （maintained in10%FBS） using Lipofectamine2000reagent （Invitrogen） according to the manufacturers’instruction.48h after transfection, virus supernatant was harvested from these cells, and then used to infect NPC cells. To attain100%percentage of infected NPC cells based on EGFP assay and FACS analysis, lentiviral infected cells were selected by2μg/ml puromycin （GIBCO） for2weeks to kill non-infected cells.2RNA isolation, reverse transcription and qRT-PCR:For miRNA and mRNA analyses, total RNA from NPC cells was extracted with Trizol Reagent （TaKaRa） according to the protocol provided by the manufacturer. Total RNA was reversely transcribed with the PrimeScript RT reagent Kit （TaKaRa）. The expression levels of mature miRNA were determined by SYBR Green quantitative PCR amplifications performed on the Stratagene Mx3000P Real-Time PCR system （Agilent Technologies, Inc., USA）. U6was used for normalization. Expression of mRNA analysis was performed using SYBR Green Master Mix （TaKaRa） as described using GAPDH for normalization on a Stratagene Mx3000P qRT-PCR System. The primers used for the amplification of the indicated genes were listed in Table S1-4. All samples were normalized to internal controls and fold changes were calculated through relative quantification （2-ΔΔCt）.3CCK-8assay:A total of800-1000cells were seeded in a96-well plate and then allowed to grow in normal medium for96hours. Cells were incubated in100ul normal+10ul CCK-8. The absorbance in each well was measured at450nm by a microplate reader. 4Colony formation assay:200cells were seeded in6-well plates and grown for2weeks. Then washed with PBS, fixed with methanol for10min, and stained with hematoxylin for10min.5Cell-cycle analysis:Pipet cell suspension （EtOH70%） over night at-20℃. Treated with RNase A to remove RNAs from the cells, cell-cycle distribution was analyzed by propidium iodide staining and flow cytometry.6Luciferase activities detection:Insert the3’-UTR into3’downstream to the luciferase reporter in the pGL3vector which carries activated promoter sequences. The association of miRNA with3’-UTR will cause the attenuation of translation of luciferase mRNA, which shows the decline of luciferase activities.7Western blot and high sensitivity chemiluminescence imaging system were used to survey the proteic level of genes.8Animal experiment:Cells were injected subcutaneously or into hepatic subcapsular of nude mice, tumors in subcutaneous were measured every2-3days, tumor volunes were calculated as follows:D×d×d/2（D meant the longest diameter and d meant the shortest diameter）. Tumors were used to extract RNA and proptein, and fixed with4%paraformaldehyde for HE staining and Immunohistochemistry.9Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package. Two-tailed Student’s t test was used for comparisons of2independent groups. Statistical significance was assessed by the Student’s t-test （*p<0.5;#p<0.01）.Part II miR-9impairs cancer stem cells through negative regulation of Hesl in NPC1Establishment of Hes1and shHes1stably over-expressed NPC cells, Western blot and Cell Immunofluorescence were used to analyze protein expression of "stemness "genes.2Luciferase activities were detected to verify Hesl was a direct target of miR-9in NPC cells after Hes1wt3’UTR （mut3’UTR） and miRNAs cotransfection.3Tumor sphere formation and ratio of stem cell populations （SP） detected by flow cytometry and hoechst33342were performed to evaluate the "sternness" of NPC cells.4Intratumoral delivery of miR-9in a nasopharyngeal cancer xenograft model: Nude mice were subcutaneously inoculated with CNE2cells and maintained until the tumor cells had formed solid, palpable tumors with an average volume of80to100mm3,7days following inoculation, miR-9or a negative control miRNA was repeatedly administered by intratumoral injections every3days. All mice were killed on day16.5Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package, Two-tailed Student’s t test was used for comparisons of2independent groups.Part Ⅲ miR-9triggers EMT and migration by repressing Klf4in NPC1Establishment of c-Myc and miR-9stably over-expressed NPC cells, Western blot and Cell Immunofluorescence were used to analyze protein expression of genes, Immunohistochemistry was used to investigate the protein expression and the definite localization of proteins.2Transwell, Scratching test and Boyden were used for migration and invasion of cells.3Fluorescently labeled phalloidin staining test and Scanning electron microscopy were used to survey the Skeleton and morphological variation of NPC cells.4PBS with0.05nM EDTA and inverted fluorescence microscope were used for adhesion ability of cells.5Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package.Two-tailed Student’s t test was used for comparisons of2independent groups; One-way ANONA was used for comparisons of several groups; Factorial ANOVA performed an analysis of variance for multiple classification variables. Part IV miR-9modulates the expression of interferon-regulated genes and MHC class I molecules in NPC1RNA extract and qRT-PCR were carried out to detect the mRNA expression of genes。2mRNA microarray analysis:Expression microarray analysis was carried out with commercially available Affymetrix Human Gene U133Plus2.0array according to the Affymetrix standard protocol, which carried47,000transcripts representing38,500well-characterized human genes. All the hybridization procedures and data analysis were performed by Capital Bio Corp.（Bejing, China）. Total RNA samples were isolated from NPC cells （CNE2cells） using Trizol reagent （Invitrogen）. Briefly, total RNA was used to synthesize cDNA in an in vitro transcription reaction, and then cDNA was fluorescently labeled by Cy5or Cy3-CPT with Klenow enzyme. Labeled cDNA was then hybridized to Affymetrix Human Gene U133Plus2.0arrays. Hybridization was processed at45℃, with rotation for16h （Affymetrix GeneChip Hybridization Oven640）. Chips were then washed and stained in the Affymetrix Fluidics Station450. Hybridization signals were scanned with a Lux-Scan3.0scanner （Capital Bio. Corporation, Beijing, China）. The resultant images were digitized with Genepix Pro6.0software （Axon Instruments, Foster City, CA, USA）.3Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package. Two-tailed Student’s t test was used for comparisons of2independent groups.ResultsPart I miR-9inhibits cell growth and tumorigenesis through repressing CCNG1in NPC1. qRT-PCR data demonstrated the miR-9expression was significantly lower in NPC cells （i.e.,6-10B,5-8F, CNE2, HNE1, HONE1, SUNE1cells） than that in NP-69cells （F=370.010, P=0.000）.2. We established miR-9-expressing NPC cell lines （i.e., CNE2, HONE1and SUNE1cells）.3. The levels of miR-9in CNE2, HONE1and SUNE1cells transfected with miR-9mimics was much higher than that in CNE2, HONE1and SUNE1cells transfected with control RNA （t=7.619,11.224,5.228, P=0.017,0.000,0.035） Moreover, the levels of miR-9in CNE2and5-8F cells transfected with miR-9inhibitor was much lower than that in CNE2and5-8F cells transfected with inhibitor control （t=10.924,14.95, P=0.000,0.004）, indicating that miR-9inhibitor efficiently down-regulated the endogenous miR-9expression in CNE2and5-8F cells.4. miR-9inhibits cell growth and affect cell-cycle distribuionCCK-8assay showed that miR-9-expressing NPC cells inhibited cell growth, whereas the proliferation of NPC cells transfected with miR-9inhibitor was enhanced （P=0.000）. Flat cloning formation experiments also indicated that the ability of proliferation was significantly inhibited in miR-9-expressing groups （t=7.721,2.744,16.545, P=0.000,0.034,0.000）. In sum, the above two experiments illustrates that miR-9overexpression suppresses the proliferation of NPC cells in vitro.We studied the effects of miR-9on cell cycle using FACS. The results showed that compare to the control group, the ratio of G0-G1phase in miR-9-expressing cells were significantly increased （P<0.05）, while the ratio of G0-G1phase in NPC cells transfected with miR-9inhibitor were significantly decreased （P<0.05）, indicating that miR-9could cease cell cycle at phase G0/G1.5. The suppressive effects of miR-9overexpression on tumorigenicity of NPC cellsTo explore the effects of miR-9on NPC tumorigenicity, miR-9-expressing CNE2cells （1×106） we firstly subcutaneously injected into the dorsal flank of nude mice. At34days after implantation, mice injected with vector-expressing CNE2cells carried large tumor burdens, while mice injected with miR-9-expressing CNE2cells did not developed tumor. Then, we increased the number of injected cells from1×106to1.5×106. At21days after implantation, all the mice injected with vector-expressing CNE2cells developed tumors, but only one of four mice injected with miR-9-expressing CNE2cells developed tumor which is much smaller than the controls. Additionally, miR-9-expressing CNE2cells （1.2×106） were transplanted subcapsularly into the livers of nude mice. At23days after implantation, all the mice transplanted with vector-expressing CNE2cells formed tumors in livers, while none of those mice injected with miR-9-expressing CNE2cells developed tumor.Nextly, miR-9-expressing SUNE1cells （1×106） cells were subcutaneously transplanted into the dorsal flank nude mice. The tumor became palpable between7and9days after inoculation, and all the mice injected with vector-expressing cells and4/5mice injected with miR-9-expressing CNE2cells formed tumors at the end of the experiment （t=4.279, P=0.003）. As early as9days postimplantation, the growth of transplanted tumors between2groups became statistically significant （P=0.021）. At19days after implantation, those mice injected with vector-expressing cells carried larger burdens. We also showed that both the staining intensity and the number of hyperproliferative BrdU and Ki67tumor cells were significantly decreased compared with control.6. CCNG1is a direct target gene of miR-9To explore the mechanism of growth inhibition induced by miR-9, we want to idenfity the target gene of miR-9. Firstly, we found that miR-9overexpression down-regulated CCNG1expression in NPC cells, while miR-9inhibitor up-regulated CCNG1expression in NPC cells. Secondly, we performed luciferase reporter assay to determine whether miR-9could directly target the3’-UTR of CCNG1in NPC cells. CNE2cells were transfected with wt or mt3’-UTR vector and miR-9mimics. The results showed a significant decrease of luciferase activity when compared with miR control （t=32.542, P=0.000）. The activity of mt3’-UTR vector was unaffected by a simultaneous transfection with miR-9mimics or miR-9inhibitor （t=0.455,0.241, P=0.673,0.821）. Moreover, cotransfection with miR-9inhibitor and wt3’-UTR vector in CNE2cells led to an increase of luciferase activity （t=6.238, P=0.003） Taken together, all these results strongly suggest that CCNG1is a direct target gene of miR-9in NPC cells. 7. miR-9affects cell proliferation and cell cycle via CCNG1in NPC cells.To elucidate whether the growth-suppressive effect of miR-9could be mediated by CCNG1in NPC cells, we firstly silenced CCNG1to investigate whether the reduced expression of CCNG1could mimic the suppressive effect of miR-9. HONE1cells were transfected with si-CCNG1, and then we examined cell proliferation rate and cell cycle distribution （F=14.134, P=0.002）. CCNG1knockdown led to significant cell growth inhibition and cell-cycle arrest, similar to those induced by miR-9（P<0.01）. Subsequently, we evaluated whether ectopic expression of CCNG1could rescue the suppressive effect of miR-9. HONE1cells overexpressing miR-9were transfected with pC3-CCNG1. We observed that the ectopic expression of CCNG1in miR-9-expressing NPC cells significantly rescued miR-9-induced cell growth inhibition and cell-cycle arrest （F=93.810, P=0.000）. In summary, the growth-suppressive effect of miR-9could be mediated by CCNG1in NPC cells.Part II miR-9impairs cancer stem cells through negative regulation of Hesl in NPC1. miR-9overexpresssion down-regulates the expression of stemness-related genes in NPC cellsWestern blot showed that Nanog, Oct4and ABCG2was down-expressed in miR-9-expressing CNE2and SUNE1cells, and up-expressed in5-8F cells transfected with miR-9inhibitor. Immunohistochemistry also showed a low-expression of Nanog and Sox2in subcutaneous xenotransplanted tumors overexpressing miR-9. We found that the ability of tumor spheres of miR-9-expressing cells reduced （t=39.436,31.577, P=0.000,0.000）, and the ratio of SP cells was decreased in miR-9-expressing cells.2. The expression of stemness-related genes between adherent cells and tumor spheresFor further study, we tested the expression of stemness-related genes between adherent cells and tumor spheres （the second generation）. Western blot data resulte indicated that the expression of Hesl, Sox2, Oct4, Nanog and ABCG2was higher in tumor spheres than in adherent cells, while miR-9expression （detected by qRT-PCR） showed no disparity between adherent cells and tumor spheres （t=0.765,0.653, P=0.487,0.533）3. Effects of Hesl on "sternness" of NPC cellsWe found a high expression of Nanog and ABCG2in Hesl-expressing （LV-Hes1） NPC cells, and low expression in Hesl-silenced （shHesl） cells. The SP cells in SUNE1overexpressing Hes1could up to8.6%compared to the control5.3%, while1.0%in Hes1-silenced cells; the SP cells in CNE2overexpressing Hes1could be up to5.3%compared to the control2.0%, while0.4%in Hesl-silenced CNE2cells. Moreover, the ability of tumor sphere formation was enhanced in Hes1-expressing CNE2and SUNE1cells （t=14.902,20.412, P=0.003,0.000）4. Hes1is a direct target of miR-9in NPC cellsAccording to the targeted gene prediction performed by TargetScan and studies reported by Siok-Lay Tan, to explore the mechanism of "sternness" inhibition induced by miR-9, we investigated whether miR-9could regulate Hesl expression in NPC cells, and we found that miR-9overexpression down-regulated Hesl expression in NPC cells, while miR-9inhibitor up-regulated Hesl expression in NPC cells.We performed luciferase reporter assay to determine whether miR-9could directly target the3’-UTR of Hesl in NPC cells. CNE2cells were then transfected with wt or mt3’-UTR vector and miR-9mimics. The results showed a significant decrease of luciferase activity when compared with miR control （t=23.089, P=0.000）. The activity of mt3’-UTR vector was unaffected by a simultaneous transfection with miR-9mimics or miR-9inhibitor （t=1.075,0.218, P=0.343,0.838）. Moreover, cotransfection with miR-9inhibitor and wt3’-UTR vector in CNE2cells led to an increase of luciferase activity （t=11.573, P=0.000）. Taken together, these results strongly suggest that Hesl is a direct target gene of miR-9in NPC cells.5. Hesl rescues the suppressive effect of miR-9on "sternness"Subsequently, we evaluated whether over-expression of Hes1could rescue the suppressive effect of miR-9. miR-9-expressing CNE2cells were infected with LV-Hes1. We showed that ectopic expression of Hesl significantly increased expression of Hesl and other genes related to "sternness", and rescued miR-9-induced reduction of SP cells and tumor spheres （F=162.277, P=0.000）6. Intraturnoral delivery of miR-9leads to regression of tumors in a NPC xenograft modelAs the in vitro and in vivo data showed an antitumorigenic role for miR-9in NPC, we examined the therapeutic potential of synthetic miR-9Agomir or mimics in vivo. In the first pre-experiment, we found that the tumor injected with miR-9Agomir formed cavities along the direction of the needles after injection with a delayed healing, but the controls healed rapidly （data not shown）. In the repeated test, we injected miR-9mimics into two tumors with MaxSuppressorTM In vivo RNA-LANCErⅡ, and found that intratumoral delivery of synthetic miR-9induced a specific inhibitory response and it formed a hollow in the center of one tumor which was interesting. Now, we are carrying out a third time repeat.Part III miR-9triggers EMT and migration by repressing Klf4in NPC1. c-Myc activates miR-9expressionWe established c-Myc-expressing NPC cells, levels of c-Myc and miR-9expression were evaluated by qRT-PCR. The result showed that c-Myc and miR-9levels in HONE1and SUNE1cells harboring c-Myc transgene was significantly higher than that in cells harboring vector control （t=5.199,4.769, P=0.035,0.041） and （t=2.536,4.690, P=0.039,0.005）. Moreover, the levels of c-Myc and miR-9in HONE1and SUNE1cells transfected with sic-Myc was much lower than that transfected with inhibitor control （t=11.545,11.744, P=0.000,0.000） and （t=6.585,10.473, P=0.003,0.000）. In summary, c-Myc efficiently up-regulated the endogenous miR-9expression in NPC cells.2. miR-9-mediated c-Myc induced EMTTo study whether c-Myc can affect EMT process in NPC cells, we detected EMT-related genes using qRT-PCR. The level of E-cadherin seemed to be variable and became higher when c-Myc was highly expressed（t=-44.880, P=0.000）, whereas there was no such relationship with N-cadherin and Vimentin （P>0.05）. Interestingly, we found expreesion of vimentin at protein level was higher when c-Myc was highly expressed, suggesting that c-Myc may have some correlation with E-cadherin and vimentin.3. miR-9-mediated c-Myc enhanced motilityAs shown above, miR-9-mediated c-Myc induced E-cadherin and vimentin alteration. Transwell assay and Boyden chamber assay displayed the enhanced ability of migration and invasion in c-Myc-expressing cells （t=12.231,10.000; P=0.000,0.000）, while miR-9inhibitor could rescue c-Myc-induced enhancement of motility （F=147.016,59.900, P=0.001,0.001）4. miR-9induces EMT-like changes in NPC cells.During culture of miR-9-expressing cells, we were surprised to find that the cells gradually lose the original cobblestone-like or neat form and appeard morphological diversification, some cells became elongated and the antennas of cells were increased. Previous studies suggested that miR-9was associated with EMT in breast cancer, further study on the role of miR-9in EMT was carried out. Western blot showed that E-cadherin and a-catenin which represent epithelial factors were down-regulated in NPC cells overexpressing miR-9, however, up-regulated by miR-9inhibitor. While the expression of N-cadherin and vimentin were up-regulated in miR-9-expressing cells, but down-regulated by miR-9inhibitor, indicating that the ectopic expression of miR-9lead to an EMT-like conversion.5. miR-9promotes cell migration and invasion in NPC.Transwell assay and Boyden chamber assay demonstrated that miR-9overexpression increased the migration and invasion of CNE2and HONE1cells （t=6.533,10.983,13.131,12.351,P=0.000,0.000,0.000,0.000）. Scratch-Migration assay also showed the increased migration of cells overexpressing miR-9as compared with controls.6. miR-9reduces the ability of cell adhesionCell adhesion was performed and analysed by counting the adherent cells, experiments showed that miR-9-expressing cells had lower rates of adhesion than the control cells （F=846.528,751.057, P=0.000,0.000） 7. Effect of miR-9on cytoskeletonWe investigated whether or not miR-9induces changes in cytoskeleton by detect ing F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin, and we also observed the microscopic morphology of the cells using scanning electron microscopy. We found an increased expression of F-actin in miR-9overexpressed cells, with increased pseudopodia.As is known, Rac and Rho regulate the formation of distinct actin filament-based structures, and Cdc42and Rac are also required for the assembly of adhesion sites to the extracellular matrix. We detected an increased expression of Rho and Rac in miR-9-expressing cells. All above results suggested that miR-9was correlated with changes in shape of cells and pseudopodia formation, which is the initial step of tumor metastasis.8. Klf4is a direct target gene of miR-9in NPC cellsWe found that Klf4promoted mesenchymal-epithelial transition （MET） and inhibited migration and metastasis of NPC cells in other studies of our group. According to the targeted gene prediction performed by microrna.org, we investigated whether miR-9could regulate Klf4expression in NPC cells. Western blot suggested a decreased expression of Klf4in miR-9-expressing NPC cells and subcutaneous xenotransplanted tumors, whereas the expression of Klf4was up-regulated by miR-9inhibitor.We performed luciferase reporter assay to determine whether miR-9could directly target the3’-UTR of Klf4in NPC cells. The target sequence of Klf43’-UTR （wt3’-UTR） or the mutant sequence （mt3’-UTR） was cloned into a luciferase reporter vector. CNE2cells were then transfected with wt or mt3’-UTR vector and miR-9mimics. The results showed a significant decrease of luciferase activity when compared with miR control （t=4.380, P=0.012）. The activity of mt3’-UTR vector was unaffected by a simultaneous transfection with miR-9mimics or miR-9inhibitor （t=0.921,0.302, P=0.409,0.777）. Moreover, cotransfection with miR-9inhibitor and wt3’-UTR vector in CNE2cells led to a increase of luciferase activity （t=13.777, P=0.000）. Taken together, all these results strongly suggest that Klf4 was a direct target gene of miR-9in NPC cells.9. miR-9-induced EMT and enhanced motility are mediated by Klf4.Subsequently, we evaluated whether over-expression of Klf4could rescue the improvement of miR-9in motility. HONE1cells overexpressing miR-9were infected with LV-Klf4. Western blot showed that ectopic expression of Klf4significantly increased expression of Klf4and E-cadherin, but decreased expression of N-cadherin. Furthermore, Transwell assay and Boyden chamber assay showed that Klf4rescued the increased ability of cell migration and invasion induced by miR-9（F=137.015, P=0.000）. In sum, we suggest that miR-9promotes EMT and cell motility by targeting Klf4.10. c-Myc promotes tumor metastasis in vivoTo fully explore the effects of c-Myc on metastasis in vivo, we injected1×106CNE2cells infected with LV-c-Myc or the control plasmid （LV-con） subcapsularly into the livers of nude mice,7mice for each group,23days after implantation, all the mice developed tumors in liver at the end of the experiment. The5per7mice in LV-c-Myc group developed lymph node metastases, while2per7mice in control group developed lymph node metastases, suggesting c-Myc can promote tumor metastasis in vivo.In summary, we have found that miR-9triggers EMT and migration in NPC by repressing Klf4.Part IV miR-9modulates the expression of interferon-regulated genes and MHC class I molecules in NPC1. miR-9altered IFN-regulated gene expression in NPC cellsMicroarray analysis of CNE2cells infected with LV-miR-9showed the induction of many IFN-regulated target genes （e.g., IFI44L, PSMB8, IRF5, PSMB10, IFI27, PSB9HUMAN, IFIT2, TRAIL, IFIT1, PSB8HUMAN, IRF1and B2M）, which was further confirmed by qRT-PCR analyses.To fully explore the effects of miR-9on IFN-regulated target genes, CNE2cells were transiently transfected with miR-9mimics or anti-miR-9, respectively. The results of qRT-PCR demonstrated that miR-9mimic upregulated the expression of some IFN-regulated genes （such as IFI44L, PSMB8, IRF5, PSMB10, IFI27, IFIT2, TRAIL, IFIT1, IRF1, B2M and GBP1） in CNE2cells （P<0.05or0.01）, and downregulated the expression of some IFN-regulated genes （such as ISG20and AIM2） in CNE2cells （P<0.05or0.01）, respectively, while anti-miR-9correspondingly decreased the expression of miR-9mimics-upregulated IFN-related genes （i.e., IFI44L, PSMB8, IRF5, PSMB10, IFI27, IFIT2, TRAIL, IFIT1, IRF1, B2M and GBP1） in CNE2cells （P<0.05or0.01）, and increased the expression of miR-9mimics-downregulated gene （i.e., ISG20and AIM2） in CNE2cells （P<0.01）, separately. Collectively, the most significant alteration after miR-9overexpression in NPC cells was the increased expression of genes involved in IFN induction, including IFI44L, PSMB8, IRF5, PSMB10, IFI27, IFIT2, TRAIL, IFIT1, IRF1, B2M and GBP1.2. Induction of the expression of MHC class I molecules by miR-9in NPC cellsMicroarray analysis of CNE2cells infected with LV-miR-9showed the up-regulated expression of MHC class I molecules （HLA-B, HLA-H, HLA-C, HLA-F, Q8WW48HUMAN, NP001004349.1, Q6ZUW0HUMAN and O19682HUMAN） and TAP1gene （encoding antigen peptide transporter1, as ATP-binding cassette （ABC） transporter）. qRT-PCR of HLA-B, HLA-F and TAP1confirmed the microarray changes.Furthermore, when CNE2cells were transiently transfected with miR-9mimics, the expression of MHC Class I molecules （such as HLA-B and HLA-F） and TAP1was significantly upregulated, while CNE2cells transfected with anti-miR-9indicated the decreased expression of HLA-B, HLA-F and TAP1（P<0.05or0.01）. In summary, miR-9plays a significant role in regulating the expression of MHC Class I molecules.3. miR-9overexpression enhanced or reduced IL-related gene expression in NPC cellsThe microarray data derived from CNE2cells infected with LV-miR-9demonstrated the significantly altered expression in human IL-related genes （for example, IL20RB, GALT, IL7, IL1B, IL11, IL1F8, ILIA, IL6and IL7R）, which was further confirmed by qRT-PCR analyses. To fully explore the effects of both miR-9upregulation and endogenous miR-9downregulation on IL-related genes, CNE2cells were transiently transfected with miR-9mimics or anti-miR-9, respectively. The results of qRT-PCR illustrated that miR-9mimics upregulated the expression of some IL-related genes （i.e., IL20RB, GALT, IL7） in CNE2cells （P<0.05or0.01）, while other interleukin-related genes （i.e., IL1B, IL11, IL1F8, ILIA, IL6and IL7R） were remarkably downregulated by miR-9mimics in CNE2cells （P<0.05or0.01）. Conversely, anti-miR-9correspondingly decreased the expression of IL20RB, GALT and IL7in CNE2cells （P<0.05or0.01）, and increased the expression of IL1B, IL11, IL1F8, IL1A, IL6and IL7R in CNE2cells （P<0.05or0.01）, separately. Collectively, the most significant alteration after miR-9overexpression was the decreased expression of IL-related genes, including IL1B, IL11, IL1F8, IL1A, IL6and IL7R.Conclusions1. miR-9inhibits cell proliferation and tumorigenesis by targeting CCNG1, while miR-9promotes EMT and migration by repressing Klf4, suggesting that miR-9plays different roles in modulating cell proliferation, and EMT, migration&metastasis by directly targets CCNG1and Klf4in NPC, respectively;2. miR-9significantly suppresses cell proliferation and tumorigenesis of NPC by directly downregulating Hesl to deplete cancer stem cells;3. miR-9modulates the expression of interferon-regulated genes and MHC class I molecules in NPC. |
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