| ObjectiveNasopharyngeal carcinoma(NPC) is a kind of head and neck squamous cell carcinoma frequently occurred in south of China. It was difficult for early diagnosis in NPC in clinical and it was reported that more than 60% diagnosed NPC patients had metastasis of neck lymph node or distant organs.Furthermore, the middle survival was less than one year if metastasis was detected.Therefore, it is very important to study the formation and metastasis mechanisms of NPC for improving curative effects and prolonging overall survivals of patients.miRNAs are small noncoding RNAs that serve as negative regulators of gene expression. Through interactions with the 3'-untranslated region (3'-UTR) of mRNA by partial sequence homology, miRNAs cause gene silencing either by mRNA degradation or translation repression. In addition to participation in embryonic neural development, diabetes and maintenance cardiac function, accumulating evidence suggests that miR-9 could be key players in regulation of tumor cell proliferation,invasion and metastasis in many kind of tumors,including Burkitt lymphoma,non-hodgkin's B cell lymphoma, ovarian cancer, gastric carcinoma, endometrial carcinoma. Increasing evidence indicates that miRNAs may function as either oncogenes or tumor suppressors in a cell type-specific manner,but there is no report about miR-9 expression and function in NPC or other head and neck carcinoma.Chemine receptor-4(CXCR4) was deeply researched by many reachers. It was reported that CXCR4 played an important role in the process of progression and metastasis in many tumors, but rarely studied in NPC.We found that miR-9 could bind with CXCR4 3'-UTR through its"seed sequence"by bioinformatics method,indicating the direct targeted regulation relationship between miR-9 and CXCR4 is to be confirmed.Our study aimed to observe the expressions of miR-9 and CXCR4 in NPC tissues with different metastatic potential and chronic nasopharyngitis tissues, normal immortalization nasopharynx epithelial cell NP69 and nasopharyngeal carcinoma cell lines; to explicit the effect of miR-9 and CXCR4 on proliferation,migration,invasion and metastasis by screening C666-1 cell line of miR-9 overexpression and CXCR4 interference;to explore whether miR-9 and CXCR4 have direct targeted regulation relationship,whether miR-9 could partially reverse the stimulative effect of CXCR4 in NPC by constructing CXCR4 3'-UTR double fluorescence report plasmid including binding sites of miR-9 and function of CXCR4 by in vivo and vitro experiment;to discuss and explicit whether lower expression of miR-9 in NPC is related with DNA methylation in promoter of its encoding genes by detection of miR-9 expression and CpG island DNA methylation in miR-9 encoding genes after 5-AZA and TSA used in C666-1.Methods1.Clinical samples collecting and groupingForty patients with NPC (20 cases with metastasis and 20 cases without metastasis) and twenty chronic nasopharyngitis cases were recruited in this study from October 2008 to December 2010. All the specimen were confirmed by HE staining method and detected the expression level of miR-9 and CXCR4,analized their expression correlation between miR-9 and CXCR4.2.Culture of NPC cell linesNPC cell lines including C666-1,Sune-1,5-8F,6-10B,CNE-1,CNE-2 and Hone-1 were maintained in RMPI-1640 medium which was supplemented with 10% heated inactivated fetal calf serum, and normal immortalization nasopharynx epithelial cell NP69 was maintained in KMSF medium,all the cell lines were digested by common conditions.3.Real Time-PCRMicroRNA and total RNA were extracted from NPC cell lines and NP69,and then reversely transcripted into cDNA and PCR amplified using taqman probes and primers,relative expression strength of miR-9 and CXCR4 were expressed by 2-ΔΔCt method.4.In situ hybridizationParaffin organizations and cells climb pieces were dewaxed and hydrated(fixed by 4% paraformaldehyde),trypsin digested,prehybridizated,hybridizated,serum closed,incubated overnight by digoxin labeled antibody at 4℃,washed by BCIP/NBT buffer,stained by BCIP/NBT and nuclear fast red,and then staining results were observed.5.IHC detectionParaffin sections of tissues prepared for detection were stained by SP method, and the results of IHC were observed by light microscope and judged:Sections were observed in 10×objective lens and the staining intensities of all cells in 5 fields of vision were counted randomly, then scores were counted.6.Vector construction and identification ①Construction and identification of pLVTHM/miR-9 and pLVTHM/ CXCR4-shRNA-miR (con) vectorpre-miR-9 transcription templates and shRNA-miR (con) DNA Oligo sequences were synthesized,annealed and double digested by MluI and Clal, connected to construct vectors:pLVTHM/miR-9 and pLVTHM/CXCR4-shRNA-miR (con)②Construction and identification of psiCHECK-2-CXCR4 3'-UTR vector and its mutantswe double digested PCR product of CXCR4 3'-UTR and psiCHECK-2 by Xhol and NotI to construct vector psiCHECK-2-CXCR4 3'-UTR,meanwhile we constructed psiCHECK-2-mt-s-CXCR4 3'-UTR (replace mutation) and psiCHECK-2-mt-d-CXCR4 3'-UTR (missing mutation) by overlap PCR method.③Construction and identification of pReceiver-CXCR4-ORF and pReceiver- CXCR4-ORF+3'-UTR vectorWe construct pReceiver-CXCR4-ORF and pReceiver-CXCR4-ORF+3'-UTR vectors using peripheral genomic DNA of normal persons as template7.Lentivirus PackageDNA-Cacl2 solution was used for lentivirus Package.8.Screening miR-9 overexpression and CXCR4 interference cell lines.We sorted C666-1 GFP+ cells by flow cytometric analysis after lentivirus infecting C666-1 cells, and named them as C666-1/miR-9 and C666-1/pLVTHM, C666-1/CXCR4-shRNA-miR and C666-1/CXCR4-shRNA-con,identificated them by RT-PCR and western-blot methods.9.Western blot:Western blot analysis was performed according to the standard method with antibodies to CXCR4 and GAPDH after total protein were extracted and quantificated by BCA method.10.Immunofluorescence assay. The expression of CXCR4 was determined by immunofluorescence assay. Cells were fixed by 4%paraformaldehyde and permeabilizated by 0.2%triton X-100 for 5mins, blocked by 10% normal goat serum for 30mins, inoculated with rabbit antihuman CXCR4 protein monoclonal antibody at 4℃for overnight and rinsed with phosphate buffered saline (PBS) with 0.2% tween 20 (PBST) three times for 10 min each. The mixture was then incubated with fluorescein isothiocyanate (FITC)-goat antimouse immunoglobulin G (IgG) antibody (1:200) at 37℃for 60 min and rinsed three times for 10 min each with PBST solution. Finally, inoculated with DAPI for 5mins.Cell pictures were obtained by fluorescence microscope in every sample and randomly selected to test cell fluorescence intensity.11.Functional studies in vitro and in vivo:①CCK-8 assay:CCK-8 assay was performed to assess the effect of miR-9 and CXCR4 on cell proliferation.Cells(1×103cell/well) were plated in a 96-well plate and maintained in RPMI1640 supplemented with 10% FBS.At 24,48,72,96 and 120h after seeding,culture medium was removed,cells were treated with 20ul CCK8 working solution for 1h at 37℃,and spectrometric absorbance at wavelength of 450nm was measured on a microplate reader.②Colony formation assay:For colony formation assay,200 cells were seeded into 6-well plate. After being cultured for 1 10-14 days, surviving colonies (>50 cells/colony) were counted with 1% crystal violet staining. Triplicate independent experiments were performed.③Cell apoptosis:Flow cytometry detection,caspase-3 activity analysis and Tunel in situ apoptosis detection were used to detect cell apoptosis status.④wound healing assays:In vitro wound healing assay was carried out to determine the ability of cells to form membrane protrusion and cell migration. When the cell confluence reached 90%,a single wound was created in the center of cell monolayer by gentle removal of the attached cells with a sterile plastic pipette tip.The debris was removed by washing the cells with serum free medium.Migration of cells into the wound was then observed after 12h..⑤Transwell migration assay:For cell migration assay, cells were starved with serum free medium for 24 hrs before the assay. Cells (5×104) were suspended in 0.5 ml serum-free medium and loaded on the upper compartment of transwell chamber. The lower compartment was filled with complete medium as chemoattractant. After 12 hrs, migration cells were fixed, stained, and counted under a microscope. Triplicate independent experiments were done.⑥Transwell invasion assaysFor invasion assay, cells were starved with serum free medium for 24 hrs before the assay.5×104 cells were suspended in 0.5 ml serum-free medium and loaded on the upper compartment of invasion chamber coated with Matrigel. The lower compartment was filled with complete medium as chemoattractant. After 12 hrs, invasive cells were fixed, stained, and counted under a microscope. Triplicate independent experiments were done.⑦Tumor formation in nude mice:For in vivo experiment,cells (4×106) in 200μL serum-free RPMI1640 were injected s.c into the right flank of 4-6 weeks-old nude mice, respectively. The tumor volume was calculated by the formula V=0.5×L×W2(L:length;W:width),and when the volme of tumor was close to 500 cm3 nude mice were killed.⑧In vivo metastasis experiment:Nude mice(BALB/C nu/nu,4-6 weeks old,18-24g in weight) were maintained under pathogen-free conditions(specific pathogen-free level).Primary tumors were established by direct injection of 2×106 cells into the liver as previously described.All the mice were sacrificed on day 21,and tissues were collected for pathologic analysis.GFP fluorescence images were observed under an in vivo fluorescence instrument.12.MiRNA target site prediction. A search for predicted target miRNAs was performed usingthe databases TargetScan (http://www.targetscan.org/),miRbase (http://www.mirbase.org) and PicTar(http://pictar.mdc-berlin.de/).equence S conservation was examined using University of California Santa Cruz genome browser(http://genome.ucsc.edu/) and then we selected interesting target gene (http://genome.ucsc.edu/).13.Dual-luciferase reporter gene assay:293FT cells, chosen based on their low endogenous expression of miRNAs (26), were grown to 80-90% confluence in white 96-well plates in DMEM supplemented with 10% FBS,1% nonessential amino acids, L-glutamine, and penicillin/straptamicin at 37℃under 5% CO2.Cells were transfected with 20 ng empty psiCHECK-2 vector or psiCHECK-2-CXCR4-3'-UTR reporter and miR- 9 mimics or its mutant-miR-9 mimics mt and negative control (miR control), miR-9 inhibitor or its negative control (miR- 9 inhibitor control)at concentrations of 20nM in reduced serum and antibiotics-free Opti-MEM with lipofectamine 2000. T he Firefly and Renilla luciferase were measured in cell lysates using a dual-luciferase reporter assay system on a fusion plate reader. Firefly luciferase activity was used for normalization and as an internal control for transfection efficiency.14.5-AZA and TSA treatment of C666-1 cells. 1×105 C666-1 cells were split 12 to 24 h before treatment. Cells were then given one of the following treatments. (i) 50uM 5-Aza-2'-deoxycytidine (5-AZA) was used for 72 h. Medium containing 5-AZA was changed every 24 h;(ii) 50uM 5-AZA was used for 72 h followed by 10nm TSA for an additional 24 h. The dose of 5-AZA (50uM) and TSA(lOnm) was chosen based on preliminary studies showing optimal reactivation of miR-9 gene expression and DNA methylation in promoter. 15.hsa-miR-9-l,hsa-miR-9-2 and hsa-miR-9-3 CpG islands methylation analysis In an attempt to determine all CpG islands and all potential transcription start sites (TSS) for hsa-miR-9-1,hsa-miR-9-2 and hsa-miR-9-3, we first proceeded with the identification of the promoter sequence. The analyses were initiated using an identified RefSeq by GenBank accession number, after which we submitted the gene sequence to a Genome BLAT Search through the UCSC Genome bioinformatics website (http://genome.ucsc.edu). We selected 2000bps of sequence extending from the 5' upstream region to 1000 bps downstream of the region of the TSS. The BLAT program returned a sequence that was first submitted to the CpGPLOT program from the European Bioinformatics Institute website (http://www.ebi.ac.uk/emboss/cpgplot) This program defines a CpG island as 200 or 300bps of sequence with 50%C+G content and 0.65 CpG observed/CpG expected.16.Bisulfite genomic sequencing:Genomic DNA was isolated from cell samples and 1000ng treated with sodium bisulfite using the DNA Methylation kit to convert unmethylated cytosine to uracil.Bisulfite-treated DNA was then amplified with specific primers for CpG islands surrounding miR-9-1,miR-9-2 and miR-9-3 genomic locations by PCR with platinum taq hifidility,finally PCR product and pMD19-T vector were connected and coated for DNA sequencing to detect DNA methylation status.17.Statistical analysis:All analyses were carried out using the SPSS 13.0 software package.A P value less than 0.05 was considered statistically significant.Comparisiong of miR-9 and CXCR4 expression level in chronic nasopharyngitis tissues,nasopharyngeal carcinoma tissues without metastasis, nasopharyngeal carcinoma tissues with metastasis was analyzed with the Kruskal-Wallis test (K independent samples test).Sperman rank correlation was used for relationship between CXCR4 protein and miR-9 expression level.Comparison of mean in different cell lines about iR-9 and CXCR4 expession was analyzed by one-way ANOVA or indepent-samples T test and Student- Neuman-keuls methos for multiple comparison.Dunnetts T3 method was exployed for heterogeneity of variance.Theχ2 test for proportion was used to analyze the vivo metastasis characteristics of different groups.Factorial analysis was utilized to analyze the results of CCK8,invasion and wound healing assay.The difference in wound migration and invasion indices between C666-1 cell clones were analyzed by the independented t test.Comparison of mean in colony formation and wound healing assay in two sample was analyzed by independent-samples T test;CCK8 assay and tumor formation in nude mice was analyzed by one-way ANOVA;χ2 test was used for rates of liver and lung metastasis among different cell lines.miR-9 exprssion level in C666-1,C666-1 5-AZA and C666-1 5-AZA+TSA groups were analyzed by One-way ANOVA and R×Cχ2 t test for BSP results.Results1.miR-9 was lower expressed in NPC tissues and cell lines.miR-9 was positively expressed in nuclei and membrane of nasophayngeal carcinoma cell and epithelial cells in chronic nasopharyngitis tissues.The expression levels of miR-9 detected by ISH in nasopharyngeal carcinoma tissues (mean rank 22.25) were significantly lower than that of chronic nasopharyngitis tissues (mean rank 47.00) (Z=-5.218, P=0.000), and the differences of miR-9 expression level among nasopharyngeal carcinoma tissues with different T stage,N stage and clinical stage were significant (χ2=20.477, P=0.000;χ2=14.423, P=0.002;χ2=17.561, P=0.001). miR-9 expression levels were down-regulated gradually as T stage,N stage and clinical stage were increased,and miR-9 expression levels in T3-T4 stage,N3 stage and IV stage NPC tissues were lower than those of T1-T2 stage, N1-N2 stage and I-III stage.Notely, miR-9 expression level in NO stage NPC tissues (mean rank 30.56) was significantly lower than that of N1-N3 stage NPC tissues (mean rank 17.58) (Z=-2.992, P=0.002),indicating that miR-9 is related with formation and metastasis of NPC. The expression level of miR-9 in NP69 (18.00±0.10) detected by Real time-PCR was significantly higher than those in C666-1 (27.19±0.11), Sune-1(25.80±0.22),5-8F(23.77±0.13),6-10B(24.76±0.07),CNE-1(24.16±0.12),CNE-2 (22.92±0.14) and Hone-1 (26.77±0.21) (F=1212.097, P=0.000), and the expreesion level of miR-9 in C666-1 was lower than those of 5-8F and CNE-2 (P=0.000),6-10B and CNE-1 (P=0.000)2. miR-9 downregulation is related with CpG island DNA methylation in promoter of hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3.①CpG island distribution in hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3 promoters:We used CpG island searcher software to analyze CpG islands in hsa-miR-9-1,hsa-miR-9-2 and hsa-miR-9-3 promoters,and found that there is a CpG island in hsa-miR-9-1 from 165bp to 1255bp(1091bp,including 18 CpG dinucleotides, GC%=62.1%, ObsCpG/ExpCpG=0.741),a CpG island in hsa-miR-9-2 from 3bp to 203bp(201bp,including 9 CpG dinucleotides,GC%=55.2%, ObsCpG/ExpCpG=0.756) and a CpG island in hsa-miR-9-3 from 87bp to 1899bp (1813bp,including 35 CpG dinucleotides, GC%=65.4%, ObsCpG/ExpCpG=0.739).②5-AZA and TSA induce miR-9 re-expression and DNA demethylation in C666-1 cell.5-AZA and TSA could induce miR-9 re-expression in C666-1,miR-9 expression level in C666-1 5-AZA group and C666-1 5-AZA+TSA group increased 11.31 and 22.63 times (F=780.280,P=0.000),and miR-9 expression level in C666-1 5-AZA group and C666-1 5-AZA+TSA group were significantly higher than that of C666-1 group (P=0.000),and the same as C666-1 5-AZA+TSA group and C666-1 5-AZA group (P=0.000).Methylation ratios of hsa-miR-9-1 in C666-1,C666-1 5-AZA and C666-1 5-AZA+TSA groups were 78.8%(67/85),32.9%(28/85) and 14.1%(12/85) respectively (χ2=77.324, P=0.000), methylation ratios of hsa-miR-9-2 were 72.2%(39/54),38.9%(21/54) and 14.8%(8/54) respectively (χ2=36.850, P=0.000), methylation ratios of hsa-miR-9-3 were 76%(133/175),32%(56/175) and 20.6%(36/175) (χ2=122.407, P=0.000) by BSP sequencing., methylation ratios of hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3 were highest in C666-1 group and lowest in C666-1-AZA+TSA group,indicating 5-AZA and TSA could change methylation status of miR-9 encoding genes in C666-1 cell, in other word,DNA methylation and acetylation in promoter were main reasons for miR-9 downregulation in NPC cell.3. The effect of miR-9 on functions of NPC.The proliferation rates of C666-1/miR-9 was significantly slow than that of C666-1/PLVTHM (F=696.168, P=0.000),The colony numbers formed by C666-1/miR-9(48.15±3.82) was significantly less than that by C666-1/PLVTHM (83.70±3.74) (t=29.746, P=0.000) indicating that miR-9 overexpression could inhibit proliferarion and colony formation ability of NPC cell;There was no obvious apoptosis induced by C666-1/miR-9 and C666-1/PLVTHM,indicating that miR-9 overexpression has no effect of NPC cell apotosis;In wound healing assay,The number of C666-1/miR-9 cells passed through the scratch (47.167±2.552) was less than that of C666-1/PLVTHM (95.917±3.118) (t=41.912, P= 0.000),and in transwell migration assay C666-1/miR-9(36.500±4.542)and C666-1/PLVTHM(93.417±5.195) (t=28.569, P= 0.000) were the same as in wound healing assay,indicating that miR-9 overexpression could inhibit migration ability of NPC cell.In transwell invasion assay,the number of C666-1/miR-9 cells penetrated Matrigel (32.667±3.257) was less than that of C666-1/PLVTHM (81.833±4.366) (t=31.270, P=0.000) indicating that miR-9 overexpression could inhibit invasion ability of NPC cell.In subcutaneous tumor formation assay,the average volume of tumor formed by C666-1/miR-9 (128.688±75.127) was less than that by C666-1/PLVTHM (208.699±151.858) (F=1472.708, P=0.000);After cells inoculated under liver capsule,the metastasis rate of intrahepatic spread in C666-1/miR-9(44.4%,4/9) was lower than that of C666-1/PLVTHM (88.9%,8/9) (χ2=4.000,P=0.046),and the metastasis rate of lung spread in C666-1/miR-9(22.2%,2/9) was lower than that of C666-1/PLVTHM (77.8%,7/9) (χ2=5.556, P=0.018) indicating that miR-9 overexpression could inhibit tumor formation and metastasis ability of NPC cell.4. miR-9 inhibits expression and function of CXCR4We predicted that miR-9 may play its role through combining its seed sequence(CUUUGGU) with 3'-UTR of CXCR4mRNA (ACCAAAG) by bioinformatics method.To confirm the hyposesis,we constructed plasmid psiCHECK-2-CXCR4 3'-UTR and its mutants psiCHECK-2-mt-d-CXCR4 3'-UTR or psiCHECK-2-mt-s-CXCR4 3'-UTR that to be transfected with miR-9 mimics,miR-9 NC,miR-9 inhibitor and miR-9 inhibitor NC into 293FT cell and then detected luciferase activity, finding that con-transfection of psiCHECK-2-CXCR4 3'-UTR and miR-9 mimics could significantly reduce luciferase activity (P<0.001), con-transfection of psiCHECK-2-CXCR4 3'-UTR and miR-9 inhibitor could significantly enhance luciferase activity(P=0.001).However,con-transfection of psiCHECK-2-mt-d-CXCR4 3'-UTR or psiCHECK-2-mt-s-CXCR4 3'-UTR with miR-9 mimics or miR-9 inhibitor even could not change luciferase activity (P>0.05),and con-transfection of mutant of miR-9mimics(miR-9-mt) and psiCHECK-2-CXCR4 3'-UTR also could not change luciferase activity,indicating that miR-9 could bind with 3'-UTR of CXCR4.Next,we constructed pReceiver-CXCR4-ORF and vectors and transfected them into 293FT cells in which CXCR4 protein is lower expressed,founding that the expression level of CXCR4 protein was almost the same,indicating the 3'-UTR of CXCR4 has no effect on protein translation,but when we con-transfected the above vectors with miR-9 mimics and miR-9 mimics mt,we found miR-9 mimics rather than miR-9 mimics mt could significantly inhibit CXCR4 protein expression after being transfected with pReceiver-CXCR4-ORF+3'-UTR, and both of miR-9 mimics and miR-9 mimics mt could not significantly inhibit CXCR4 protein expression after being transfected with pReceiver-CXCR4-ORF,indicating miR-9 has no effect on CXCR4 protein expression no matter whether the sequence of miR-9 is mutanted when 3'-UTR of CXCR4 is lost;next,we detected miR-9 and CXCR4 expression in C666-1/miR-9 and C666-1/PLVTHM by Q-PCR and western-blot,and found that CXCR4 protein rather than CXCR4 mRNA was lower in C666-1/miR-9 than C666-1/PLVTHM,but CXCR4 protein expression could be recovered to some extent,indicating miR-9 could inhibit CXCR4 protein rather than CXCR4 mRNA expression,which was consistent with results by immune fluorescence.Finally, in order to explore whether miR-9 could partial reverse the function of CXCR4 in cell proliferation,migration,invasion and metastasis,we screened C666-1/CXCR4+PLVTHM and C666-1/CXCR4+miR-9 cells,and found that the proliferation of C666-1/CXCR4+miR-9 was significantly slower than that of C666-1/CXCR4+PLVTHM (F=2392.535, P=0.000),and colony numbers formed by C666-1/CXCR4+miR-9(64.90±2.29) were significantly less than that of C666-1/CXCR4+PLVTHM(115.20±2.44) (t=67.185, P=0.000),indicating that miR-9 could partial reverse the function of CXCR4 in cell proliferation and colony formation;C666-1/CXCR4+PLVTHM and C666-1/CXCR4+miR-9 have no effect on cell apoptosis.The number of C666-1/CXCR4+miR-9 cells passed through scratches (53.000±7.520) was much less than that of C666-1/CXCR4+PLVTHM (236.083±10.423) (t=49.348, P= 0.000),and the number of C666-1/CXCR4+miR-9 cells penetrated transwell membrane (56.667±3.229) was also much less than that of C666-1/CXCR4+PLVTHM (115.833±5.524) (t= 32.033, P=0.000),the number of C666-1/CXCR4+miR-9 cells penetrated Matrigel (45.250±3.745) was also much less than that of C666-1/CXCR4+PLVTHM (106.500±3.802) (t=39.760,P=0.000), indicating that miR-9 could partial reverse the function of CXCR4 in cell migration and invasion.The average volumes of subcutaneous tumor in C666-1/CXCR4+PLVT HM(249.404±143.100) group were greater than that of C666-1/CXCR4+miR-9 group(134.357±74.782) (F= 659.513, P=0.000);The intrahepatic transfer rate in C666-1/CXCR4+PLVTHM group (100%,9/9) was higher than that of C666-1/CXCR4+miR-9 group (55.6%,5/9) (x2=5.143,P=0.023),and the pulmonary metastasis rate in C666-1/CXCR4+PLVTHM group (88.9%,8/9) was higher than that of C666-1/CXCR4+miR-9 group (33.3%,3/9) (χ2=5.844, P=0.016), indicating that miR-9 could partial reverse the function of CXCR4 in tumor formation and metastasis in vivo.Conclusion①miR-9 was down-regulated in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cells.②The lower expression of miR-9 in NPC is related with CpG island DNA methylation in promoter of miR-9 encoding genes hsa-miR-9-l,hsa-miR-9-2 and hsa-miR-9-3.③miR-9 overexpression could inhibit the proliferation,colony formation, migration and invasion, subcutaneous tumor formation and liver, lung metastasis ability of C666-1 cell,indicating miR-9 acted as tumor suppressor gene in NPC.④miR-9 inhibited the proliferation, migration and invasion ability of C666-1 cell by regulating expression of CXCR4 protein and partially reversing the function of CXCR4 in NPC.
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