| BackgroundNasopharyngeal carcinoma (NPC) has a remarkably unusual ethnic and geographic distribution in Southern China and Southeast Asia, especially those of Cantonese origin. Three major etiological factors of NPC include genetic susceptibility, environmental factors and Epstein-Barr virus (EBV) infection, but the molecular mechanism of its pathogenesis is not yet fully understood. The characteristics are highly malignant local invasion and early distant metastasis. The radiotherapy proves to be the most sensitive and effective treatment, but the average 5-year survival rate for NPC patients remains at 70%. About 30-40% of patients diagnosed with advanced nasopharyngeal carcinoma and a considerable proportion of patients have recrudesce after treated about four years. Until now, the underlying mechanisms of NPC tumorigenesis are not completely understood. Therefore, further study the pathogenesis of NPC extremely urgent.MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs of 19～25 nucleotides in length that modulate gene expression post-transcriptionally. miRNAs are mostly transcribed from a hairpin intermediate of about 70-100 nucleotides called pre-miRNA, and undergo further processing by the ribonucleases Dicer. The mature miRNA targets the 3’untranslated region (3’UTR) of its target mRNA, and induces mRNA degradation. When miRNA and its target mRNA sequence show imperfect complementarities, translation into a protein is blocked. Most microRNA genes are found in intergenic regions or in anti-sense orientation to genes and contain their own miRNA gene promoter and regulatory units. As miRNAs involve in biological development, cell proliferation, differentiation and apoptosis.Tumorigenesis is a complicated process which depends largely on the interaction of different genes. Besides protein-coding genes, miRNAs are regarded as a new player in cancer. It has been revealed that the deregulation of miRNAs is associated with a variety of cancers, and involved in the initiation and progression of cancer cells. Their abnormal expression plays a key role in the hallmarks of cancer, such as cell proliferation, differentiation, apoptosis, migration, invasion, metastasis and angiogenesis. These miRNAs have been confirmed to downregulate the expression of many cancer-related genes and function as oncogenes and tumor suppressor genes.The present study shows that miRNAs exist not only in tissues and cells but also in many body fluids, including serum, plasma, saliva, urine and amniotic fluid. Plasma or serum miRNA usually considered to be circulating miRNAs. miRNAs can be expressed in the presence of body fluids noninvasive cancer biomarkers. Since the early events of miRNA expression deregulated in tumorigenesis, the cycle miRNA levels can also be used for early diagnosis and prediction of cancer treatment response and potential new patient selection criteria of the role in clinical trials.2008, It was the first time to miR-21 is upregulated in serum in diffuse large B-cell lymphoma and is closely related to recurrence-free survival of patients. It is possible to distinguish these patients with prostate cancer in healthy pepole accroding to serum levels of miR-141. In the report, fifteen serum miRNAs were upregulated in prostate cancer, such as miR-16, miR-92a and miR-92b and so on. Tsujiura’s and other studies have shown that, serum miR-21 and miR-106B was significantly higher in patients with gastric cancer than in healthy pepole. However, serum miR-21 and miR-106B expression were reduced after treated, compared with preoperative. Since then, over 100 studies in different types showed that serum or plasma miRNAs wre considered as possible cancer biomarkers.In nasopharyngeal carcinoma, microRNAs expression were dysregulated, such as miR-26a, miR-9, miR-378, miR-10b, miR-144, miR-214 and so on. Studies have shown that these microRNAs involved in the occurrence and development of NPC. miR-26a were down-regulated in NPC cell lines and nasopharyngeal tissues and inhibited proliferation and invasion of NPC by targeting EZH2. The same, miR-9 was downregulated in NPC and nasopharyngeal carcinoma cells and regulated cell proliferation, migration and invasion by targeting CXCR4. However, miR-378 were upregulated and promoted cell proliferation, metastasis and invasion by targeting TOB2 in NPC. miR-10b were high expression, and inhibited the proliferation and invasion of nasopharyngeal carcinoma cells. As of 2015, about 32 microRNAs were studied in NPC.miRNAs play an important role in the development of NPC, and the circulating miRNAs was as clinical noninvasive biomarker detection. However, in NPC, few reports circulating miRNA research. Our previous studies suggested miRNA-124 downregulated in NPC plasma by array screening and real time PCR validation. However the molecular pathogenesis of nasopharyngeal carcinoma specific regulation has not been studied.miRNAs play an important role in the development of NPC, and the circulating miRNAs are considered as clinical noninvasive biomarker. However, circulating miRNA research has not been reported in NPC. In 2013, our group found the plasma miR-124 expression was downregulated in the tissue of nasopharyngeal carcinoma compared with nasopharyngeal non-cancerous tissue using microRNA-array to screen plasma miRNAs expression profiles change and validated by qPCR. The expression of miR-124 was downregulated in different types of cancer, but the expression of miR-124 and the role of miR-124 in nasopharyngeal carcinoma have not been reported.miR-124 is full length 21nt and firstly cloned from the mouse, and then the expression level was confirmed in human embryonic stem cells. In 2007 years, Visvanathan J et al found miR-124 was involved in neurite formation in the embryonic nervous system; while Makeyev EV et al demonstrated that miR-124 promoted the development of the nervous system. In cancer research, Silber J was first discovered that miR-124 was low expression significantly in glioblastoma multiforme and brain tumor stem cells, and also confirmed that miR-124 inhibited cell proliferation. miR-124 was down-regluated expreesion and inhibited cell proliferation, metastasis, invasion capabilities in non-small cell lung cancer, breast cancer, liver cancer, bladder cancer and so on. But miR-124 has not been reported in the literature in NPC. However, the role of miR-124 in NPC and the molecular mechanisms in which miR-124 exerts its functions remain largely unknown.Based on the above findings, the aim of this study is to examine the expression of miR-124, identify its possible role in NPC pathogenesis, and elucidate the molecular mechanisms of its suppressive or oncogenic activities on NPC. We hope that this study will improve the better understanding of NPC pathogenesis and the development of novel effective therapies for NPC.Materials and Methods1. Cell cultureThe human NPC cell lines 5-8F,6-10B, CNE1, CNE2, C666-1, HONE1, and SUNE-1 were cultured in RPMI-1640 with 10% FBS and 1% Penicillin-Streptomycin antibiotic solution. An immortalized nasopharyngeal epithelial cell NP69 was cultured in Keratinocyte-SFM supplemented with bovine pituitary extract. HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). All cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37℃.2. Clinical specimens178 primary NPC biopsy specimens and 55 normal biopsies of thenasopharynx were obtained from Nanfang Hospital (Southern Medical University, Guangzhou, China). Both tumor and normal tissues were histologically confirmed by H&E (hematoxylin and eosin) staining. All NPC specimens were classified as undifferentiated nonkeratinizing type. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of Nanfang Hospital.3. Vector construction[1] pLVTHM/miR-124 This recombinant lentiviral vector can stably overexpress miR-124 which was constructed successfully by real time PCR and gene sequencing.[2] psicheck.2-wt Foxql 3’UTR and psicheck.2-mut Foxql 3’UTR A 185-bp fragment of Foxql 3’UTR was amplified by PCR and cloned downstream of the firefly luciferase gene in psicheck.2 vector. The vector was named wild-type (wt) 3’UTR. Site-directed mutagenesis of the miR-124 binding site in Foxq1 3’UTR was performed using GeneTailor Site-Directed Mutagenesis System and named mutant (mt) 3’UTR.[3] Lv-Foxql and Lv-siRNA These two lentiviral vectors were purchased from Genechem (Shanghai, China).4. Cell transfection[1] miRNA transfection:The transfection was performed using Lipofectamine 2000 reagent, and a working concentration of miRNA was 100 nmol in a 6-well plate.[2] Lentivirus infection:The packaged lentivirus were used to infect NPC cell lines. At 48h transfection, the cells were observed using a fluorescence microscope to detect green fluorescent protein (GFP). The GFP-positive cells were isolated, and the cell lines were established, which could stably overexpress miR-124, overexpress Foxql respectively.[3] miRNA and plasmid co-transfection The co-transfection was performed using Lipofectamine 2000 reagent, and a working concentration of miRNA was 50 nmol, a working concentration of psicheck.2-wt Foxql 3’UTR and psicheck.2-mut Foxq1 3’UTR was 100ng.5. Real-time PCRTotal RNA and small RNA were extracted from cells and tissues. The RNA was reversely transcribed into cDNA. Quantitative real-time PCR (qPCR) was performed using SYBR Green PCR master mix. All samples were normalized to internal controls and fold changes were calculated through relative quantification (2-△△Ct).6. Western blotingTotal protein was isolated and quantitated using BCA assay. The protein lysates were separated by 10% SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies and detected by chemiluminescence.7. Luciferase reporter assayThe cells were seeded in 24-well plates and cotransfected with psicheck.2 constructs with or without miR-124 inhibitor or precursor for 24h. At 24h post-transfection, the activity of firefly luciferase was measured by using the dual-luciferase reporter assay system as described by the manufacturer (Promega Corporation). Relative luciferase activity was normalized with Renilla luciferase activity.8. Cell biology experiments[1] CCK-8 array Cells were seeded into 96 wells plates at a density of 1×103 cells/well with five replicate wells of each condition. Cell counting kit 8 (keyGEN.BioTECH,Nanjing,China) was added to the maintenance cell medium, and all cells were incubated at 37 C for an additional 2h before being measured. Absorbance values were measured daily for four consecutive days at a wavelength of 450 nm.[2] Colony formation assay The cells were plated in 6-well plates at 2×102 per well and grown for 2 weeks. After 2 weeks, the cells were washed twice with PBS, fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet. The number of colonies was counted under the microscope.[3] Cell-cycle analysis The cell-cycle distribution was analyzed by propidium iodide (PI) staining and flow cytometry.[4] Cell migration assay The cells were resuspended in serum-free RPMI-1640 medium.Then the cells were added to each transwell chamber with 10% fetal bovine serum. After cultured for 24h, the cells fixed and stained under the microscope.[5] Cell invasion assay Matrigel was added into the Transwell chamber uniformly after thawing (50ul) in 37℃ foster 12h. The remaining operation was the same cell migration assay.9. Animal experiment[1] Tumor xenograft model in vivo:A total of 5×105 lv-Ctrl/5-8F or lv-miR-124/5-8F cells were injected subcutaneously into the dorsal flank of nude mice. Each group contained 6 mice and the experiment was repeated 3 times. Tumor size was measured every 2 days. When subcutaneous tumors reached the volume of 500mm3, mice were sacrificed and tumors were dissected. Tumor volumes were calculated as follows:volume=(Dxd2)/2, where D meant the longest diameter and d meant the shortest diameter.[2] Tumor metastasis assay in vivo:To establish this model, we inoculated NPC cells into the liver as a single nodule, which would subsequently metastasized to other parts of the liver and the lung. Ten nude mice were randomly divided into two groups. 1×106 lv-miR-124/5-8F or lv-miR-Ctrl/5-8F cells were injected into the liver of each mouse. The mice were killed and autopsied on day 25, and the incidence of lung or liver metastasis were recorded.10. ImmunohistochemistryFormalin-fixed, paraffin-embedded tissues of transplanted tumors were sectioned at 4-mm thickness and analyzed for anti-Foxq1 primary antibody (Abcam Ltd, Cambridge, UK), anti-Ki-67 primary antibody, and anti-PCNA primary antibody (Cell Signaling Technology, Inc.USA). Visualization was achieved using the EnVisionp peroxidase system (Dako). A sample was considered positive if more than 50% of the tumor cells retained nuclear staining, and 5 fields were randomly selected according to semiquantitative scales. The intensity of staining was scored manually: negative (0), weak positive (1), medium positive (2), strong positive (3). Data recording and analysis by 2 independent experienced pathologists, and only tumor cells were scored.11. Statistical analysisStatistical analyses were conducted using spss13.0 statistical software. All experiments were performed for three times. The data are shown as the mean±SEM unless otherwise noted. Two-tailed Student’s t test was used for comparison of two independent groups. And more than three or three sets of data were compared using ANOVA, when homogeneity of variance, using ANOVA, multiple comparisons using the LSD method. Approximate F test Welch method was used when heterogeneity of variancemultiple and multiple comparisons using Dunnett T3. MiR-124 and Foxql expressions between tumor and control specimens were analyzed by a Mann-Whitney U test. The relationship between miR-124 and Foxql was analyzed using Spearman’s correlation analysis. P values of <0.05 were considered statistically significant.Results1. The expression level of miR-124 in NPC cells and tissuesApanal of human NPC cell lines (5-8F,6-10B, CNE1, CNE2, C666-1, HONE1 and 6-10B) was firstly analyzed to quantitate the expression level of miR-124. The results of qRT-PCR showed that the expression of miR-124 was decreased in all seven NPC cell lines examined, compared with the immortalized nontumorigenic cell line NP69. We further tested the expression level of miR-124 in 178 primary NPC tissues and 55 non-cancer nasopharyngitis biopsy samples to analyze the clinicopathologic significance of miR-124. The relationship between the miR-124 expression level and clinicopathologic characteristics in NPC patients were summarized. MiR-124 was not significantly associated with age and gender of the patients. Consistent with the result of the NPC cell lines, the average expression level of miR-124 was decreased in NPC tissue compared with non-cancer biopsy samples (t=4.906, P<0.001). We found that miR-124 expression was higher in stage I, whereas stages Ⅱ-Ⅳ had lower levels, showing a significant correlation of miR-124 with clinical stages. Furthermore, we found that the miR-124 expression was higher in stage T1, whereas stages T2-T4 had lower levels, showing a significant correlation of miR-124 with T stage. In addition, we found that the level of miR-124 was lower in distance metastasis tissues compared with local metastasis tissues. Taken together, these data provided strong evidence that miR-124 expression was closely related to the progression and clinicopathologic features of NPC. On the basis of these results, we focused on miR-124 for further functional studies to evaluate its roles in NPC pathogenesis.2. The growth-inhibitory effect of miR-124 on NPC cells in vitro and in vivoWe selected the undifferentiated 5-8F and low-differentiated 6-10B cells, which excellently exhibited similar clinical phenotypes of NPC, as the most suitable model for further experiments. To explore the effect of miR-124 on cell growth,5-8F and 6-10B cells were transiently transfected with miR-124 mimics. The results showed that the expression of miR-124 was upregulated by 114.15-fold in 5-8F and by 60.99-fold in 6-10B cells. All these data demonstrated that the transient transfection could effectively upregulate the expression of miR-124.The results of CCK-8 assay displayed that miR-124 overexpression inhibited cell growth in 5-8F cells by 36.5% and in 6-10B cells by 40.3% at 96h post-transfection (5-8F; F=214.753,P<0.001; 6-10B; F=220.420, f<0.001)。The Resuits of cell cycle distribution showed at 5-F cells transfected with miR-124 displayed an mcieased percentage of cells in G1-phase(t=25.860,P<0.001) and fewer cells in S phase(t=19.452,p<0.001). The results of cel] cycle distribution showed that 6-10B cells transfected wkh miR-124 displayed an increased percentage of cells in G1-phase(T=20.086,P<0.001) and Fewer cells in Sphase(t=14.769,P<0.001). These results suggested that the growth-suppressive effect of miR-124 was partly due to a G1-phase arrest.After analyzing the proliferation capacity of miR-124, we investigated the cell migration and invasion capacities. Migration arrays demonstrated that miR-124 overexpression inhibited cell migration compared with miR-Ctrl in 5-8F cells by 76% (t=30.948, P<0.001) and in 6-10B cells by 54%(t=20.100, P<0.001). Invasion arrays demonstrated that miR-124 overexpression inhibited cell invasion compared with miR-Ctrl in 5-8F cells by 65.8%(t=26.929, P<0.001) and in 6-10B cells by 62.6%(t=19.759,P<0.001)We next stably restored the expression of miR-124 in 5-8F and 6-10B cell using lentiviral vectors. Following fluorescence activated cell sorting, the GFP positive cells were isolated, and cell lines lv-miR-124/5-8F and lv-miR-124/6-10B were established to stable overexpress miR-124. In colony formation assay, the colony formation efficiency was reduced by 46%(t=7.190,P=0.002) in lv-miR-124/5-8F and 33%(t=4.051, P=0.015) in lv-miR-124/6-10B cells respectively. We also performed tumor growth assay in vivo. The results showed that as ealy as sevene days post implantation, the growth of transplanted tumors between lv-miR-124/5-8F and control cells became statistically significant. At two weeks after implantation, those mice injected with lv-miR-124/5-8F carried larger burdens. As compared with control, the average tumor volume of the lv-miR-124/5-8F group was markedly reduced by more than lv-Ctrl (t=6.149, P<0.001). Additionally, both the staining intensity and the number of hyperproliferative Ki-67 positive and PCNA positive tumor cells were significantly decreased compared with control (Ki67:Z=3.878, P<0.001; PCNA:Z=5.300, P<0.001).To further investigate the metastatic effect of miR-124 in vivo, primary tumors were established by direct injection of lv-miR-124/5-8F or lv-miR-Ctrl/5-8F cells into the liver. Twenty-five days after transplantation, the mice were killed and the livers and lungs were dissected for macroscopic and microscopic histology. Livers and lungs of mice bearing miR-124-expressing 5-8F tumors harbored statistically significantly fewer microscopic and macroscopic metastases than those of mice bearing mock-infected 5-8F tumors.All these results demonstrated that the abnormal expression of miR-124 is associated with cell proliferation and metastases of NPC cells, and it functions as a potential tumor suppressor in NPC pathogenesis.3. The molecular mechanisms of growth-inhibition induced by miR-124We used three databases (PicTar, TargetScan and PITA) to predict the target gene of miR-124 in NPC cells, we focused on 27 possible target genes of mir-124 by utilizing bioinformatic analysis. The selected target genes have been verified that could affect tumor growth or metastasis in the website (http://www.ncbi.nlm.nih.gov/nucleotide/). The selected targets were validated by RT-qPCR in lv-miR-124/5-8F cells. The result of RT-qPCR showed that the expression level of Foxq1 decreased more significantly, compared with other 26 possible target genes. Finally, we selected Foxq1 gene as the target gene of miR-124. Foxq1 is highly conserved among different species, whose 3’-UTR of mRNA contained a complementary site for the seed region of miR-124. Real-time PCR and western blot was performed to detect the expression level of Foxql, and the results showed that Foxq1 was up-regulated in 7 NPC cell lines compared with NP69. Then we analyzed the the mRNA and protein level of Foxql after transfected lv-miR-124 into 5-8F and 6-10B cell lines. Foxq1 was down-regulated after transfected lv-miR-124.To further confirm that Foxq1 was a direct target of miR-124, dual-luciferase assay was performed. The target region sequence of Foxql 3’-UTR (wt 3’-UTR) or the mutant sequence (mut 3’-UTR) was cloned into a luciferase reporter vector. These recombinant vectors were co-transfected with miR-124 mimics or miR-Ctrl into 5-8F cell line. The results showed that miR-124 could down-regulated the luciferase activity of the Foxql wt 3’-UTR construct (t=7.744,P<0.001).The activity of mut3’-UTR vector was unaffected by a simultaneous transfection with miR-124. Moreover, miR-Ctrl did not significantly affect the luciferase activity of either the wt or mut 3’-UTR construct. In summary, these results strongly suggested that miR-124 directly regulated Foxql in NPC cell lines.To explore the function of Foxql in NPC cell lines, Foxql-siRNAs were transfected into 5-8F cell lines. Real-time PCR was confirmed that the Foxq1 mRNA level expression was down-regulated compared with miR-Ctrl after transfected. CCK8 array showed that Foxql silencing inhibited the proliferation (F=43.89, P<0.001). Furthermore, migration and invasion arrays obtained the same results that knockdown of Foxql repressed the migration (t= 13.155, P<0.001) and invasion (t=17.13, P<0.001) capacities of 5-8F cells. According to these results, Foxql functioned as a potential oncogene in NPC cell lines.To elucidate whether the suppressive effect of miR-124 was mediated by repression of Foxql in NPC cells, we performed gain-of-function and loss-of-function studies. First, we silenced Foxql to investigate whether the reduced expression of Foxql could mimic the suppressive effect of miR-124.5-8F cells were transfected with siRNA-Foxql or miR-124 mimics. Then, we examined cell proliferative, migratory and invasive capacities. Foxql knockdown led to significant suppressive effects, similar to those induced by miR-124 The similar results were obtained in 6-10B cell lines. Subsequently, we evaluated whether ectopic expression of Foxql could rescue the suppressive effect of miR-124. The lv-miR-124/5-8F cell line after transfected lv-Foxql was named lv-miR-124+lv-Foxql/5-8F. The expression level of Foxql was tested using Real-time PCR and Western bolt. CCK8 array and colony-forming assay showed that Foxq1 could partially abrogate the effects mediated by miR-124 in lv-miR-124+lv-Foxql/5-8F cells. At the same time, the migration and invasion arrays showed that Foxql could partially restore the migration and invasion activity compared with the lv-Ctrl/5-8F cells. The similar results were obtained in lv-miR-124+lv-Foxq 1/6-10B cell lines. Therefore, we showed that Foxql could partially rescue the suppression of miR-124 in NPC cells.4. MiR-124 and Foxq1 were inversely correlated in NPC tissuesTo further investigate the expression of Foxq1,178 clinical human primary NPC tissues and 55 non-cancer nasopharyngitis biopsy samples were tested for analyzing the clinicopathologic significance. We found that the average expression level of Foxql was up-regulated in NPC tissue compared with non-cancer biopsy samples (t=16.910, P<0.001). Further analysis found that the Foxql expression of clinical I stage was the lowest in the clinical stages. In addition, we found that Foxql expression was lower in stage T1, whereas stages T2-T4 had higher levels, showing a significant correlation of Foxql with T stage. We then correlated Foxql with the miR-124 expression levels in the same NPC specimens. Significant inverse correlation was observed when Foxq1 expression levels were plotted against miR-124 expression levels (2-tailed Spearman’s correlation, r=-0.565; p<0.05).Conclusions1. The expression level of miR-124 is downregulated in NPC cells and tissues. miR-124 were negatively correlated with the clinical stage and T stage.2. The abnormal expression of miR-124 is associated with cell proliferation and metastases of NPC cells. Meanwhile, miR-124 inhibited cell proliferation in transplanted tumors in nude mice experiments and inhibited metastasis of nasopharyngeal carcinoma in liver cancer lung metastasis model, which functions as a potential tumor suppressor in NPC pathogenesis.3. Overesperss Foxql promoted cell growth, migration and invasion in NPC cell lines. miR-124 inhibits the growth and migration and metastasis by targeting Foxql expression in NPC cells.4. The expression level of Foxql was upregulated in NPC cells and tissues and correlated with the clinical stage and T stage. MiR-124 and Foxql were inversely correlated in NPC tissues. |
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