microrna-124-3p inhibits the growth and metastasis of

ONCOLOGY REPORTS 35: 1385-1394, 2016

Abstract. The present study investigated the effects of microRNA-124-3p (miR-124-3p) expression on nasopharyn-geal carcinoma (NPC) cells and its relevant mechanism. A total of 90 NPC tissues and 85 postnasal catarrh tissues were collected. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect tissue samples and expression of miR-124-3p in CNE1, CNE2, SUNE1, H0NE1, 5-8F, 6-10B and C666-1 NPC cell line and immortalized nasopharyngeal epithelial cells line (NP69). Overexpressed miRNA-124-3p in CNE-2 was downregulated, and low-expressed miRNA-124-3p in C666-1 was upregulated by liposome-mediated transfec-tion. Cell Counting Kit-8 (CCK-8), flow cytometry, the scratch test, Transwell migration assay and Boyden chamber assays were used to detect cell proliferation, apoptosis, migration and invasion. The target gene of miRNA-124-3 calculated by bioinformatics was further determined using dual-luciferase system. Protein levels of the signal transducers and activa-tors of transcription 3 (STAT3), phospho-STAT3 (p-STAT3), mouse anti-human cyclin D2 (CCND2) and matrix metal-loproteinase-2 (MMP-2) were tested by western blotting. miRNA-124-3p expression in NPC was markedly down-regulated compared to postnasal catarrh tissues (P<0.001); miRNA-124-3p expression showed close linkage with clinical stages, regional lymph node involvement and T stages (all P<0.001). miRNA-124-3p expression was lower in the 7 NPC cell lines than NP69 cells (all P<0.05). After upregulation of miR-124-3p, proliferation, apoptosis, migration and invasion of C666-1 cells were suppressed; while after downregula-tion of miR-124-3p, CNE2 cells were increased (all P<0.05). Expression of STAT3, p-STAT3, CCND2 and MMP-2 in

C666-1 cells was decreased after transfection with miRNA-124-3p, and the above protein expression in CNE-2 cells was increased after inhibition of miRNA-124-3p (all P<0.05). To sum up, this study shows that miR-124-3p may negatively regulate the transcription of the STAT3 by interfering with its 3'UTR, and the degradation of STAT3 affects its downstream expression of such as p-STAT3, CCND2 and MMP-2, thereby promoting NPC cells apoptosis and inhibiting proliferation, migration and invasion of NPC cells.

Introduction

Nasopharyngeal carcinoma (NPC), a malignance arising from the epithelium of the nasopharynx, shows a special geographic and demographic variation (1,2). As a globally common cancer, ~84,000 cases were diagnosed with NPC annually, and >80% of them were reported from Southeast Asia, China, and some Asian countries (3,4). The etiology of NPC is multifactorial such as Epstein-Barr viral (EBV) infection, genetic suscepti-bility and environmental factors such as cigarette smoking, and occupational exposure to dusts (5-7). Although many improve-ments in diagnostic imaging, radiation therapy, and adjuvant chemotherapy were made in NPC management, many patients developed distant metastases; in this regard, understanding of the molecular mechanisms is urgently needed (8,9).

MicroRNAs (miRNAs) are a class of small non-coding RNAs, which have been implicated in several essential biological processes by direct interaction with their target mRNAs in the 3'-untranslated regions (3'-UTR) (10). miRNAs plays a vital role in regulation human transcriptome; moreover, the dysregulation of miRNAs contributes to the development of human diseases including cancer (11). Previous research illustrated that miRNAs can function as tumor suppressors or oncogenes during tumor development and progression suggesting their potential as biomarkers for cancer diagnosis and therapy (12,13). Many miRNAs have been reported to be dysregulated in NPC, such as miR-10b, miR-26a, miR-9, miR-144, miR-214 and miR-124-3p (also named miR-124) which is involved in the development and progression of NPC (14-18). The miR-124-3p, the most abundant miRNA in the brain, is inevitable in neurogenesis and its decreased expression is associated to carcinogenesis (19,20). The epigen-etic silencing of miR-124-3p is documented to be a tumor

MicroRNA-124-3p inhibits the growth and metastasis of nasopharyngeal carcinoma cells by targeting STAT3SHAN XU1, NiNG ZHAO1, LiAN HUi1, MiN SONG2, Zi-WEi MiAO3 and XUE-JUN JiANG1

Departments of 1ENT and 2Pathology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001; 3Developmental Biology Teaching and Research Section,

China Medical University, Shenyang, Liaoning 110001, P.R. China

Received July 22, 2015; Accepted September 17, 2015

DOi: 10.3892/or.2015.4524

Correspondence to: Dr Xue-Jun Jiang, Department of ENT, The First Affiliated Hospital of China Medical University, 155 Nanjing North Street, Shenyang, Liaoning 110001, P.R. ChinaE-mail: [email protected]

Key words: microRNA-124-3p, proliferation, apoptosis, migration, invasion, nasopharyngeal carcinoma

XU et al: miR-124-3p AND NPC1386

suppressor in many malignances by targeting genes such as Salt Overly Sensitive 1 (SOS1) and Clock in glioma, catalytic subunit α of phosphatidylinositol 3-kinases (PiK3CA) in hepatocellular carcinoma (HCC), the transmembrane 4 super-family member (CD151) in breast cancer, androgen receptor in prostate cancer, sphingosine kinase 1 (SPHK1) in gastric cancer, extracellular-regulated protein kinases (ERK) in cutaneous squamous cell carcinoma, signal transducers and activators of transcription 3 (STAT3) and inhibitory member of the apoptosis stimulating proteins of p53 family (iASPP) in colorectal cancer (21-27). While the role of miR-124-3p in NPC as well as the mechanisms remain largely unknown.

STAT3, a member of transcription factor family located on chromosome 17q21, is known as a DNA-binding protein in response to epidermal growth factor; the phosphorylation of STAT-3 could cause its dimerization, translocation into the nucleus and DNA binding, thus it was able to regulate cell proliferation, differentiation, and apoptosis (28,29). it has been reported that STAT3 activation (phospho-STAT3, p-STAT3) has been discovered in over 75% of NPC tumors (30). Of interest, STAT3 is highly expressed and activated in several human cancers including NPC, whereas, the tumor suppres-sant miRNA-124-3p was frequently downregulated (18,31). However, the correlations of STAT3 and NPC are not adequately delineated and need further elaboration. in this regard, we are interested in seeking the association between miRNA-124-3p and STAT3, which could provide a clear understanding of the carcinogenesis mechanisms in NPC.

Materials and methods

Ethics statement. This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University. All study participants provided written informed consents.

Collection of specimens. A total of 90 patients with locally recurrent NPC were diagnosed and received treatment at the Department of Otorhinolaryngology of the First Affiliated Hospital of China Medical University between October 2012 and October 2014. NPC group enrolled patients with non-kera-tinizing carcinoma in pars nasalis pharynges by pathological diagnosis; postnasal catarrh group included patients with chronic inflammation in pars nasalis pharynges. All patients had complete clinical data and had not received radiotherapy and chemotherapy before operation; other 85 postnasal catarrh tissues were collected as control group. informed consent was obtained from all individuals before biopsy. All specimens were fixed using 4% polysorbate, embedded and sliced, more-over, specimens were further determined by Department of ENT, the First Affiliated Hospital of China Medical University and met the conclusion criteria: patients diagnosed as partially differentiated non-keratinizing NPC (NPC group), and post-nasal catarrh patients were the chronic nasopharyngitis group. The exclusion criteria were as follows: patients with recurrent NPC; NPC patients who received preoperative radiotherapy or chemotherapy. The NPC were graded according to the American Joint Committee on Cancer (AJCC) and the Union international Centre le Cancer (UiCC) tumor node metastasis (TNM) classification (32).

Cell culture. Human NPC C666-l, Sune-l, 5-SF, 6-10B, CNE-l, CNE-2, Hone-l cells and the immortalized nasopharynx epithelium cell line NP69 were preserved in Cancer Research institute, China Medical University (Shenyang, China). Cell lines were cultured in medium RPMi-1640 (Gibco, USA) containing 10% fetal calf serum at 37˚C in 5% CO2 in satu-rated humidity.

MicroRNA-124-3p expression estimated using quantitative real-time polymerase chain reaction (qRT-PCR). Total DNA was extracted based on TRizol reagent (invitrogen, USA), Expression of NPC tissue specimens, chronic nasopharyngitis tissue specimens and miRNA-124-3p in NPC cell lines were tested applying SYBR Prime Script miRNA RT-PCR kit (Takara). Reaction systems (20 µl) were as follows: SYBR Premix Ex Taq ii (2X) 10 µl; PCR forward primer (10 µM) 0.8 µl; Uni-miR qPCR primer (10 µM) 0.8 µl; ROX Reference Dye ii (50X) 0.4 µl; cDNA template 2 µl; ddH2O 6 µl. The PCR condition was 40 cycles of pre-denaturation at 95˚C for 10 min; denaturation at 95˚C for 10 sec, annealing at 60˚C for 10 sec, and elongation at 72˚C for 15 sec. Primers were synthe-tized by Shanghai Bio-Engineering Company (Shanghai, China): miR-124-3p forward, 5'-CTCAACTGGTGTCG TGGAGTCGGCAATTCAGTTGAGGGCATTCA-3' and reverse, 5'-ACACTCCAGCTGGGTAAGGCACGCGGTGA ATGCC-3'; and U6 forward, 5'-CTCGCTTCGGCAGC ACA-3' and reverse, 5'-AACGCTTCACGAAnTGCGT-3'.miRNA-124-3p expression was expressed as 2-ΔΔCt. U6 snRNA expression was used as the reference gene.

Cell translation. Highly expressed miRNA-124-3p NPC cell line CNE-2 was downregulated by transfection and lipo-some-mediating, whereas, downregulated the miRNA-124-3p NPC cell line C666-1 was upregulated; blank control group, inhibitor negative control (NC) group and mimics NC group were also evaluated. Well-developed cells, were digested one day before seeding in a 6-well plate and transfected in cell density of 30% the next day. The mixtures were transfected based on Lipofectamine 2000 reagent (invitrogen, USA): 10 µl miRNA-124-3p mimics, inhibitor and 5 µl Lipofectamine 2000 reagent was transferred using needle without non - RNA enzyme to an Opti-MEM medium. Mixture was placed into a 6-well plate and then shaken carefully; after 6 h culture at 37˚C, medium and mixture were discarded; RPMI-1640 medium containing 10% fetal bovine serum (FBS) was added into each well.

Dual-luciferase system. Cells were obtained after 48 h trans-fection and washed once using PBS; 1X PLB (100 µl/24-well plate) was put into an Eppendorf (EP) tube containing cells, the mixture was shaken gently; 10 µl lysates were added into a 96-well plate; Stop&Glo was then absorbed to estimate Renilla values.

Cell proliferation. After transfection and liquid change at 16 h, cells were digested with 0.25% pancreatin and then collected; after centrifugation, cells were placed into a fresh culture medium and seeded in a 96-well plate (0.8x103 cells/well, 100 µl/well); 5 duplicate wells were set in each group. The detection was done using Cell Counting Kit-8 (CCK-8; Dojindo

ONCOLOGY REPORTS 35: 1385-1394, 2016 1387

Laboratory, Japan). Two hours before detection, 10 µl CCK-8 was added per well; the mixture was shaken carefully and developed 2 h at 37˚C; absorbance at 450 nm of the microtiter wells was measured with an enzyme-linked immunosorbent assay (ELiSA) plate reader (650 nm as reference). Cell prolif-eration activity was estimated using CCK-8 in 0, 24, 48, 72 and 96 h after transfection, and corresponding growth curve was drawn.

Cell apoptosis detection using flow cytometry. Cells were stained with propidium iodide (Pi; Nanjing Kaiji Biological Technology Development Co., Ltd.) to detect cell apoptosis. The specific steps were as follows: cells were washed using 1X buffer A once (centrifugation at 2,000 rpm, 5 min) and collected; then cell concentration was adjusted to 1x106/ml; 70% alcohol, was added to the cells at -20˚C for more than 12 h; cells were obtained after centrifugation; alcohol were removed applying 1X buffer A to wash cells; cells were then placed in 500 µl buffer A; RNase A was added to make the final concentration to be 0.25 mg/ml and reacted at 37˚C for 30 min; 5 µl Pi was added away from direct sunlight for 30 min; then flow cytometry with excitation at 488 nm was applied to test cell apoptosis.

The scratch test. Cells of each group were seeded into a 12-well plate, after 12 h of cell transfection, when the degree of fusion was 80-90%, a wound was created along the bottom of the plate with 200 µl micropipette; cells were slightly washed once with PBS, and then incubated with serum-free medium. Cell movement in 0, 12, 24 and 36 h was observed under inverted microscope and photographed; distance of the change in the wound was recorded to show the migration rate.

The Transwell migration assay. After 48 h of transfec-tion, cells in each group were digested using pancreatin to single-cell suspension; cells were washed twice with serum-free medium, and counted, then cell concentration was adjusted to lx105/ml. Cell suspension (100 µl) was slowly dropped into the well; the inner membrance was made of polycarbonate (PC), aperture of the well was 8.0 µm; 500 µl complete medium containing 10% FBS was added to lower chamber of the Transwell. After seeding, cells were incu-bated at 37˚C with 5% CO2 and under saturated humidity for 20-22 h. The invasive ability was observed under an inverted microscope. The upper chamber was washed 3 times using D-Hanks; non-migratory cells in the upper chamber were washed and then removed with cotton bud; cells were fixed using methanol for 15 min. Cells were dyed with hematoxylin for 10 min; after air-dried, polycarbonate membrane was cut carefully and put on the slide, then sealed with neutral balsam. The invasive cells were counted, and imaged imme-diately under an upright fluorescence microscope with 5 high power fields.

Boyden chamber assays. The final concentration of Matrigel was adjusted to 24 µg/ml by using serum-free medium; 45 µl liquid was extracted and placed in each well. After transfection for 48 h, cells were digested using pancreatin to be single-cell suspension; the concentration of cells was

adjusted to lx105/ml. Cell suspension (100 µl) was slowly added into the well containing Matrigel; 500 µl complete medium containing 10% FBS was added to lower chamber. After seeding, cells were cultured at 37˚C with 5% CO2 under saturated humidity for 20-22 h. The migration ability of cells was then observed under inverted microscope. The upper chamber of the well was washed 3 times using D-Hanks; non-migratory cells in the upper chamber were washed and then removed with cotton bud; cells were fixed using methanol for 15 min. Cells were stained with hematoxylin for 10 min; then air-dried, polycarbonate membrane was put on the slide and then sealed. The migrated cells were counted, and imaged immediately under an upright fluorescence microscope.

Western blotting. Cell precipitates were obtained and washed twice using PBS; the supernatant was discarded; RiPA lysate was added, the liquid was place in ice-bath for 30 min and developed at 4˚C with 12,000 rpm for 15 min; the supernatant was obtained and put in a new EP tube. Total protein concen-tration was determined using BCA protein assay kit. Protein samples were separated. The condition for electrophoresis: spacer gel in constant voltage at 60 V; separation gel in constant voltage at 100 V; Filter papers, PVDF membranes and sponge pads were prepared 20 min before electrophoresis ended. The gel was soaked using electroporation buffer for 10 min; membranes were blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% dried skimmed milk and incubated for 30 min in room temperature. Blocked PVDF membranes were incubated in solution (containing primary antibody [rabbit anti-human STAT3, phospho-STAT3 (p-STAT3) and matrix metalloproteinase-2 (MMP-2) polyclonal antibody] and mouse anti-human cyclin D2 (CCND2) monoclonal antibody (Cell Signaling Technology, USA) for 2 h; eluted PVDF membranes were cultured in solu-tion which was diluted using secondary antibody (Jinqiao Biotechnology Co., Ltd., Beijing, China) for 1 h, and reacted applying ECL chemiluminescence and then developed using X-ray.

Statistical analysis. All information was analyzed using SPSS 19.0 (SPSS, Chicago, iL, USA). Differences in measurement data were applied using mean ± standard devia-tion analysis. Comparison was analyzed using t-test between two groups and one-way ANOVA among groups. P<0.05 was considered statistically significant; P<0.01 as highly statisti-cally significant.

Results

MicroRNA-124-3p expression in nasopharyngeal carcinoma tissues and cell lines. miRNA-124-3p expression in NPC and postnasal catarrh tissues were tested using qRT-PCR. The results demonstrated that miRNA-124-3p expression in NPC tissues was significantly downregulated compared to that in postnasal catarrh tissues (Fig. 1A, P<0.001). As shown in Table i, miRNA-124-3p expression showed no correlation with gender, age and distant metastases (all P>0.05); while miRNA-124-3p expressions were closely correlated with size and extent of tumor (T stages, P<0.001), regional lymph node involvement (N stages, P<0.001) and clinical stages


(i-ii stages/iii-iV stages, P<0.001). As shown in Fig. 1B, miRNA-124-3p expression in seven NPC cells were obvi-ously decreased compared to NP69 cells (both P<0.05); and miRNA-124-3p expressions were lowest in C666-1 cells, and highest in CNE-2 cells (all P<0.05) in the NPC cells. in this regard, we aim to downregulate miRNA-124-3p expression in the NPC cell line CNE-2 and upregulate miRNA-124-3p expression in NPC cell line C666-1 in further research.

Analysis on target gene of microRNA-124-3p. Target gene of miRNA-124-3p was screened using TargetScan

(http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de/) and miRiad (http://www.biomfo.mochsl.org.br/miriad/); the results showed that a 3'UTR sequence of STAT3 matched the miRNA-124-3p (Fig. 2A). As shown in Fig. 2B, over-expression of miRNA-124-3p significantly inhibited the activity of firefly luciferase in cells transfected with wild-type pMiR_STAT3_WT plasmid; as compared with NC group (Scramble), the luciferase activity decreased to ~50% (P<0.01), while overexpression of miRNA-124-3p showed no influence on mutant pMiR_STAT3_MUT. These results revealed that miRNA-124-3p could directly combine with 3'UTR of STAT3 to inhibit the STAT3 transcriptional activity in cells.

Effects of microRNA-124-3p expression on proliferation of nasopharyngeal carcinoma cells. As in Fig. 3A, after overex-pression of miRNA-124-3p in C666-1 cells, cell proliferation in miRNA-124-3p mimics group was obviously decreased, and lower than that in the mimics NC group and blank control group at 24, 48, 72 and 96 h (all P<0.05); while cell proliferation in mimics NC group and blank control group demonstrated no significant differences at each time-point (all P>0.05). As shown in Fig. 3B, after inhibition of miRNA-124-3p expression in CNE-2 cells, cell proliferation in miRNA-124-3p inhibitor was increased, and higher than that in inhibitor NC group and blank control group at 24, 48, 72 and 96 h (all P<0.05); however, cell proliferation in inhibitor NC group and blank control group showed no significant differences at each time-point (all P>0.05).

Effects of microRNA-124-3p expression on apoptosis of nasopharyngeal carcinoma cells. As shown in Fig. 4, the apoptosis ratio of C666-1 cells was (30.7±2.5%) in miRNA-124-3p mimics group, (14.9±1.3%) in blank control group, (14.7±1.2%) in mimics NC group, and increased significantly (all P<0.05). However, the apoptosis ratio of C666-1 cells showed no differences between blank control group and mimics NC group (P>0.05). The apoptosis ratio of CNE-2 cells was (2.9±0.56%) in miRNA-124-3p inhibitor group, and lower than that in inhibitor NC group (8.9±1.3) and blank control group (9.1±1.2) (all P<0.05). While the apoptosis ratio

Figure 1. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect microRNA-124-3p expression in nasopharyngeal carcinoma tis-sues, postnasal catarrh tissues and cell lines. (A) microRNA-124-3p expressions in nasopharyngeal carcinoma tissues and postnasal catarrh tissues (**P<0.001, compared with control group); (B) microRNA-124-3p expression in nasopharyngeal carcinoma cell lines.

Table i. MicroRNA-124-3p expression in different clinical variables of nasopharyngeal carcinoma tissues.

MicroRNA-124-3pVariables n expression P-value

Gender Male 60 1.532±0.496 0.862 Female 30 1.551±0.472Age (years) >45 52 1.546±0.481 0.841 ≤45 38 1.525±0.498T stages T1-T2 56 1.827±0.313 <0.001 T3-T4 34 1.047±0.331N stages N0-N1 41 1.951±0.273 <0.001 N2-N3 49 1.182±0.345Distant metastases Metastasis 6 1.604±0.295 0.716 No metastasis 84 1.527±0.508Clinical stages i-ii 34 2.017±0.252 <0.001 iii-iV 56 1.237±0.355


of CNE-2 cells showed no differences between inhibitor NC group and blank control group (P>0.05).

Effects of microRNA-124-3p expression on migration of naso-pharyngeal carcinoma cells. As shown in Fig. 5, the scratch

Figure 2. STAT3 as the target gene of microRNA-124-3p. (A) Sequences in STAT3 3'UTR were matched with that of miRNA-124-3p; (B) Nasopharyngeal carcinoma cells transfected with reporter gene plasmids of STAT3 3'UTR or target-site deletion and reference, and negative control (NC) or microRNA-124-3p mimics. After 48 h, dual-luciferase system was conducted.

Figure 3. Effects of miR-124-3p expression on proliferation of C666-1 and CNE-2 cells. (A) Overexpressed microRNA-124-3p suppressed C666-1 prolifera-tion; (B) inhibition of microRNA-124-3p increased CNE-2 proliferation; *P<0.05 compared with negative control (NC) group and blank control group.

Figure 4. CNE-2 and C666-1 apoptosis after transfection with microRNA-124-3p inhibitor/mimics. (A) C666-1 apoptosis in microRNA-124-3p mimics group was higher than negative control (NC) group and blank control group, *P<0.05 compared with microRNA-124-3p mimics group; (B) CNE-2 apoptosis in miRNA-124-3p inhibitor group was lower than inhibitor negative control (NC) group and blank control group, *P<0.05, compared with microRNA-124-3p inhibitor group.


assay results indicated that after overexpression of miRNA-124-3p, migration rate of C666-1 cells was decreased as compared with that in blank control group and the mimics NC group (all P<0.05); while no significant difference existed between blank control group and mimics NC group (P>0.05). After inhibition of miRNA-124-3p expression, migration rate of CNE-2 cells increased significantly as compared with that in inhibitor NC group and blank control group (all P<0.05); while the differences of migration rate between inhibitor NC group and blank control group showed no significance (P>0.05). Transwell assay results demonstrated that together with the increase in miRNA-124-3p expression, the number of migrated C666-1 cells decreased obviously as compared with that in mimics NC group and blank control group

(all P<0.05); while the number of migrated cells showed no differences between blank control group and mimics NC group (P>0.05). With the inhibition of miRNA-124-3p expression, the number of migrated CNE2 cells increased compared to that in inhibitor NC group and blank control group (all P<0.05); however, the number of migrated cells presented no differences between blank control group and inhibitor NC group (P>0.05, Fig. 6).

Effects of microRNA-124-3p expression on the cell inva-sive ability. As shown in Fig. 7, with the overexpression of miRNA-124-3p, the number of invasive C666-1 cells was decreased as compared with that in blank control group and mimics NC group (all P<0.05); no difference was revealed

Figure 5. Cell migration detected by the scratch test (magnification, x100). The first image shows the results for the C666-1 cell wound scratch assay, which has shown that following miRNA-124-3p overexpression, the cell migration rate of C666-1 cells was decreased compared with untreated group and mimics NC group. The second image shows the results for the CNE-2 cell wound scratch assay, which has demonstrated that following inhibition of miRNA-124-3p expression, the migration rate of CNE-2 cells increased significantly as compared with that in the inhibitor NC group and the untreated group.


between blank control group and mimics NC group (P>0.05). After the inhibition of miRNA-124-3p expression, the inva-sive number of CNE2 cells increased compared to that in inhibitor NC group and blank control group (all P<0.05); but no difference existed between inhibitor NC group and blank control group (P>0.05).

Protein levels detected by western blotting. Fig. 8 shows that, STAT3, p-STAT3, CCND2 and MMP-2 levels of C666-1 cells were significantly inhibited in miRNA-124-3p mimics group compared to those in blank control group and mimics NC group (all P<0.05); however, no difference of protein levels was found (P>0.05). STAT3, p-STAT3, CCND2 and MMP-2 levels of CNE2 cells were significantly increased in miRNA-124-3p inhibitor group as compared with those in blank control group and inhibitor NC group (all P<0.05); while no difference of protein levels existed between blank control group and inhibitor NC group (P>0.05).

Discussion

In this study, we identified that miR-124-3p was significantly downregulated in NPC cells; moreover, miR-124-3p down-regulates the transcription of the STAT by interfering with its 3'UTR, and the degradation of STAT3 influences expres-sion of p-STAT3 CCND2 and MMP-2 thereby promoting NPC cell apoptosis and inhibiting proliferation, migration and invasion of NPC cells. The above results supported that STAT3 was a direct target gene of miR-124-3p.

Deregulated gene expression is one of the chief hallmarks of cancer cells, correlated with a myriad of mechanisms resulting in overexpression of tumor-promoting genes or downregulation of tumor-suppressing genes, accordingly promoting tumor progression (33). Previous studies have revealed that the miR-124-3p is a tumor suppressant and commonly downregulated in glioma, hepatocellular carci-noma, oral squamous cell carcinomas as well as breast

Figure 7. Cell invasion detected using Boyden chamber assay (magnification, x200). (A) With the overexpression of miRNA-124-3p, the number of invasive C666-1 cells was decreased as compared with that in the blank control group and the mimics NC group. *P<0.05 compared with the miRNA-124-3p mimics group. (B) Following inhibition of miRNA-124-3p expression, the invasive number of CNE2 cells increased compared to that in the inhibitor NC group and the blank control group. *P<0.05 compared with miRNA-124-3p inhibitor.

Figure 6. Cell migration tested by Transwell migration assay (magnification, x200). (A) Together with the increase in miRNA-124-3p expression, the number of migrated C666-1 cells evidently decreased compared with that in the mimics NC group and the blank control group. *P<0.05 compared with the miRNA-124-3p mimics group. (B) With the inhibition of miRNA-124-3p expression, the number of migrated CNE2 cells increased compared to that in the inhibitor NC group and the blank control group. *P<0.05 compared with the miRNA-124-3p inhibitor group.


cancer (34-37). Evidence revealed that tumor-specific silencing of miR-124-3p was a common molecular event in HCC resulting in cell cycle arrest at the G1-S check-point as well as apoptosis in HCC cells (35). in this study, the evidence indicated that miR-124-3p could inhibit the proliferation, migration and invasion of NPC cells. The suppressive capability of miR-124-3p indicated its function as a tumor-suppressive microRNA in NPC. Several studies have illustrated that miR-124-3p was downregulated and negatively associated with clinical characteristic and prog-nosis in glioblastoma and breast cancer (38,39). Shi et al demonstrated that miR-124-3p was a potential tumor-suppressive miRNA and downregulated in prostate cancer causing inhibition of prostate cancer cell proliferation by

targeting the androgen receptor (25). These results revealed a crucial role for miR-124-3p in the proliferation as well as metastasis of different cancers. Furthermore, our results were consistent with previous observations of Peng et al that the downregulation of miR-124-3p in NPC tissues was closely related with clinical stages, regional lymph node involvement and T stages (18). Consequently, miR-124-3p could be used as an independent biomarker for diagnosis and treatment of NPC patients with different clinical characteristics.

The expression of STAT3, p-STAT3, CCND2 and MMP-2 levels were significantly suppressed after trans-fection of miR-124-3p mimics; while after inhibition of miRNA-124-3p expression, STAT3, p-STAT3, CCND2 and MMP-2 levels were notably increased. STAT3, an oncogene,

Figure 8. STAT3, p-STAT3, CCND2 and MMP-2 expressions detected applying western blotting. (A) STAT3, p-STAT3, CCND2 and MMP-2 levels of C666-1 cells were significantly inhibited in the miRNA-124-3p mimics group compared to those in the blank control group and the mimics NC group. *P<0.05 compared with the miRNA-124-3p mimics group. (B) STAT3, p-STAT3, CCND2 and MMP-2 levels of CNE2 cells were significantly increased in the miRNA-124-3p inhibitor group as compared with those in the blank control group and the inhibitor NC group. *P<0.05 compared with miRNA-124-3p inhibitor group.


is a vital regulator for multiple cellular processes, including cell growth, metastasis, apoptosis, differentiation and epithe-lial-mesenchymal transition, and plays an important role in NPC carcinogenesis (31). EBV infection showed strong asso-ciation with NPC. A previous study by Lo et al discussed the linkage between EBV infection and STAT3 activation in NPC cells; the results demonstrated that introduction of the EBV genome into NPC cells lead to STAT3 activation (P-STAT3), indicating a potential role of STAT3 in NPC carcinogen-esis (40). P-STAT3 (STAT3 activation) was found to be in most of NPCs to be crucial in driving NPC progression and metastasis (30). CCND2 and MMP-2, downstream proteins of Stat3, play crucial roles in the regulation of cell cycle progression and metastasis (41). CCND2, a member of the cyclin families, activates cyclin-dependent kinases (CDKs), and controls the cell cycle at key checkpoints; furthermore, overexpression of CCND2 has been reported in many tumors including NPC (42,43). MMPs, a family of zinc metallo-endopeptidases, digest extracellular matrix (ECM) molecules, and were closely correlated with cancer development and progression (44). MMP-2, a 72-kDa type iV collagenase, has been suggested to be a vital factor in facilitating the metastasis of NPC (45). Our results revealed that miR-124-3p expression negatively regulated STAT3, and correspondingly influence p-STAT3 expression and downstream expression of CCND2 and MMP-2 in NPC cells, suggesting an important role of miR-124-3p in NPC biology.

This study revealed that miR-124-3p negatively regulated the transcription of the STAT3 by interfering with its 3'UTR, and the degradation of STAT3 affects downstream expression of p-STAT3, CCND2 and MMP-2, thereby promoting NPC cells apoptosis and inhibiting proliferation, migration and invasion of NPC cells. This study contributes to our under-standing of molecular mechanisms, and in the search for new molecular therapeutic targets in NPC.

Acknowledgements

We would like to acknowledge the reviewers for their useful comments on this paper.

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