MicroRNA-185 inhibits cell proliferation while promoting apoptosis and autophagy through negative regulation of TGF- β 1/mTOR axis and HOXC6 in nasopharyngeal carcinoma

Abstract

This article has been retracted, and the online PDF replaced with this retraction notice. doi: 10.3233/CBM-220951

1. Introduction

Nasopharyngeal carcinoma (NPC) is a distinct type of malignant head and neck cancer and has a striking geographic and ethnic distribution, particularly in Southeast Asia and Southern China [1]. Multiple risk factors contributing to NPC include Epstein-Barr virus (EBV) infection, cigarette smoking, alcohol consumption nitrosamines in salted, pickled foods, as well as genetic susceptibility [2, 3, 4]. Although the primary tumor was controlled by the treatment with intensity-modulated radiation therapy (IMRT) and its combination with chemotherapy temporarily, local recurrence and distant metastases are still major obstacles to achieve successful treatment for NPC treatment [5]. Most patients with NPC are diagnosed at later stages, approximately 90% of them are diagnosed with lymph node metastases, and 5–10% of them manifested with distant metastases [6]. Many patients are still afflicted with the disease due to the poor response to chemotherapy, particularly in the advanced stages [7]. However, the molecular mechanism responsible for NPC onset and progression is still in its infancy [8]. In this respect, many tumor suppressor microRNAs (miRs) have been reported to participate in NPC progression and therapeutic response, such as miR-451 and miR-3188 [9, 10].

MiRs, non-coding RNAs affect almost every cellular process, such as proliferation, apoptosis, differentiation, invasion, and metabolism [11, 12]. MiR-185-3p is recently considered as a candidate in the abnormal profile of radio-resistant miRNA and modulates NPC radio-resistance, while its specific role in NPC remains a matter of controversy [13, 14]. It is presumed that miR-185 can bind with the 3’-untranslated region (UTR) region of homeobox C6 (HOXC6) gene in the biological prediction site microRNA.org. Interestingly, a previous study also investigates that HOXC6 is identified as being overexpressed in human head and neck squamous cell carcinoma [15]. At the same time, HOXC6 has been reported to be an oncogene that promotes tumor cell proliferation by the inhibition of autophagy and activation of mTOR pathway [16]. Recent studies indicate that the activation of mTOR pathway could promote NPC cell proliferation and tumorigenesis [17, 18]. Furthermore, transforming growth factor $\beta$ 1 (TGF- $\beta$ 1) has been reported as a key regulator inducing epithelial to mesenchymal transition (EMT) in head and neck squamous cell carcinoma [19]. These data lead us to hypothesis that miR-185 manifests itself as a tumor suppressor in NPC. We also analyzed the relativity of the miR-185 in relation to TGF- $\beta$ 1/mTOR axis via HOXC6 gene in NPC in vivo and in vitro.

2. Materials and method

2.1 Microarray analysis

“Nasopharyngeal carcinoma” served as the key word was retrieved in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), andGSE13597 and GSE64634 were selected. GSE13597 included 3 normal control samples and 25 NPC samples, and GSE64634 included 4 normal control samples and 12 NPC samples. The datasets were processed with differential analysis using Limma packageof R language. $|$ logFC $|$ $>$ 2 and $p$ value $<$ 0.05 were set as the threshold to screen out differentially expressed genes (DEGs). The pheatmap package of R language was employed to analyze the first 30 DEGs in the results of two datasets to construct the heat maps of expression. Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to construct Venn map of the first 50 DEGs to find the intersection between two datasets. Besides, GSE12425 dataset was found in GEO database, and it contained 10 normal control samples and 31 NPC samples. To further analyze the HOXC6 expression in GSE12425 dataset to identify the screening results. Moreover, “NPC” and “microRNA (miR)” served as the key words were retrieved in the GEO database. The finding showed that GSE70970 dataset included 17 normal control samples and 246 NPC samples. GPL20699 miRNA was used as sequencing Platform. The GSE70970 was possessed with differential analysis using the abovementioned methods.

2.2 Analysis of miRNAs that regulate HOXC6

The miRs that regulate HOXC6 were retrieved in the TargetScan (http://www.targetscan.org/vert_71/) and microRNA.org (http://34.236.212.39/microrna/home.do) databases with human as selection. Meanwhile, GSE70970 differential miR was analyzed. The Venn diagrams website was employed to select intersection between the predicted results in the two databases and results of GSE70970 differential analysis.

2.3 Ethics statement

The experiment was approved by the Institutional Review Board of the Second Hospital of Jilin University. Written informed consent was obtained from each participant. And all experimental procedures were accordance with the requirements committee of the Second Hospital of Jilin University.

2.4 Study subjects

A total of 126 patients (81 males and 45 females with an average age of 45.14 $\pm$ 13.72 years) of NPC confirmed by pathology in the Second Hospital of Jilin University during the 2014 $\sim$ 2016 years were enrolled. The tumor-node-metastasis (TNM) classification allocated 83 patients to stage I/II, 43 to stage III/IV, 47 to lymph node metastasis (LNM), 79 to non-LNM, 43 to high differentiation, 29 to moderate differentiation, 154 to low differentiation [20]. All patients were not treated with radiotherapy and chemotherapy. Meanwhile, the paracancerous tissues from 126 patients with NPC in the Second Hospital of Jilin University were selected as the control group. Exclusion criteria were as follows: patients accepted radiotherapy or chemotherapy or have past history of malignancy in addition to NPC, congenital or acquired immunodeficiency disease. The patients never had infection according to the Karnofsky (KPS) $<$ 70 and hemoglobin is less than 90 g/L.

2.5 Follow-up information

Outpatient follow-up, combination with telephone and letters were used to follow up the patients. The first follow-up was performed at one month after treatment for all patients. The follow-up deadline was December 2017. Routine chest radiography, abdominal B ultrasound and laryngoscopy were performed during outpatient review. Nasopharyngeal computerized tomography (CT) scans, magnetic resonance imaging (MRI) examination, or whole-body skeletal ECT scans were selected to perform for these patients. The follow-up time of 126 cases of NPC was 5 $\sim$ 48 months, 121 cases were successfully followed up, and the follow-up rate was 96.03%. And 5 cases were lost to follow-up, and the loss rate was 3.9%. The survival time was calculated from the end of treatment to the patient’s death or the last follow-up date.

2.6 Immunohistochemistry

The tissues were dehydrated, embedded in paraffin and cut into 4 $\mu$ m sections. The sections were added with citrate buffer (pH 6.0). After dewaxing and hydration, endogenous peroxidase (PO) was blocked with 30 mL hydrogen peroxide (H ${}_2$ O ${}_2$ ), closed with sheep serum and incubated with rabbit anti-human HOXC6 (ab151575, 1/1000) at 4 ${}^{\circ }$ C. After washing, samples was added with biotin-labeled goat anti-rabbit secondary antibody immunoglobulin G (IgG) and incubated at 37 ${}^{\circ }$ C for 30 min. Subsequently, after washing, samples were reacted with streptavidin-horseradish peroxidase (SA-HRP) compounds, stained using diaminobenzidine (DAB), and counterstained by hematoxylin. Ten high-magnification fields ( $\times$ 400) were randomly selected in each section and scored individually. The evaluation standards are as follows: staining intensity score: negative, 0 points; very weak positive, 1 point; weak positive, 2 points; positive, 3 points. Percentage of positive NPC cells: Positive cells $⩽$ 5%, 0 points; 6%–25%, 1 points; 26%–50%, 2 points; 51%–75%, 3 points; Positive cells $⩾$ 75%, 4 points. Score of each visual field $=$ staining intensity score $\times$ percentage of positive NPC cells (or nasal mucosa cells). The final score of the section was the average score of 10 visual fields.

2.7 Screening of NPC cell lines

NPC cell lines (5-8F, HNE-1, HNE-2) were purchased from the cell center of Xiangya School of Medicine (Central South University, Changsha, China), and (CNE-1, CNE-2) were purchased from American Type Culture Collection (ATCC Manassas, VA, USA). The five cell lines were maintained at 37 ${}^{\circ }$ C in a humidified atmosphere of 5% CO ${}_2$ in RMPI-1640 media and supplemented with 10% fetal bovine serum (FBS). The inoculum was replaced every 24 h for subculture and cell line with the highest expression of miR-185 was selected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) for further experiment.

Table 1

The primers for RT-qPCR

Genes	Primer sequences
miR-185	Forward: 5’-CATGGAGAGAAAGGCAGT-3’
	Reverse: 5’-GTGCAGGGTCCGAGGT-3’
HOXC6	Forward: 5’-CACCGCCTATGATCCAGTGAGG
	CA-3’
	Reverse: 5’-GCTGGAACTGAACACGACATTC
	TC-3’
TGF- $\beta$ 1	Forward: 5’-CCCAGCATCTGCAAAGCTC-3’
	Reverse: 5’-GTCAATGTACAGCTGCCGCA-3’
mTOR	Forward: 5’-TGTTCCGACGAATCTCAAAGC-3’
	Reverse: 5’-TCATATGTTCCTGGCACAGCC-3’
Cyclin D1	Forward: 5-’GCTGCGAAGTGGAAACCATC-3’
	Reverse: 5’-CCTCCTTCTGCACACATTTGAA-3’
PCNA	Forward: 5’-CCTGCTGGGATATTAGCTCCA-3’
	Reverse: 5-’CAGCGGTAGGTGTCGAAGC-3’
bcl-2	Forward: 5’-CTGCACCTGACGCCCTTCACC-3’
	Reverse: 5’-CACATGACCCCACCGAACTCAA
	AGA-3’
Bax	Forward: 5’-AAGAAGCTGAGCGAGTGTC-3’
	Reverse: 5’-CGGCCCCAGTTGAAGTTGC-3’
caspase-3	Forward: 5’-GGCTTTGACCACGACCTTTGA
	AGA-3’
	Reverse: 5’-GGAAGGGCACTTCAAACCAGT
	GAA-3’
LC3	Forward: 5’-GATGTCCGACTTATTCGAGAGC-3’
	Reverse: 5’-TTGAGCTGTAAGCGCCTTCTA-3’
Beclin1	Forward: 5’-TGTCACCATCCAGGAACTCA-3’
	Reverse: 5’-CTGTTGGCACTTTCTGTGGA-3’
U6	Forward: 5’-GTACAAAATACGTGACGTAGA
	AAG-3’
	Reverse: 5’-GGTGTTTCGTCCTTTCCAC-3’
GAPDH	Forward: 5’-CAACTACATGGTTTACATGTTC-3’
	Reverse: 5’-GCCAGTGGACTCCACGAC-3’

Note: RT-qPCR, reverse transcription quantitative polymerase chain reaction; miR-185, microRNA 185; HOXC6, homeobox C6; TGF- $\beta$ 1; transforming growth factor $\beta$ 1; PCNA, proliferating cell nuclear antigen; Bcl-2, B cell lymphoma 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

2.8 Cell transfection and grouping

The shRNA sequence of HOXC6 gene was designed by Thermo Fisher’s (Thermo Fisher Scientific Inc., NY, USA) shRNA design software: CAC CGCAGGACTGCTCTAGATTAAGCGAACTTAATC TAGAGCAGTCCTGCAAAA. Connection and cleavage endonuclease of liner between DNA fragment were encoded by shRNA-HOXC6 and blank plasmid pRNAT-CMV3.2/Neo (SD1264, Beijing China Ocean Co., Ltd., Beijing, China), and then recombinant shRNA-HOXC6 expression plasmid was transferred into the competent Escherichia coli, and marked gene and screened the positive clones. CNE-2 cells were divided into six groups, including the blank (without any treatment), NC (negative control, transfected with empty plasmid), miR-185 mimic (transfected with miR-185 mimic plasmid), miR-185 inhibitor (transfected with miR-185 inhibitor plasmid), shRNA-HOXC6 (transfected with HOXC6 shRNA plasmid) and miR-185 mimic $+$ shRNA-HOXC6 groups (transfected with HOXC6 shRNA and miR-185 mimic plasmid). Target plasmid was purchased from Dharmacon Research, Inc. (Lafayette, CO, USA). Then CNE-2 cells in the logarithmic growth were seeded in 6-well plates at a concentration of 3 $\times$ 10 ${}^5$ cells per well. When the cell density reached 80%, lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) was used for cell transfection. Target plasmid with 4 $\mu$ g and 10 $\mu$ L Lipofectamine 2000 were diluted by 250 $\mu$ L serum free Opti-MEM (Gibco, CO, USA), respectively. After fixed at room temperature for 5 min, the mixture was well distributed. After fixed for 20 min, mixture was added into culture under a saturated humidity condition of 5% CO ${}_2$ at 37 ${}^{\circ }$ C. After 6 h incubation, cells were replaced with complete medium, cultured for 48 h, followed by collection of cells.

2.9 Dual-luciferase reporter gene assay

The bioinformatics prediction website (http://www.microRNA.org) was used to analysis target gene of miR-185, and dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-185 and HOXC6. The full length of 3 UTR region of HOXC6 amplified gene and PCR products were cloned into downstream of the multiple cloning sites pmirGLo (Promega, WI, USA) luciferase vector by using the utilize endonuclease site SpeI and Hind III build the HOXC6-wild type (Wt) vector. Targetscan database was used to predict the binding sites of miR-185 and its target genes. Polymerase chain reaction (PCR) was used to structure HOXC6-Mutant (Mut) vector. Renilla luciferase expression vector pRL-TK (TaKaRa, Dalian, China) was served as internal reference. miR-185 mimic/NC and luciferase reporter vector were co-transfected into cells and dual-luciferase reporter gene assay was performed based on Promega Dual-Luciferase Reporter Gene Assay System (Promega, Madison, WI, USA). The program was conducted and the data was collected at the end of fluorescence readings. Then the ratio of firefly luciferase expression and renilla luciferase expression was calculated. The experiment was repeated for 3 times.

2.10 RT-qPCR

Total RNA was extracted by Trizol Kit (No.16096 020, Thermo Fisher, NY, USA). The concentration and purity of RNA were determined by ultraviolet spectrophotometer. Five $\mu$ g RNA was reversely transcripted into complementary deoxyribo nucleic acid (cDNA) according to RT-qPCR Kit (ABI company, Forst City, CA, USA), Conditions for reverse transcription reaction were as follows: reverse transcription at 37 ${}^{\circ }$ C for 30 min, and inactivation of reverse transcriptase at 85 ${}^{\circ }$ C for 5 s. Based on SYBR ${}^{\mathrm{\circledR }}$ Premix Ex TaqTM II Kit (RR820A, Xingzhi Biological Technology, Guangzhou, China) instruction, RT-qPCR was performed in a reaction system of 50 $\mu$ L. Reaction system was as follows: 25 $\mu$ L SYBR ${}^{\mathrm{\circledR }}$ Premix Ex TaqTM II (2 $\times$ ), 2 $\mu$ L PCR upstream primer, 2 $\mu$ L PCR downstream primer, 1 $\mu$ L ROX Reference Dye (50 $\times$ ), 4 $\mu$ L DNA template, 16 $\mu$ L double-distilled water (ddH ${}_2$ O). Reacted in ABI PRISM ${}^{\mathrm{\circledR }}$ 7300 (Prism ${}^{\mathrm{\circledR }}$ 7300, KunKe, Shanghai, China) system. The amplification conditions were as follows: Pre-denaturation at 94 ${}^{\circ }$ C for 5 min, denaturation at 94 ${}^{\circ }$ C for 30 s, followed by 30 cycles of annealing at 54.5 ${}^{\circ }$ C for 30 s, extension at 72 ${}^{\circ }$ C for 30 s, at 72 ${}^{\circ }$ C for 10 min, and then reserved at 4 ${}^{\circ }$ C. The primer sequences for RT-qPCR are presented in Table 1 (Synthesized by Shanghai Jierui bioengineering Co., Ltd., China), U6 was served as internal reference of miR-185, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was served as the internal reference of other target genes. 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ was used to calculate the relative expression of the targeted genes. The formula was as follows: $\mathrm{\Delta }\mathrm{\Delta }\text{Ct}=\mathrm{\Delta }{\text{Ct}}_{\text{experimental group}}-\mathrm{\Delta }{\text{Ct}}_{\text{control group}}$ and $\mathrm{\Delta }\text{Ct}={\text{Ct}}_{\text{target gene}}-{\text{Ct}}_{\text{GAPDH}}$ . Ct is referred to amplified cycles when the real time fluorescence intensity reached the set threshold. Each experiment was performed in triplicates.

2.11 Western blot analysis

Cells were extracted and homogenated by 5 times volume of cell homogenate in ice for 10 min in a homogenizer. Cells were transferred into centrifuge tube, disrupted by ultrasonic wave 15 s for 3 times and centrifuged for 20 min at 25764 $\times$ g at 4 ${}^{\circ }$ C. The supernatant was extracted and total protein concentration was measured with a the bicinchoninic acid (BCA) kit, and the final product was stored at $-$ 20 ${}^{\circ }$ C, followed by separated 10% separation gel and 5% lamination gel by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation of proteins by polyacrylamide gel electrophoresis, the protein was transferred to nanocrystalline (NC) membrane by wet rotation method, and the membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Then the membranes were incubated overnight at 4 ${}^{\circ }$ C with diluted rabbit polyclonal antibody HOXC6 (ab151575, 1:1000), TGF- $\beta$ 1 (ab92486, 1:100), mTOR (ab2732, 1:2000), caspase-3 (ab13847, 1:500), Bax (ab32503, 1:1000), BCL 2 (ab32124, 1:1000), PCNA (ab92552, 1:1000). Cyclin D1 (ab134175, 1:10000), LC3A/B (ab128025, 1:500), Beclin1 (ab210498, 1:1000). All of the antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). The membranes were washed with butylene succinate-co-butylene terephthalate (PBST) for 3 times, 10 min per time. After secondary Goat-anti-rabbit (ab205718, Abcam, Cambridge, MA, USA) polyclonal antibody was diluted by 5% skim milk, second antibody was incubated, placed at shaking table, and shaken at room temperature for 1 h, followed by rinsing using phosphate buffer saline (PBS) for 3 times, 15 min per time. The developer was added and bio-Rad gel imaging system (MG8600, Thmorgan, Beijing, China) was used to photograph, IPP7.0 software (Media Cybernetics, Singapore) was used for quantitative analysis. The experiment was repeated 3 times.

2.12 Counting Kit-8 (CCK-8) assay

After transfection for 12 h, CNE-2 cells were inoculated in 96-well plates at a density of 2000 cells per well. Then 100 $\mu$ L cells were added into cell-culture medium and cultured at 37 ${}^{\circ }$ C. Cell vitality was evaluated at 24 h, 48 h, and 72 h after transfection. Each well of the plate was supplemented with 10 $\mu$ L of CCK8 kit (C0037, Beyotime, Shanghai, China) and incubated in an incubator for 2 h at 37 ${}^{\circ }$ C. The optical density (OD) value of each well was evaluated at 450 nm by using a multimode microplate reader (Multiskan FC, Thermo Fisher, NY, USA). Three replicates were prepared. The experiment was conducted three times to obtain the average. The curve of cell viability was drawn with time as the horizontal axis, and the OD value as the vertical axis. The experiment was repeated for 3 times.

2.13 Flow cytometry

Based on the instruction of Annexin-V-FITC apoptosis detection kit (C1065, Beyotime, Shanghai, China), Annexin-V-FITC, propidium iodide (PI), 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES)were equipped with a Annexin-V-FITC/PI dye liquid buffer on the proportion 1:2:50. After transfection for 48 h, every 100 $\mu$ L dye liquor was re-suspended at a density of 1 $\times$ 10 ${}^6$ cells, mixed and incubated at room temperature for 15 min, followed by added with 1 mL HEPES buffer and mixed. The flow cytometry was used to record the excitation wavelength at 488 nm, 525 nm and 620 nm bandpass filters to detect the fluorescence of FITC, PI, and cell apoptosis, respectively. Each group had 3 samples and repeated for 3 times.

After digestion, digestive juice was abandoned and the cells shrank and became round under a microscope. Cells were added with serum-containing culture fluid to terminate the digestion. The cells were then dissociated into suspension. Suspension was centrifuged at 1000 r/min for 5 min, and then the supernatant was removed. Cells was washed with balanced salt solution PBS for 2 times and 60 $\mu$ m aperture filter was used to filter cells, then cells were fixed for 30 min with 70% precooling ethanol, centrifuged and collected. After rinsed with PBS, cells were added with 1% iodinated PI containing RNA enzyme and dyed for 30 min. PI was rinsed with PBS 2 times. The equilibrium PBS was used to balance the salt solution and adjust volume to 1 mL, and the cells were filtered with 60 $\mu$ m aperture filter. Cell cycle were detected using BD-Aria flow cytometry (FACSCalibur, Beckman Kurt, USA), with 3 samples per group, and the experiment was repeated 3 times.

2.14 Transwell assay

Fifty mg/L matrigel (Sigma, SF, USA) was diluted at the proportion of 1:8. Each chamber were laid with 60 $\mu$ L matrigel on the upper surface of the basement membrane and air dried at room temperature. The residual liquid from the culture plate was absorbed, added with 50 $\mu$ L serum-free culture medium containing 10 g/L BSA, and fixed at 37 ${}^{\circ }$ C for 30 min. After transfection for 12 h, the cells of each group were cultured to logarithmic growth phase and those cells were selected in each group. Serum-free culture containing 10 g/L BSA was used to adjust the cells density to 1 $\times$ 10 ${}^5$ /mL. A total of 200 $\mu$ L cell suspension was added in each transwell. A total of 500 $\mu$ L medium containing 100 mL/L FBS was added into the lower chamber plate and transwell was cultured in 5% CO ${}_2$ incubator at 37 h for 24 h. After removing the cell, cotton swab was used to wipe the photovoltaic power forecasting (PVPF) membrane close to the inner surface of the chamber, and then fixed at room temperature with 95% alcohol for 30 min, transfected by crystal violet (Sigma, SF, USA) for 20 min and rinsed by clean water for 3 times. Finally, images were acquired with an inverted microscope (CKX41SF inverted optical microscope, OLYMPUS, Japan).

2.15 Morphological observation of autophagy

CNE-1 cells density was adjusted from logarithmic growth phase to the density of 1.5 $\times$ 10 ${}^5$ /well after cell counting. The culture medium was removed after cultured for 24 h in a 6-well plate, then cultured 48 h with 2 mL medium containing 0.1% dimethyl sulfoxide (DMSO). The original culture solution was removed and added with 0.5 mL fixed solution. After cultured for 10 min, the waste fixative was removed and rinsed with PBS for one time, eliminated the waste solution for 15 min avoiding exposure to light. The waste liquid was rinsed with PBS for 3 times and stored avoiding exposure to light. The morphology of CNE-2 cells in each group was observed under a fluorescence microscope, and the ultrastructural changes in each group were observed under a transmission electron microscope.

2.16 Statistical analysis

Statistical analysis was conducted by using SPSS 21.0 (IBM Corp., Armonk, NY, USA). All the parameters of the experimental data were expressed by mean $\pm$ standard deviation. Differences between NPC tissues and paracancerous tissues were compared by $t$ test. Multiple groups were compared by analysis of variance, and by the Kolmogorov-Smirnov test when there was equal variance across groups. The LSD test was used for comparisons of two groups, and Wilcoxon rank-sum test was used when variances were heterogeneous. Inspection level $\alpha =$ 0.05, the Kaplan-Meier method was used for survival analysis. A $p<$ 0.05 was considered significant.

Figure 1.

NPC is modulated by miR-185 via regulating HOXC6 by microarray analysis. A, analysis results of GSE13597 chip data; B, analysis results of GSE64634 chip data. The abscissa refers to sample number, and the ordinate refers to names of DEGs. The upper right histogram refers to color gradation, and the color changes from top to the bottom means that the expression value changes from high to low. Each block represents the expression level of gene in one sample, and each column represents the expression of all genes in samples. The left dendrogram refers to the cluster analysis results of different genes in different samples. The transverse line at the top represents the types of samples: the blue refers to the control sample and the red refers to the NPC sample. C, Venn diagrams of GSE13597 and GSE64634 chip data. The blue on the left refers to analysis results of GSE13597 chip data, red on the right refers to analysis results of GSE64634 chip data, and red in the middle represents the intersection between GSE13597 and GSE64634 datasets; D, HOXC6 expression in the GSE12425 dataset. The abscissa refers to the types of samples, and the ordinate refers to names of genes and of selected datasets. The grey box represents the HOXC6 expression in control samples, and green box represents the HOXC6 expression in NPC samples. The $p$ situated in the top left corner refers to be statistically different between two datasets. E, analysis results of miR dataset GSE70970 chip data in NPC. The abscissa refers to sample number, and the ordinate refers to names of miRs. The meaning of histogram, color gradation, and dendrogram was as the same as A and B. F, the analysis results of miRs that regulate HOXC. The blue circle refers to predicted results of microRNA.org database, the red circle represents differential analysis of GSE70970 chip data, the green circle means predicted results of TargetScan database, and the arrow in the middle refers to the intersection among three results.

Figure 2.

HOXC6 is positively expressed in NPC tissues. A, the expression of HOXC6 in paracancerous and NPC tissues; B, HOXC6 positive expression in paracancerous and NPC tissues; the measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by paired $t$ test; the experiments were repeated 3 times; * $p<$ 0.05 vs. the paracancerous tissues; NPC, nasopharyngeal carcinoma; HOXC6, homeobox C6.

3. Results

3.1 miR-185 is identified to modulate NPC by regulating HOXC6 by microarray analysis

The NPC datasets GSE13597 and GSE64634 were possessed with differential analysis. The results displayed that there were 602 DEGs in GSE13597 dataset, and 198 DEGs in the GSE64634 dataset. The heat map of the first 30 DEGs in the results of two datasets were constructed (Fig. 1A and B). To further screen the genes related to NPC, the Venn maps of the first 50 DEGs in the results of two datasets were constructed to select the intersection between two datasets (Fig. 1C). The results showed that in the first 50 DEGs in the two datasets, HOXC6 showed obvious difference in the two datasets, and HOXC6 was highly expressed in NPC samples in the two datasets. To further identify the HOXC6 expression in NPC, GSE12425 was screened from GEO database, and HOXC6 expression in the GSE12425 dataset was analyzed (Fig. 1D). The findings revealed that in GSE12425 dataset, HOXC6 was also activated in NPC, which further verified the HOXC6 expression in NPC. HOXC6 might be closely related to NPC. The potential axiss related to NPC were also retrieved. Several studies have reported that the TGF/mTOR axis are closely correlated with NPC [21, 22, 23, 24]. However, whether HOXC6 could regulate NPC through the TGF/mTOR axis remains unclear. To further understand the mechanism of HOXC6, miRNA chip data related to NPC were retrieved in GEO database. Moreover, miRNA chip data GSE70970 was conducted differential analysis, and 23 differential miRs were found (Fig. 1E). The TargetScan and microRNA.org were adopted to predict the miRs that regulate HOXC6. The results showed that there were 24 miRs in microRNA.org database. Through analysis of HOXC6 in TargetScan database, there were 368 potential miRs and the first 50 miRs were selected for further analysis. The intersection between the predicted results in the two databases and results of GSE70970 differential analysis was selected (Fig. 1F). The finding indicated that only does has-miR-185 simultaneously exist in three independent analysis results. All these results showed that has-miR-185 could regulate NPC development by modulating HOXC6.

3.2 Up-regulated HOXC6 expression exhibited in NPC tissues

As shown in Fig. 2, immunohistochemistry method was performed to detect the positive expression rate of HOXC6 in NPC and paracancerous tissues. The results revealed that positive protein level of HOXC6 was presented of light yellow to dark yellow which diffusely distributed in the cytoplasm, and partially arranged in lumps or clusters. The positive expression rate of HOXC6 protein was (6.24 $\pm$ 0.61)% in NPC tissue, which was significantly higher than that in paracancerous tissue (3.11 $\pm$ 0.20)% ( $p<$ 0.05). The findings indicate that HOXC6 was overexpressed in NPC tissues.

Figure 3.

Poorly expressed miR-185 and highly expressed HOXC6 contributes to NPC progression. *, $p<$ 0.05 vs. the paracancerous tissues; the measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by paired $t$ test; the experiments were repeated 3 times; miR-185, microRNA 185; HOXC6, homeobox C6; TGF- $\beta$ 1, transforming growth factor $\beta$ 1; PCNA, proliferating cell nuclear antigen; Bcl-2, B cell lymphoma 2.

Table 2

The relationship of miR-185 and HOXC6 with the baseline characteristic of NPC patients

Item	Case	miR-185 expression	$p$	HOXC6 expression	$p$
Gender			0.075		0.668
Male	81	0.37 $\pm$ 0.03		2.35 $\pm$ 0.25
Female	45	0.36 $\pm$ 0.03		2.37 $\pm$ 0.25
Age			0.174		0.873
$<$ 60	106	0.37 $\pm$ 0.03		2.36 $\pm$ 0.27
$⩾$ 60	20	0.36 $\pm$ 0.03		2.35 $\pm$ 0.15
Histological type			0.1		0.794
Highly differentiated	11	0.39 $\pm$ 0.02		2.33 $\pm$ 0.20
Moderately differentiated	24	0.37 $\pm$ 0.03		2.34 $\pm$ 0.26
Poorly differentiated	91	0.37 $\pm$ 0.03		2.37 $\pm$ 0.25
TNM stage			$<$ 0.001		0.004
I/II stage	37	0.39 $\pm$ 0.03		2.26 $\pm$ 0.22
III/IV stage	89	0.36 $\pm$ 0.03		2.40 $\pm$ 0.25
LNM			$<$ 0.001		0.003
Yes	92	0.36 $\pm$ 0.03		2.40 $\pm$ 0.25
No	34	0.39 $\pm$ 0.03		2.25 $\pm$ 0.23

Note: miR-185, microRNA 185; HOXC6, homeobox C6; NPC, nasopharyngeal carcinoma; TNM, tumor node metastasis; LNM, lymph node metastasis.

Figure 4.

High expression of miR-185 and low expression of HOXC6 increase the survival time of NPC patients. A, the survival time of patients with low expression of miR-185 in NPC tissue was significantly lower than that of patients with high expression of miR-185; B, the survival time of patients with HOXC6 overexpression in NPC tissue was significantly lower than that of patients with low expression of HOXC6; miR-185, microRNA-185; HOXC6, homobox C6.

3.3 Low expression of miR-185 and high expression of HOXC6 is associated with NPC progression

RT-qPCR was used to detect the expression of miR-185, HOXC6, TGF- $\beta$ 1, mTOR, Cyclin D1, PCNA, Bcl-2 in NPC and paracancerous tissues. Meanwhile, we also evaluated whether there is a relationship between miR-185, the expression of HOXC6 and the clinical pathological features of patients with NPC. Compared with paracancerous tissue, the expression of miR-185 in NPC tissue was remarkably declined ( $p<$ 0.05) (Fig. 3). By means of analysis of the relationship between the expression levels of miR-185, HOXC6 and clinical pathological features of NPC patients, leads to the conclusion that miR-185 and HOXC6 expressions in patients has no significant relationship with gender, age and histological type ( $p>$ 0.05). The expression of miR-185 in patients with stage III/IV and lymph node metastasis was significantly lower than that in patients with stage I/II and without lymph node metastasis, the mRNA expression of miR-185 is opposite (Table 2). These results suggested that the expression of miR-185 and HOXC6 was closely related to the clinical stage and lymph node metastasis in NPC patients, which indicated that low expression of miR-185 and high expression of HOXC6 was associated with NPC progression.

Figure 5.

miR-185 targets HOXC6. A, it is presumed that miR-185 binds to the 3’UTR of HOXC6 on the microRNA.org; B, the luciferase activity is decreased in the CNE-2 cells treated by a combination of miR-185 mimic and HOXC6-3’UTR-wt, suggesting that miR-185 binds to the 3’UTR of HOXC6; the measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by $t$ test; the experiments were repeated 3 times; *, wt, wild-type; mut, mutant; miR-185, microRNA-185; NC, negative control; $p<$ 0.05 vs. the paracancerous tissues; HOXC6, homobox C6.

Figure 6.

miR-185 is poorly expressed in CNE-2. The measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by one-way ANOVA; the experiments were repeated 3 times; miR-185, microRNA 185; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

3.4 High expression of miR-185 and low expression of HOXC6 have longer survival time of NPC patients

In order to investigate the relationship between miR-185 and HOXC6 expression and survival time in patients with NPC, the Kaplan-Meier method was performed. Among the 121 patients with NPC, 119 were followed up with a follow-up rate of 96.03%. Five patients were lost to follow-up and the loss rate was 3.9%. The follow-up time was 5 $\sim$ 48 months. At the end of the last follow-up, 39 patients died. Among the 126 patients, 63 patients had relatively high expression of miR-185 (greater than average), and 63 patients had relatively low expression of miR-185 (less than average). The average survival time of patients with high expression of miR-185 was 45.8 months, while the survival time of patients with low expression of miR-185 was 21.2 months. The survival time of patients with low expression of miR-185 in NPC tissue was significantly lower than that of patients with high expression of miR-185 ( $p<$ 0.05) (Fig. 4A). Among the 126 patients, 61 patients had relatively high expression of HOXC6 (greater than average), and 65 patients had relatively low expression of HOXC6 (less than average). The mean survival time of patients with high expression of HOXC6 was 30.3 months, while the survival time of patients with low expression of HOXC6 was 40.9 months. The survival time of patients with HOXC6 overexpression in NPC tissue was significantly lower than that of patients with low expression of HOXC6 ( $p>$ 0.05) (Fig. 4B). These findings indicate that overexpressed miR-185 and silenced HOXC6 could longer the survival time of patients.

Figure 7.

The overexpression of miR-185/inhibition of HOXC6 dampens TGF-1/mTOR axis by RT-qPCR and western blot analysis. A, overexpressed miR-185 could inhibit HOXC6 and TGF- $\beta$ 1/mTOR axis; B, overexpressed miR-185 or silencing HOXC6 could suppress the expression of TGF-1/mTOR axis related genes and Cyclin, D1, PCNA, Bcl-2 and other genes while promoted the expression of Bax, Caspase-3, LC3, Beclin1 and other genes; the measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by one-way ANOVA; the experiments were repeated 3 times; *, $p<$ 0.05 vs. the blank group; #, $p<$ 0.05 vs. the miR-185 mimic and shRNA-HOXC6 groups; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR-185, microRNA-185; HOXC6, homeobox C6; TGF- $\beta$ 1, transforming growth factor $\beta$ 1; PCNA, proliferating cell nuclear antigen; Bcl-2, B cell lymphoma 2; NC, negative control; p-mTOR, content of mTOR phosphorylation.

3.5 HOXC6 is a target gene of miR-185

The bioinformatics prediction website (http://www.microRNA.org) was used to analysis target gene of miR-185, and dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-185 and HOXC6. According to the online bioinformation analysis software, miR-185 targeted the sequence of 3’-UTR region of HOXC6 gene (Fig. 5A). Results of the dual luciferase reporter gene assay show that luciferase activity was significantly decreased in cells co-transfected with miR-185 and HOXC6-Wt compared with that co-transfected with NC ( $p<$ 0.05). However, in cells carrying the HOXC6-Mut plasmid, luciferase activity did not differ significantly between the two groups ( $p>$ 0.05).These findings provide evidence that miR-185 could specifically bind to HOXC6 and down-regulate the gene expression (Fig. 5B).

Figure 8.

Cell viability is inhibited by overexpressed miR-185. The measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by ANOVA; the experiments were repeated 3 times; *, $p<$ 0.05 vs. the blank group; #, $p<$ 0.05 vs. the miR-185 mimic and shRNA-HOXC6 groups; NC, negative control; miR-185, microRNA-185; HOXC6, homobox C6.

Figure 9.

Overexpression of miR-185 reduces cell proliferation and induces cell apoptosis determined by PI single staining test, Annexin V-FITC staining, and flow cytometry. A and B, the PI single staining test result showed that miR-185 could inhibit the proliferation of NPC cells by targeting HOXC6 gene; C and D, the Annexin V-FITC staining and flow cytometry showed that overexpressed miR-185 could promote NPC cell apoptosis; the measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by one-way ANOVA; the experiments were repeated 3 times; *, $p<$ 0.05 vs. the blank group; #, $p<$ 0.05 vs. the miR-185 mimic and shRNA-HOXC6 groups; NC, negative control; miR-185, microRNA-185; HOXC6, homobox C6.

3.6 CNE-2 cell line exhibits the lowest miR-185 expression among CNE-1, CNE-2, HNE-1, HNE-2, and 5-8F cell lines

RT-qPCR was used to select the cell line with the highest expression of miR-185 from NPC cell lines (CNE-1, CNE-2, HNE-1, HNE-2, 5-8F). The result of the RT-qPCR experiment showed (Fig. 6) that compared with other four cells, the miR-185 expression in CNE-2 cells was the lowest, so the CNE-2 cell line was used for subsequent experiments.

Figure 10.

Cell invasion is inhibited by overexpressed miR-185. A, the basolateral chamber of each group under the inverted optical microscope ( $\times$ 400); B, the result of the transwell assay revealed that miR-185 could inhabit invasion of NPC cells by targeting HOXC6; the measurement data were all expressed by the average value $\pm$ standard deviation, the result of which was verified by one-way ANOVA; the experiments were repeated 3 times; the scale is 25 um; *, $p<$ 0.05 vs. the blank group; #, $p<$ 0.05 vs. the miR-185 mimic and shRNA-HOXC6 groups; NC, negative control; miR-185, microRNA-185; HOXC6, homobox C6.

3.7 Up-regulated miR-185 inhibits HOXC6 and TGF- $\beta$ 1/mTOR axis

RT-qPCR and western blot analysis were performed to detect the expressions of miR-185, HOXC6, TGF- $\beta$ 1, mTOR, p-mTOR, Cyclin D1, PCNA, Bcl-2, Bax, caspase-3, LC3 II, Beclin1 with the aim to investigate the mechanisms and functions of miR-185 and HOXC6 in NPC. Result of the RT-qPCR shows that (Fig. 7) there was no significant difference in the expressions of miR-185, HOXC6, TGF- $\beta$ 1, mTOR, Cyclin D1, PCNA, Bcl-2, Bax, caspase-3, LC3 II, Beclin1 and the content of mTOR phosphorylation between the blank and NC groups (all $p>$ 0.05). Compared with the blank group, the expression of miR-185, and the mRNA and protein expressions of Bax, caspase-3, LC3 II, Beclin1 were remarkably increased in the miR-185 mimic group, while the mRNA and protein expressions of HOXC6, TGF- $\beta$ 1, mTOR, Cyclin D1, PCNA, Bcl-2 and the content of mTOR phosphorylation were significantly declined (all $p<$ 0.05). The condition in the miR-185 inhibitor group was contrary to that in the miR-185 mimic group. MiR-185 expression exhibited no significant difference between the blank and shRNA-HOXC6 groups. Compared with the blank group, the shRNA-HOXC6 group showed elevated mRNA and protein expressions of Bax, caspase-3, LC3 II, Beclin1, and diminished mRNA and protein expressions of HOXC6, TGF- $\beta$ 1, mTOR, Cyclin D1, PCNA, Bcl-2 and content of mTOR phosphorylation (all $p<$ 0.05). While the miR-185 mimic $+$ shRNA-HOXC6 group had ascended expressions of miR-185, Bax, caspase-3, LC3 II, Beclin1, but descended expressions of HOXC6, TGF- $\beta$ 1, mTOR, Cyclin D1, PCNA, Bcl-2, as well as the content of mTOR phosphorylation (all $p<$ 0.05). These finding shows that overexpressed miR-185 or silencing HOXC6 could suppress the expression of TGF-1/mTOR axis related genes and Cyclin, D1, PCNA, Bcl-2 and other genes while promoted the expression of Bax, Caspase-3, LC3, Beclin1 and other genes.

3.8 Up-regulated miR-185 expression inhibits cell viability

In the following experiments, we mainly investigated the ability of miR-185 to influence the biological functions of NPC cells by CCK8 method. The results of the CCK8 show that there had differences in cell proliferation in each group. Cell reproductive capacity in 24 h and viability between the NC and blank groups had no obvious difference at different time points ( $p>$ 0.05). However, compared with the blank group, cell viability was markedly decreased in the miR-185 mimic and shRNA-HOXC6 groups, but increased in the miR-185 inhibitor group (all $p<$ 0.05). Compared with the miR-185 mimic and shRNA-HOXC6 groups, cell viability was significantly descended in the miR-185 mimic $+$ shRNA-HOXC6 group (all $p<$ 0.05) (Fig. 8). Taken together, these results suggest that miR-185 inhibited NPC cell proliferation by down-regulating HOXC6.

Figure 11.

Cell autophagy is promoted by overexpression of miR-185 ( $\times$ 10000). miR-185, microRNA-185; HOXC6, homeobox C6; NC, negative control.

Figure 12.

MiR-185 promotes autophagy of NPC cells by inhibition of the TGF- $\beta$ 1/mTOR axis through suppression of HOXC6. NPC, nasopharyngeal carcinoma; miR-185, microRNA-185; HOXC6, homobox C6.

3.9 Up-regulation of miR-185 reduces cell cycle entry (cell cycle progression) but induces cell apoptosis

PI single staining test was performed to evaluate the cell cycle distribution. The PI single staining test result showed (Fig. 9A–B) that there was no distinct difference between the blank and NC groups. Compared with the blank group, the changes of cycle in the miR-185 mimic and shRNA-HOXC6 group were mainly manifested in shortening of G0/G1 phase (increased of cell proportion) and longing of S phase (decreased of cell proportion), the miR-185 inhibitor group was the opposite (all $p<$ 0.05). Compared with the miR-185 mimic and shRNA-HOXC6 groups, the miR-185 mimic $+$ shRNA-HOXC6 group had shorter G0/G1 phase and longer S phase (all $p<$ 0.05). In conclusion, results shows miR-185 could inhibit the proliferation of NPC cells by targeting HOXC6 gene.

Annexin V-FITC staining and flow cytometry were used to determine the apoptosis of CRC cells. Experimental results of apoptosis indicate (Fig. 9C–D) that there was no significant difference between the NC and blank groups after transfection ( $p>$ 0.05). Compared with the blank group, the rate of apoptosis was higher in the miR-185 mimic and shRNA-HOXC6 groups, but lower in the miR-185 inhibitor group ( $p<$ 0.05). The rate of apoptosis in the miR-185 mimic $+$ shRNA-HOXC6 group was markedly increased when compared with the miR-185 mimic and shRNA-HOXC6 groups ( $p<$ 0.05). These results indicate that overexpressed miR-185 could promote NPC cell apoptosis.

3.10 Up-regulated miR-185 decelerated the invasion of NPC cells

Besides, transwell assay was performed to investigate the effects of miR-185 on NPC cell invasion. The result of the transwell assay reveals (Fig. 10) that the blank and NC groups showed no notable difference on the cell invasion ( $p>$ 0.05). Compared with the blank group, the cell invasion was significantly declined in the miR-185 mimic and shRNA-HOXC6 groups, but elevated in the miR-185 inhibitor group ( $p<$ 0.05). Compared with the miR-185 mimic andshRNA-HOXC6 groups, the cell invasion was notably decreased in the miR-185 mimic $+$ shRNA-HOXC6 group ( $p<$ 0.05). These results suggest that miR-185 could inhabit invasion of NPC cells by targeting HOXC6.

3.11 Up-regulated miR-185 promoted autophagy in NPC cells

Lastly, we observed the cell autophagy under the microscope. Ultrastructural observation measured by electron microscope (Fig. 11) shows that compared with the blank and NC groups, the miR-185 mimic $+$ shRNA-HOXC6 group presented many large bright vacuoles and troponin induced autologous solutes. The nuclear membrane was not clear enough, the chromatin presented fine granular, and the nucleolus disappeared. The miR-185 mimic and shRNA-HOXC6 groups had increased vacuoles in the cytoplasm, swelled and denatured mitochondria, and formed vacuoles around the organelles and partly surrounded by bilayer membranes, as well as formed autophagic bodies. These finding shows that overexpressed miR-185 could promote autophagy in NPC cells.

4. Discussion

Previous studies have demonstrated that miRs could act functionally as “tumor suppressor genes” or “oncogenes” in tumorigenesis and modulate multiple cellular processes in the course of NPC progression [25, 26]. A recent study found that miR-185-3p expression was significantly declined following radiotherapy in radioresistant cases [13]. However, another study presents a contrary opinion that miR-185-3p contributed to the NPC radioresistance by targeting WNT2B [14]. Considering many potential targets governed by miRs, one challenge in understanding functional miR contributions in cancer cell behaviors depends on the identification of bona fide molecular targets. In this study, we aim to find out the mechanism by which biological function of miR-185 affects NPC cells. Consequently, this study demonstrates that miR-185 blocks TGF- $\beta$ 1/mTOR axis by inhibiting HOXC6, thus regulates the apoptosis, proliferation, autophagy of NPC cells.

Initially, our results found that miR-185 was reduced in patients with stage III-IV NPC or without LNM, which indicated miR-185 has an important role in the NPC initiation and progression. Previous findings revealed that up-regulated miR-185 promotes radiation-induced apoptosis and inhibition of proliferation in tumor by inhibiting ataxia telangiectasia and Rad3-related protein (ATR) pathway [27]. Interestingly, another study found that inhibition of ATM/ATR axis induces apoptosis and inhibits proliferation and radioresistance of NPC cells [28]. miR-185 has been found to inhibit breast cancer cell proliferation while inducing apoptosis by modulating the expression of c-Met [29], and another study exhibited high c-Met expression in 82.9% of NPC tissues as well as a significant correlation between high c-Met expression and LNM [30]. Additionally, in clear cell renal cell carcinoma, miR-185 was shown to inhibit cell proliferation and induce apoptosis by transcriptional repression of encoding vascular endothelial growth factor A (VEGFA). Moreover, inhibition of VEGFA promoted NPC cell apoptosis [31]. Hence, we suggest that overexpressed miR-185 may promote the apoptosis and inhibit proliferation in NPC cells. Furthermore, we also demonstrated that overexpressed miR-185 and silenced HOXC6 could longer the survival time of patients suffering with NPC. Previous study reported that the combination of low miR-185-3p and miR-324-3p was associated with poor overall survival, as well as the recurrence-free survival [13]. Patients with upregulated HOXC6 had a shorter overall survival rate in patients suffering with NPC, indicating that inhibited HOXC6 could longer the survival time of NPC patients [32, 33]. In the subsequent experiments, we demonstrated the function of miR-185 on NPC cells was achieved via TGF- $\beta$ 1/mTOR axis by targeting HOXC6.

Based on the target prediction program and the luciferase activity determination, we found that HOXC6 is a putative target gene of and negatively governed by miR-185. In our results, when HOXC6 was knocked down, NPC cell growth was inhibited and the apoptosis was promoted by up-regulated miR-185. HOXC6 is deregulated in human head and neck squamous cell carcinoma (HNSCC) and knockdown of HOXC6 using siRNA contributed to a repression of Bcl-2 expression and induced caspase-3-dependent apoptosis in HNSCC cells [15]. HOXC6 expression was found to regulate cell proliferation in NPC [32]. In addition, in our study, we demonstrated that the inhibition of HOXC6 inhibits NPC cell proliferation and promotes apoptosis inhibits NPC cell proliferation and promotes apoptosis was associated with blocked TGF- $\beta$ 1/mTOR axis [34, 35]. Consistent with our study, the capsaicin was reported to induce autophagy and apoptosis in human NPC cells by inhibiting the mTOR axis [36]. Another study also found that upregulated TRIM29 promotes NPC cell proliferation and metastasis via the mTOR axis [18]. TGF- $\beta$ 1 is one of members of TGF- $\beta$ superfamily has been demonstrated promote the invasion and metastasis of NPC with flotillin-1 [37]. Hence, we suggest that HOXC6 may regulate TGF- $\beta$ 1/mTOR axis, which is controlled by miR-185.

Given our discovery that miR-185 is downregulated in NPC patients, in relation to NPC progression, outlining a potential molecular target for NPC treatment. The polymorphic nucleotide variations of miR-185 have been discovered in breast cancer, chronic lymphocytic leukemia and colorectal cancer [38, 39]. It would be interesting to demonstrate whether miR-185 single nucleotide polymorphisms exists and functions in NPC. Interestingly, we demonstrate for the first time that HOXC6 is a target of miR-185 in NPC. However, due to the limited simple size and experimental conditions, further studies are needed to define the detailed mechanisms of miR-185 on the development of NPC through the HOXC6-mediated TGF- $\beta$ 1/mTOR signaling pathway so as to deeply expound our result from clinical pathology, cell experiment and animal experiment. The tumorigenic activity of miR-185 is mediated by inhibition of the oncogenic TGF- $\beta$ 1/mTOR axis through inhibition of HOXC6, as shown in Fig. 12. Thus, we speculate that miR-185 may be a promising therapeutic target in NPC.