MicroRNA-124-3p inhibited progression of nasopharyngeal carcinoma by interaction with PCDH8 and the inactivation of PI3K/AKT/mTOR pathway

Nasopharyngeal carcinoma (NPC) is characterised by distinct geographical distribution and is particularly prevalent in Asian countries. But the mechanisms related to the progression of nasopharyngeal carcinoma (NPC) are not completely understood. MiR-124-3p functions as a tumor suppressor in many kinds of human cancers. Here, we explored the effects and mechanism of miR-124-3p on the proliferation and colony formation in NPC. In our study, we reported that miR-124-3p was significantly downregulated in NPC tissues and cell lines. Overexpression miR-124-3p decreased NPC cell proliferation and colony formation abilities. Meanwhile, knockdown miR-124-3p increased proliferation and colony formation abilities. Additionally, dual-luciferase assay showed that miR-124-3p could positively regulated PCDH8 by targeting its 3'-UTR. Overexpression of PCDH8 could partially rescue the proliferation and colony formation role of miR-124-3p inhibitor. Our study indicated that miR-124-3p played a tumor suppressor by directly interacting with PCDH8 and inhibiting the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Overall, we found that miR-124-3p inhibited the activation of the PI3K/AKT/mTOR signaling pathway in NPC by interacting with PCDH8. Thus, PCDH8 may be a potential molecular target that impeded NPC proliferation and colony formation.


Introduction
Nasopharyngeal carcinoma (NPC) is a malignant type of head and neck squamous cell carcinoma occurring in epithelial cells of the nasopharynx [1,2]. NPC is associated with distinct geographical distribution and racial differences, and it is highly prevalent in Asian countries [3,4]. NPC is prone to invasion and distant metastasis at an early stage, and more than 70% of patients are diagnosed with advanced NPC during their initial assessment [5]. NPC is sensitive to radiotherapy; thus, this treatment strategy has greatly improved local control of lesions and the 5-year survival rate in patients with NPC [5][6][7][8]. However, frequent local recurrence, metastasis, and poor prognosis remain major challenges in the clinical treatment of NPC because the mechanisms related to NPC progression are not completely understood [9][10][11].
MicroRNAs (miRNAs) play critical roles as oncogenes or tumor suppressors in NPC. MiRNAs are a category of small non-coding RNAs (~19-25
Protocadherin-8 (PCDH8) is predicted to be a target of miR-214-3p using targetscan (http://www.targetscan.org). PCDH8 is an integral membrane protein that acts as a tumor suppressor in several types of cancer [29][30][31] and is involved in the metastasis of gastric cancer [32]. Additionally, PCDH8 has been found to be regulated by miR-217, which promotes apoptosis in small cell lung cancer [33]. PCDH8 also plays tumor-suppressive roles in NPC [34].
In this study, function assays found that overexpression of miR-124-3p decreased NPC cell proliferation and colony formation abilities, meanwhile knockdown of miR-124-3p increased proliferation and colony formation abilities. Mechanism assays indicated that miR-124-3p played a tumor suppressor by directly interacting with PCDH8 and inhibiting the activation of PI3K/AKT/mTOR signaling pathway.

Cell transfection
Hsa-miR124-3p mimics and inhibitor lentiviral packaging constructs were obtained from GenePharma Biotechnology Co., Ltd. (Shanghai, China). For cell transfection, cells were seeded into six-well plates, and lentivirus (50 µL "lv-mir124-3p mimic" or 50 µL "lv-mir124-3p inhibitor") and 3 µL polybrene were added to each well. Puromycin (2.0 mg/mL) was then added to each well, and plates were incubated for 14 days to select cells with stable expression of constructs. Cells infected with lentiviral nonsense scramble control miRNA were used as the control.

Cell Counting Kit-8 (CCK-8) assay
The proliferative activity of cells was detected by CCK-8 assays (Dojindo Laboratories, Kumamoto, Japan). NPC cells were collected during the logarithmic phase, and then cell suspension was incubated in a 96-well plate (3 ×10 3 cells/well) at 37°C in 5% CO2 overnight, until all cells adhered to the wall.After cell attachment, transfection was performed using Lipofectamine 3000 and P3000 (Invitrogen) according to the manufacturers instructions. After transfection for 0, 24, 48, 72, 96, or 120 h, 10 μL CCK-8 solution was added to each well, and the cells were incubated for 2 h at 37°C. The absorbance of each well was detected at 450 nm using a multifunctional microplate reader.

Colony formation assay
The colony-forming activity of cells was examined by colony formation assay. NPC cells of logarithmic growth phase were collected and plated into 12-well plates (100 cells/well. All cells were cultured and maintained at 37°C in an incubator with 5% CO2. Twelve days after seeding, the cells were washed with PBS thrice and fixed with methanol for 10 min. After fixation, cells were washed three times with PBS. Then the colonies containing more than 50 cells was counted after staining with crystal violet.

Western blotting
Lysis buffer containing a protease inhibitor was used for processing NPC cell protein extracts. The protein concentration of each sample was measured with a bicinchoninic acid protein assay kit (KenGEN BioTECH, Nanjing, China). First, 30 μg of each protein sample was separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 2 h at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies were used for western blotting (Supplementary table S1). The membranes were washed three times with Tris-buffered saline and Tween-20 and then incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse; Cell Signaling Technology). β-Tubulin was used as an endogenous control to compare the protein expression. A chemiluminescence system (Tanon 5200; Tanon Science & Technology Co., Ltd., Shanghai, China) was used to visualize the proteins on the membranes.

Tumor growth in xenograft model
All animal experiments were conducted under protocols approved by the Animal Experimentation Ethics Committee of Guangzhou Medical University (No.2005DKA21300). 6~8-week old immunodeficient BABL/c nude mice were used assay as a xenograft model for tumor growth. Cells were counted (5×10 6 ) and injected into the right dorsal flank of BABL/c nude mice. The weight of mice and volume of tumor were measured every 4 days, tumor volume was calculated using the formula: Tumor maximum diameter (L) × the right-angle diameter to that axis (W) 2 /2. After 4 weeks of injection, mice were sacrificed according to institutional ethical guidelines, the xenograft tumors were dissected and weighted comparing to control groups in vivo.

In situ hybridization (ISH)
In situ hybridization was performed using single-strand RNA probes comprising antisense or sense sequences of decorin or biglycan. Digoxigenin (DIG)-labeled RNA probes were synthesized by in vitro transcription with microRNA ISH Buffer using a DIG RNA Labeling Kit (Roche). Chip HLugA180Su01 were fixed and hybridized with RNA probes. Hybridization signals were detected immunohistochemically by anti-DIG-AP Fab fragments. The reagents used are shown in Supplementary table S2.

Immunohistochemistry (IHC)
IHC for target molecules (PCDH8) was performed on sections from tumor tissues of nude mice xenografts. Tissue sections were deparaffinized, subjected to antigen retrieval using 0.01M sodium citrate solution, and incubated with immunohistochemical ultrasensitive kit (MXB Biotechnologies, Fuzhou, China) and primary antibodies against PCDH8. Then sections were incubated with 3,3-Diaminobenzidine (DAB). Quantification of PCDH8 was measured by intensity and extent of staining.

Statistical analysis
Data were analyzed using Statistical Product and Service Solutions version 20.0 (IBM Corp., Armonk, NY, USA). The differences between two groups were compared with Student's t-tests or Pearson's χ 2 -tests. One-way analysis of variance was used for comparisons of multiple groups. P value of <0.05 was considered as a statistically significant difference.

miR-124-3p was expressed at low levels in NPC tissues and cell lines
The miRbase website (http://mirbase.org/) showed that the expression of miR-124-3p in head and neck squamous cell carcinoma tissues was significantly downregulated (Fig. 1A), which indicated miR-124-3p may play a tumor suppressor role in tumorigenesis. To further evaluate the expression of miR-124-3p, qPCR showed that the expression of miR-124-3p was significantly downregulated in NPC cell lines, especially 6-10B and CNE2 cell lines (Fig. 1B). To further explore the importance of miR-124-3p in clinically, NPC tissue microarray (Chip HLugA180Su01) was used in situ hybridization. In order to explore the relationship between miR-124-3p and clinical parameters of NPC patients, NPC patients were divided into two groups: high expression group and low expression group according to the median expression of miR-124-3p. The correlation between the expression of miR-124-3p and clinicopathological data was analyzed. We analyzed the effect of high and low expression of miR-124-3p on the overall survival (OS) and recurrence-free survival (RFS) in patients with NPC. The results showed that compared with patients with lower expression of miR-124-3p, NPC patients with higher expression of miR-124-3p had significantly longer 5-year OS and 5-year RFS ( Fig. 1 C and D). Cox and multivariate cox regression analysis showed that miR-124-3p, clinical stage was correlated with prognosis ( Table 1, Table 2 and 2A).

Upregulation of miR-124-3p decreased proliferation and colony formation in NPC cells
To explore the potential role of miR-124-3p in NPC, cells were transfected with miR-124-3p mimic, accordingly, miR-124-3p could be upregulated in 6-10B and CNE2 cells ( Fig. 2A). To know the function of miR-124-3p, function assays were further explored. As shown in Fig. 2B and 2C, after overexpression of miR-124-3p, cell proliferation were significantly decreased in the miR-124-3p mimic group compared with those in the mimic negative control (NC) group (P < 0.05). Similarly, after overexpression of miR-124-3p, colony formation was significantly decreased in the miR-124-3p mimic group compared with those in the mimic negative control (NC) group (P < 0.05; Fig. 2D and 2E). To further evaluation the role of miR-124-3p expression on proliferation, western blotting was used to detect the proliferation related protein. As shown in Fig. 2F, overexpression of miR-124-3p in CNE2 and 6-10B cells caused a decrease in cyclin D3 and cyclin-dependent kinase 4 protein levels (CDK4). Thus, these results indicated that miR-124-3p affected cell progression by regulating the expression of cyclin D3 and CDK4.

Knockdown miR-124-3p increased proliferation and colony formation in NPC cells
In similar opposite way, to explore the potential role of miR-124-3p in NPC, cells were transfected with miR-124-3p inhibitor, miR-124-3p could also be knockdown in S18 and HK1 cells (Fig. 3A). The function of miR-124-3p assays were also further explored. As shown in Fig. 3B and 3C, after down expression of miR-124-3p, cell proliferation were significantly increased in the miR-124-3p mimic group compared with those in the mimic negative control (NC) group (P < 0.05). Similarly, after down expression of miR-124-3p, colony formation were significantly increased in the miR-124-3p inhibitor group compared with those in the mimic negative control (NC) group (P < 0.05; Fig. 3D and 3E). To further evaluation the role of miR-124-3p expression on proliferation, western blotting was used to detect the proliferation related protein. As shown in Fig. 3F, down expression of miR-124-3p in S18 and HK1 cells caused a decrease in P27 and an increase in cyclin-dependent kinase 6 protein levels (CDK6). Thus, these results indicated that miR-124-3p affected cell progression by regulating the expression of P27 and CDK6.

miR-124-3p positively regulated PCDH8 by targeting its 3'-UTR
To explore the potential target of miR-124-3p, the target genes of miR-124-3p were investigated using TargetScan (http://www.targetscan.org/). The results showed that the 3′-UTR sequence in PCDH8 may be a binding target of miR-124-3p (Fig. 4A). Dual-luciferase assay showed that, compared with that in the NC group, after transfected with the PCDH8-WT plasmid in 293T cells, the firefly luciferase activity of miR-124-3p significantly increased. However, cells transfected with the PCDH8-MUT plasmid, the firefly luciferase activity was significantly changed (Fig. 4B). And we found the same phenomenon in S18 cells (Fig. 4C). Moreover, qPCR and western blotting showed that overexpression of miR-124-3p significantly increased the mRNA and protein level of PCDH8 (P < 0.01; Fig.  4D and 4E), whereas miR-124-3p inhibition could decrease the mRNA and protein of PCDH8 (P < 0.01; Fig. 4F and 4G). The expression of PCDH8 in CNE2 and 6-10B cells were significantly upregulated in cells transfected with miR-124-3p mimic compared with those in the mimic NC group. These results indicated that PCDH8 miR-124-3p, miR-124-3p may suppress the NPC proliferation by regulating PCDH8 transcriptional activity.

PCDH8 rescued tumor suppressor role of miR-124-3p
PCDH8 plasmids were transfected into NPC cell lines to explore the relationship between PCDH8 expression and miR-124-3p. Treatment with miR-124-3p inhibitor markedly promoted cell proliferation in S18 (Fig. 5A) and HK1 cells (Fig. 5B) endogenously expressing PCDH8. To determine whether ectopic overexpression of PCDH8 could rescue the suppressive effects of miR-124-3p on cell proliferation, lv-miR-124-3p inhibitor was transfected into S18 and HK1 cells. We observed that the cell proliferation and colony formation were inhibited in the infected miR-124-3p inhibitor and PCDH8 plasmid group compared with those in the inhibitor vector control and inhibitor NC groups. Further, transfection with PCDH8 and mir-124-3p inhibitors impaired the ability of PC cell clone formation. (Fig. 5C and D). CCK8 and colony-forming assays showed that PCDH8 could partially reduce the miR-124-3p inhibitor-mediated activity in S18 and HK1 cells. To further explore the importance of PCDH8 in clinical survival, NPC tissue microarray (Chip HLugA180Su01) was used in immunohistochemistry. In order to explore the relationship between PCDH8 and clinical parameters of NPC patients, NPC patients were divided into two groups: high expression group and low expression group according to the median expression of PCDH8. The correlation between the expression of PCDH8 and clinicopathological data was analyzed. We analyzed the effect of high and low expression of PCDH8 on the OS and RFS in patients with NPC. The results showed that compared with patients with lower expression of PCDH8, NPC patients with higher expression of PCDH8 had significantly longer 5-year OS and 5-year RFS (Fig. 5 E and F). PCDH8 and clinical parameters were analyzed by cox univariate and multivariate regression analysis to explore the prognostic factors. Clinically regression analysis demonstrated that PCDH8, clinical stage was also correlated with good OS and RFS (Table 3, Table  4 and 4A). Conjointly, these results suggested that PCDH8 could be used as potential biomarker to predict the prognosis of patients with NPC.

miR-124-3p suppress tumor formation of NPC cells in mouse xenograft models
To further investigate the effect of miR-124-3p on the proliferation of NPC cells in vivo. CNE2 cell with high expression of miR-124-3p were injected into the subcutaneous of nude mice. Compared with the control group, miR-124-3p overexpression resulted in a smaller size of the subcutaneous tumor in mice (Fig.  6A). And the mean tumor volume for miR-124-3p overexpression group was significantly smaller than NC group (Fig. 6B). Additionally, the weight of tumor tissue was lower in the miR-124-3p overexpression group than that of the NC group (Fig. 6C). Moreover, qRT-PCR analysis suggested that miR-124-3p expression was obviously increased in tumor tissue of miR-124-3p overexpressed nude mice. (Fig. 6D), and Western blotting assay showed that the protein of PCDH8 was increased when miR-124-3p overexpressed (Fig. 6E). Collectively, these results above suggested that miR-124-3p suppress proliferation of NPC cell in vivo.

Discussion
In this study, we found that miR-124-3p was significantly downregulated in NPC tissues compared with that in healthy tissues. Moreover, miR-124-3p expression inhibited the proliferation and growth of NPC cells in vitro and affected the G 1 /S transition. Overall, our findings demonstrated that miR-124-3p could inhibit the proliferation and growth of NPC cells by targeting and positively regulating PCDH8.  The abnormal expression of miR-124 is related to the regulation of important processes in various types of cancer, including cell proliferation, growth, apoptosis, and metastasis. For example, miR-124-3p directly targets the 3′-UTR of the mRNA encoding the branched chain amino acid transaminase-1, which suppresses the growth and invasion of esophageal squamous cell carcinoma [20]. Similarly, miR-124-3p blocks cell migration and invasion by affecting the combined integrin subunit alpha-3 signaling in bladder cancer [35]. Furthermore, Luo et al. reported that miR-124-3p suppressed the aggressiveness of glioma by targeting Fos-related antigen-2, suggesting potential applications of this miRNA in the treatment of this disease [21]. Liu et al. found that abnormal overexpression of miR-124-3p in gastric cancer cells could inhibit cell viability, colony formation, and tumor growth in vivo; therefore, miR-124-3p may act as a potential marker and inhibit tumor growth in gastric cancer [36]. Additionally, Idichi et al. showed that miR-124-3p expression was low in pancreatic ductal adenocarcinoma tissues and that abnormal expression of miR-124-3p suppressed cancer cell migration and invasion in these tissues [37]. In our study, we found that miR-124-3p expression was low in most NPC cell lines, but overexpression of miR-124-3p significantly inhibited proliferation and colony formation in these cell lines. Therefore, miR-124-3p may act as a tumor suppressor in NPC.  In a clinical trial, PCDH8 was inhibited in tumor tissues when compared with that in non-tumor tissues, particularly in patients with advanced hypopharyngeal carcinoma, suggesting that PCDH8 could be a prognostic biomarker and target for the treatment of this type of cancer [29]. Wang et al. found that PCDH8 was regulated by miR-217, which is involved in promoting apoptosis in small cell lung cancer [33]. Moreover, Cao et al. also showed that PCDH8 was involved in the metastasis of gastric cancer and that PCDH8 expression was lower in ovarian cancer tissues than in corresponding adjacent tissues, and contributed to the advanced progression of ovarian cancer [38]. Our findings showed that miR-124-3p positively regulated PCDH8 expression through interactions with its 3′-UTR. Moreover, when NPC cells were transfected with a PCDH8 plasmid and then treated with miR-124-3p inhibitor, cell proliferation and colony formation decreased compared with that in the control group. These results indicated that overexpression of PCDH8 could partially rescue the suppressive activity of miR-124-3p and that PCDH8 may also act as a tumor suppressor in NPC.
In conclusion, miR-124-3p expression was low in NPC tissues and was associated with the tumor suppressor of NPC. Over expression of miR-124-3p could decrease the abilities of proliferation and colony formation in NPC cells. Meanwhile, knockdown miR-124-3p increased proliferation and colony formation abilities. PCDH8 could partially rescue the proliferation and colony formation role of miR-124-3p inhibitor. Our results confirmed that miR-124-3p overexpression significantly decreased the proliferation and colony formation of NPC cells by interacting with PCDH8 and inhibiting the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway. Thus, miR-124-3p and PCDH8 maybe potential molecular targets that impeded NPC proliferation and colony formation.

Availability of data and materials
The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate
This study was approved by the Affiliated Cancer Hospital & Institute of Guangzhou Medical University.

Author Contributions
JY participated in the design of the study and drafted the manuscript. QL and XZ revised the manuscript and performed the western blotting and dual-luciferase reporter experiments. CL and YD performed the cell culture, cell transfection, CCK-8, and cell colony formation experiments. XL revised the manuscript and conducted the statistical analysis. LZ assisted with the design of the study and revised the manuscript. YY and TG conceived the study, participated in its design and coordination, and helped revise the manuscript. All authors read and approved the final version of the manuscript.