MicroRNA-299-3p/FOXP4 Axis Regulates the Proliferation and Migration of Oral Squamous Cell Carcinoma.

MicroRNAs are noncoding RNAs of 21 to 23 nucleotides in length that play important roles in almost all biological pathways. The roles of microRNA-299-3p in the development and progression of oral squamous cell carcinoma remain unclear. Expression level of microRNA-299-3p in oral squamous cell carcinoma cell lines was analyzed. Then, the effects of microRNA-299-3p on oral squamous cell carcinoma cell proliferation and migration were investigated. Moreover, bioinformation algorithm and Western blot were conducted to explore whether forkhead box P4 was a direct target of miR-299-3p. We showed that microRNA-299-3p expression was significantly reduced in oral squamous cell carcinoma cell lines. Next, overexpression of microRNA-299-3p was found to inhibit oral squamous cell carcinoma cell proliferation and migration but promote apoptosis. In addition, forkhead box P4 was identified as a functional target of microRNA-299-3p. Our results provide a new perspective for the mechanisms underlying the progression of oral squamous cell carcinoma and a novel target for the treatment of oral squamous cell carcinoma.

Introduction

Oral squamous cell carcinoma (OSCC) ranks as the sixth leading solid tumor malignancy worldwide and is estimated to have 0.3 million new cases each year.[1] Although the treatment methods for OSCC has been improved, the 5-year overall survival rate of patients with OSCC did not significantly increased.[2] Therefore, it is still imperative to widen our understanding concerning the mechanism underlying OSCC progression with the purpose to identify novel treatment biomarkers.

MicroRNAs (miRNAs) are small RNAs to effectively modulate gene expression mainly by complementary 3′-untranslated region (3′-UTR) binding.[3] A large sum of evidence suggested that miRNAs are abnormally expressed in human cancers including OSCC, and they are reported to play crucial roles in tumor initiation, progression, and metastasis.[4,5] The role of miRNAs in human cancer progression is complex as 1 miRNA can regulate the expression of multiple target genes to function as either tumor suppressor or oncogene.[4,5] MicroRNA-299-3p was documented to have dual roles in regulating human cancer proliferation, migration, and invasion.[6-8] MicroRNA-299-3p was reported as oncogenic miRNA in ovarian cancer and acute promyelocytic leukemia.[6] MicroRNA-299-3p expression was significantly upregulated in ovarian cancer and its overexpression promotes cell proliferation, migration, invasion, and at the same time inhibits apoptosis through targeting OCT4.[6] In contrast, miR-299-3p was shown to function as tumor suppressor in hepatocellular carcinoma and colon cancer.[7,8] For instance, low miR-299-3p expression was significantly associated with tumor size, venous infiltration, Edmondson-Steiner grading, TNM stage, and worse 3-year overall survival in hepatocellular carcinoma.[7] In addition, it was found that miR-299-3p inhibits hepatocellular carcinoma cell migration, invasion, and proliferation via directly targeting SIRT5.[7] Moreover, miR-299-3p was also found to target vascular endothelial growth factor A (VEGFA) to inhibit colon cancer cell proliferation and invasion.[8] However, up to date, we still not able to understand whether miR-299-3p has a role in the progression of OSCC.

In this study, we showed miR-299-3p expression was decreased in OSCC cell lines compared with the normal cell line. Overexpression of miR-299-3p inhibits OSCC cell proliferation and migration. We also validated forkhead box P4 (FOXP4), an oncogene, as a direct functional target of miR-299-3p in OSCC. Moreover, we showed miR-299-3p regulates OSCC cell behaviors through targeting the expression of FOXP4.

Materials and Methods

Cell Culture and Transfection

Normal human oral cell line Hs 680.Tg and 2 OSCC cell lines (SCC-4, SCC-9) were purchased from American type culture collection (Manassas, Virginia). These cell lines were incubated in Dulbecco modified Eagle medium (Invitrogen, Thermo Fisher Scientific, Inc, Waltham, Massachusetts) supplemented with 10% fetal bovine serum (Invitrogen) at a 37°C humidified incubator containing 5% of CO2.

The miR-299-3p mimic and the corresponding negative control miRNA were purchased from GenePharma (Shanghai, China). The pcDNA3.1 vector with or without the open reading frame of FOXP4 was purchased from GenScript (Nanjing, China). Lipofectamine 2000 (Invitrogen) was used to transfect the synthetic miRNAs or expression vectors into the cell lines according to the manufacturer’s instructions.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction

Trizol regent (Invitrogen) was employed to extract the total RNA from cell lines according to the provided protocols. M-MLV Reverse Transcriptase (Invitrogen) was used to reverse the transcribed RNA into complementary DNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on Applied Biosystems 7500 (Applied Biosystems, Foster City, California) using SYBR Premix Ex Taq (TaKaRa, Dalian, China) with the following primers: miR-299-3p: 5′-ACACTCCAGCTGGGTATGTGGGATGGTAAAC-3′ (forward) and 5′-GTGCAGGGTCCGAGGT-3′(reverse); U6 small nuclear RNA (snRNA): 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse). Relative miR-299-3p expression levels were calculated using 2−ΔΔCt method with U6 snRNA as internal control.

Protein Extraction and Western Blot

Radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitor (Beyotime, Haimen, Jiangsu, China) was used as extract total proteins. After quantified using BCA kit (Beyotime), protein samples with equal amount were separated on 10% Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to polyvinylidene fluoride (PVDF) membrane (Beyotime). The membranes were incubated with primary antibodies (rabbit anti-FOXP4: ab17726, rabbit anti-ki67: ab16667, rabbit anti-E-cadherin: ab40772, rabbit anti-Vimentin: ab193555, and rabbit anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH): ab181602; Abcam, Cambridge, Massachusetts) for overnight at 4°C after blocked with fat-free milk. Then, the membranes were incubated with horseradish peroxidase conjugated secondary antibody (ab6721, Abcam) at 37°C for 2 hours. The blot was visualized using BeyoECL kit (Beyotime) and analyzed with Image J version 1.42 software (glyceraldehyde-3-phosphate dehydrogenase (NIH), Bethesda, Maryland).

Cell Proliferation Assay

Cells (5 × 103 cells/well) were incubated in 96-well plates and cultured for 0, 24, 48, and 72 hours, respectively. Cell proliferation rate was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, 20 μL MTT solutions with the concentration of 5 mg/mL were added to each well and further incubated for 4 hours. Subsequently, optical density was measured at 570 nm after adding 150 μL of dimethyl sulfoxide to each well.

Cell Migration Assay

Cell migration was assessed by wound healing assay. Cells (5 × 105 cells/well) were seeded to a 6-well plate. A wound was created at cell surface using sterile pipette tip. After incubation for 24 hours, wound images were captured under an inverted microscope and wound closure was analyzed.

Cell Apoptosis Assay

Cells were trypsinized, washed with phosphate buffered saline, and resuspended in Annexin V Binding Buffer, followed by staining with Fluorescein isothiocyanate (FITC) Annexin V and propidium iodide (Beyotime) at dark for 30 minutes. Cells were analyzed using FACSCalibur flow cytometer (BD Biosciences, San Jose, California) to measure cell apoptosis percentage.

Bioinformatic Analysis

TargetScan (http://www.targetscan.org/vert_72/) and miRDB (http://www.mirdb.org/cgi-bin/search.cgi) were used to predict the potential targets of miR-299-3p. These analyses showed FOXP4 might be a putative target for miR-299-3p.

Luciferase Activity Reporter Assay

The wild type (wt) or mutate type (mt) of FOXP4 3′-UTR was built based on pmiRGLO vector (Promega, Madison, Wisconsin) and named as wt-FOXP4 or mt-FOXP4. Cells were cotransfected with synthetic miRNAs and wt- or mt-FOXP4 using Lipofectamine 2000. After transfection for 48 hours, luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega).

Statistical Analysis

Data analysis was performed using SPSS version 18.0 software (SPSS Inc, Chicago, Illinois). Differences were analyzed using Student t test (only 2 groups) or 1-way analysis of variance post-Tukey test (more than 2 groups); P < .05 was regarded as significant difference.

Results

MicroRNA-299-3p and FOXP4 Expression in OSCC Cell Lines

We measured miR-299-3p expression in OSCC cell lines by qRT-PCR. Compared with the normal cell line Hs 680.Tg, miR-299-3p expression was significantly reduced in OSCC cell lines SCC-4 and SCC-9 (Figure 1A). Next, we analyzed FOXP4 expression in OSCC cell lines by Western blot. As shown in Figure 1B, FOXP4 expression was significantly increased in SCC-4 and SCC-9 cell lines compared with Hs 680.Tg cell line (Figure 1B). These results indicated that downregulation of miR-299-3p may have some connection with upregulation of FOXP4 in OSCC.

Figure 1.

Downregulation of miR-299-3p and upregulation of FOXP4 in OSCC cell lines. (A) MicroRNA-299-3p expression level was examined by qRT-PCR and (B) FOXP4 protein level was examined by Western blot in normal human oral cell line Hs 680.Tg and 2 OSCC cell lines (SCC-4, SCC-9; ***P < .001). FOXP4 indicates forkhead box P4; miR-299-3p, microRNA-299-3p; OSCC, oral squamous cell carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction.

FOXP4 Was a Direct Target of miR-299-3p

By using TargetScan and miRDB analyses, we found FOXP4 contains a binding site for miR-299-3p in its 3′-UTR (Figure 2A). To further confirm this prediction, luciferase activity reporter assay was conducted. Results showed that miR-299-3p overexpression could inhibit the luciferase activity of cells transfected with wt-FOXP4 but not mt-FOXP4 (Figure 2B and C). These results indicated that FOXP4 was a direct target of miR-299-3p.

Figure 2.

Forkhead box P4 (FOXP4) was a direct target of miR-299-3p. (A) Putative binding site between miR-299-3p and the 3′-UTR of FOXP4. Relative luciferase activity in (B) SCC-4 and (C) SCC-9 cells transfected with miR-299-3p mimic or NC-miR and wt-FOXP4 and mt-FOXP4 (ns, not significant, ***P < .001). miR-299-3p indicates microRNA-299-3p; mt, mutant type; NC-miR, negative control miRNA; 3′-UTR, 3′-untranslated region; wt, wild type.

MicroRNA-299-3p Regulates OSCC Cell Behaviors Through Targeting FOXP4

Then, we investigated the biological functions of miR-299-3p and FOXP4 in OSCC. When synthetic miRNAs were transfected into OSCC cell lines, it was found miR-299-3p mimic transfection significantly increased the expression levels of miR-299-3p (Figure 3A). Western blot showed that FOXP4 expression could be increased by pcDNA-FOXP4 but decreased by miR-299-3p mimic (Figure 3B). Meanwhile, the inhibitory effect of miR-299-3p mimic on FOXP4 expression could be partially reversed by pcDNA-FOXP4 (Figure 3B). The MTT assay revealed that cell proliferation can be enhanced by pcDNA-FOXP4 but suppressed by miR-299-3p mimic (Figure 3C). The examination of expression level of ki67 confirmed the results of MTT assay (Figure 3D). Wound-healing assay showed stimulation effect of pcDNA-FOXP4 and inhibition effect of miR-299-3p mimic on cell migration (Figure 3E). Results of Western blot on the expression level of E-cadherin and Vimentin validated the results of wound-healing assay (Figure 3F). Furthermore, we showed cell apoptosis can be enhanced by miR-299-3p mimic but inhibited by pcDNA-FOXP4 (Figure 3F). In the meantime, we showed the suppression effects of miR-299-3p mimic on cell proliferation, migration, and apoptosis can be partially reversed by pcDNA-FOXP4 (Figure 3C-G). These results suggested that miR-299-3p functions as a tumor suppressor in OSCC through targeting the expression of FOXP4.

Figure 3.

MicroRNA-299-3p regulates OSCC cell proliferation and migration through targeting FOXP4. (A) MicroRNA-299-3p expression level was examined by qRT-PCR in OSCC cells transfected with miR-299-3p mimic or NC-miR. (B) Forkhead box P4 expression, (C) cell proliferation, (D) ki67 expression, (E) cell migration, (F) E-cadherin and Vimentin expression, and (G) cell apoptosis in cells transfected with miR-299-3p mimic, NC-miR, pcDNA-FOXP4, pcDNA3.1, or pcDNA-FOXP4 and miR-299-3p mimic. (*P < .05, **P < .01, ***P < .001). FOXP4 indicates forkhead box P4; miR-299-3p, microRNA-299-3p; NC-miR, negative control miRNA; OSCC, oral squamous cell carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction.

Discussion

MicroRNAs were found to be abnormally expressed in OSCC, and their dysregulation has been implicated to function as crucial roles by regulating tumor-related biological behaviors.[9-11] For instance, miR-1297 expression was found to be repressed while gene of phosphate and tension homology deleted on chromsome ten (PTEN) expression was found to be activated in the progression of OSCC.[9] The overexpression of miR-1297 or silencing of PTEN inhibited OSCC cell growth, indicating miR-1297 may drive OSCC progression through targeting PTEN.[9] MicroRNA-134 could target programmed cell death 7 to inhibit E-cadherin expression and therefore to enhance OSCC progression.[10] In addition, miR-218-5p was found downregulated in OSCC and negatively regulated cell invasion through targeting the CD44-ROCK signaling pathway.[11]

MicroRNA-299-3p has been studied in several human cancers to function as dual roles.[6-8] In this study, we analyzed miR-299-3p expression levels in OSCC cell lines. We showed miR-299-3p levels were reduced in OSCC cell lines compared with normal cell line. Functional assays showed that miR-299-3p overexpression could inhibit OSCC cell proliferation and migration but promote apoptosis in vitro. The results of MTT assay and wound-healing assay were further confirmed by investigating the expression level of ki67, E-cadherin, and Vimentin in these transfected cells. These results indicated that miR-299-3p functions as a tumor suppressor in the progression of OSCC, which is similar to its role in hepatocellular carcinoma and colon cancer.[7,8]

OCT4, Sirtuin 5, and VEGFA have been revealed to be direct targets of miR-299-3p.[6-8] Validation of the direct targets of miR-299-3p is essential to explore its biological functions and may be useful in identifying promising therapeutic targets. In this work, by using TargetScan and miRDB, we found FOXP4 was a putative target of miR-299-3p as it contains a conserved binding site in its 3′-UTR. Luciferase activity reporter assay revealed miR-299-3p could directly bind to the 3′-UTR of FOXP4. Western blot revealed that FOXP4 expression could be downregulated by miR-299-3p mimic transfection. Rescue experiments showed that FOXP4 overexpression could partially reversed the inhibitory effects of miR-299-3p mimic on OSCC cell proliferation and migration. Here, we for the first time revealed the connection of miR-299-3p and FOXP4 in OSCC, indicating miR-299-3p has a role in the progression of OSCC. Further analysis on human tissues should be conducted to validate the findings here and to investigate the prognostic value of miR-299-3p in OSCC.

Forkhead box P4 belongs to the FOXP family and encodes a 685-amino acid protein.[12] Forkhead box P4 is essential for normal T-cell cytokine recall responses to antigen following pathogenic infection but did not affect T cell development.[13] Upregulation of FOXP4 has been identified in non-small cell lung cancer, and knockdown of FOXP4 by specific short hairpin RNA (shRNA) significantly decreased cancer cell growth, invasion, and at the same time arrested cell cycle.[14] Importantly, it was shown that FOXP4 expression could be regulated by miRNAs including miR-338-3p and miR-491-5p.[15,16]

In this study, we found remarkedly decreased miR-299-3p expression and increased FOXP4 expression in OSCC cell lines. Overexpression of miR-299-3p inhibits proliferation and migration of OSCC cells through targeting FOXP4. These findings supported the theory that downregulation of miR-299-3p promotes OSCC progression through a FOXP4-induced signaling pathway.