MicroRNA-363-3p inhibits tumor cell proliferation and invasion in oral squamous cell carcinoma cell lines by targeting SSFA2

Abstract

The aim of the present study was to evaluate the expression levels of microRNA (miR)-363-3p and its underlying physiological function in oral squamous cell carcinoma (OSCC). miR-363-3p expression levels were measured in OSCC cell lines using reverse transcription-quantitative PCR. The role of miR-363-3p in OSCC cells was examined using gain-of-function assays in vitro. Cell proliferation was assessed using Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine assays and flow cytometry. Cell migration and invasion were evaluated in wound-healing and Transwell Matrigel assays. In addition, bioinformatics analysis predicted binding sites of miR-363-3p on sperm-specific antigen 2 (SSFA2). Luciferase reporter and RNA pull-down assays were conducted to test whether miR-363-3p interacted with SSFA2. miR-363-3p expression was downregulated in OSCC cell lines compared with that in the normal epithelial cell line (NHOK). Additionally, miR-363-3p overexpression suppressed OSCC cell proliferation, migration and invasion in vitro. SSFA2 was verified as a direct target of miR-363-3p, and SSFA2 overexpression partially counteracted the inhibitory effects of miR-363-3p on cell proliferation, migration and invasion in OSCC cell lines. Thus, miR-363-3p may serve as a tumor suppressor via targeting SSFA2 and may represent a potential therapeutic target for OSCC.

Keywords: microRNA-363-3p; oral squamous cell carcinoma; sperm-specific antigen 2.

Figure 1

miR-363-3p inhibits the proliferation of OSCC cell lines. (A) Expression levels of miR-363-3p in OSCC cell lines and the HOK normal epithelial cell line. ^**P<0.05 vs. NHOK cells. (B) Reverse transcription-quantitative PCR was performed to assess transfection efficiency. (C) Cell Counting Kit-8 and (D) EdU assays were conducted to examine cell proliferation. Magnification x200. (E) Effects of miR-363-3p on cell cycle progression were examined by flow cytometry. (F) Protein expression levels of cyclin D1 and PCNA were measured in OSCC cell lines by western blotting. Data are presented as the mean ± SD; ^**P<0.01 vs. NC mimic. EdU, 5-ethynyl-2'-deoxyuridine; miR, microRNA; NC, negative control; OD, optical density; OSCC, oral squamous cell carcinoma; PCNA, proliferating cell nuclear antigen.

Figure 2

miR-363-3p inhibits OSCC cell migration and invasion. (A) Wound healing assays were carried out to measure cell migration rates. (B) Cell migration and invasion was evaluated using Transwell and Matrigel assays, respectively. (C) Western blotting assays were carried out to assess the protein expression levels of Cox-2, MMP2 and MMP9. Magnification, x200. Data are presented as the mean ± SD; ^**P<0.01 vs. NC mimic. Cox-2, cyclo-oxygenase-2; miR, microRNA; MMP, matrix metallopeptidase; NC, negative control; OSCC, oral squamous cell carcinoma.

Figure 3

miR-363-3p targets SSFA2. (A) Predicted target sites of miR-363-3p in the SSFA2 3' untranslated region. (B) Luciferase reporter assay was conducted to verify the relationship between miR-363-3p and SSFA2. (C) RNA pull-down assay was carried out to confirm the interaction between miR-363-3p and SSFA2. ^**P<0.01 vs. Bio-NC. (D) Reverse transcription-quantitative PCR and (E) western blotting analyses of SSFA2 expression levels in OSCC cell lines following transfection with the miR-363-3p mimic. Data are presented as the mean ± SD; ^**P<0.01 vs. NC mimic. miR, microRNA; mut, mutant; NC, negative control; OSCC, oral squamous cell carcinoma; SSFA2, sperm-specific antigen 2; WT, wild-type.

Figure 4

Effect of miR-363-3p in OSCC cell proliferation is mediated by SSFA2. (A) Reverse transcription-quantitative PCR analysis of SSFA2 expression levels in SCC-9 cells following transfection with the pcDNA3.1 or pc-SSFA2. ^**P<0.05 vs. pcDNA3.1. (B) Reverse transcription-quantitative PCR analysis of SSFA2 expression levels in SCC-9 cells following transfection with the miR-363-3p mimic or NC mimic and SSFA2 overexpression vector or pcDNA3.1. (C) Western blotting assay was applied to detect the SSFA2 expression level. (D) Cell Counting Kit-8 and (E) EdU assays were performed to evaluate cell proliferation. Magnification, x200. (F) Effects of miR-363-3p and SSFA2 on cell cycle progression were examined using flow cytometry. Data are presented as the mean ± SD; ^**P<0.01 vs. NC mimic + pcDNA3.1; ^##P<0.01 vs. NC mimic + pc-SSFA2. ⁺⁺P<0.01 vs. miR-363-3p mimic + pcDNA3.1. EdU, 5-ethynyl-2'-deoxyuridine; miR, microRNA; NC, negative control; OD, optical density; OSCC, oral squamous cell carcinoma; SSFA2, sperm-specific antigen 2.

Figure 5

miR-363-3p inhibits OSCC cell migration and invasion by targeting SSFA2. (A) Wound healing assay were performed to determine the migration potential of SCC-9 cells following transfection. (B) Transwell Matrigel assays were applied to explore the migratory ability of OSCC cells. (C) Transwell Matrigel assay were performed to measure the invasive potential of SCC-9 cells following transfection. Magnification, x200. Data are presented as the mean ± SD; ^**P<0.01 vs. NC mimic + pcDNA3.1; ^##P<0.01 vs. NC mimic + pc-SSFA2. ⁺⁺P<0.01 vs. miR-363-3p mimic + pcDNA3.1. miR, microRNA; NC, negative control; OSCC, oral squamous cell carcinoma; SSFA2, sperm-specific antigen 2.