MicroRNA-18a regulates the metastatic properties of oral squamous cell carcinoma cells via HIF-1α expression

Abstract

Background: Rapid metastasis of oral squamous cell carcinoma (OSCC) is associated with a poor prognosis and a high mortality rate. However, the molecular mechanisms underlying OSCC metastasis have not been fully elucidated. Although deregulated expression of microRNA (miRNA) has a crucial role in malignant cancer progression, the biological function of miRNA in OSCC progression remains unclear. This study aimed to investigate the function of miRNA-18a in OSCC metastatic regulation via hypoxia-inducible factor 1α (HIF-1α).

Methods: miRNA-18a-5p (miRNA-18a) expressions in patients with OSCC (n = 39) and in OSCC cell lines (e.g., YD-10B and HSC-2 cells) were analyzed using quantitative real-time polymerase chain reaction. HIF-1α protein expressions in OSCC cells treated with miRNA-18a mimics or combined with cobalt chloride were analyzed using western blotting. The miRNA-18a expression-dependent proliferation and invasion abilities of OSCC cells were analyzed using MTT assay, EdU assay, and a Transwell® insert system.

Results: miRNA-18a expression was significantly lower in OSCC tissue than in the adjacent normal tissue. In OSCC cell lines, HIF-1α expression was significantly decreased by miRNA-18a mimic treatment. Furthermore, the migration and invasion abilities of OSCC cells were significantly decreased by miRNA-18a mimics and significantly increased by the overexpression of HIF-1α under hypoxic conditions relative to those abilities in cells treated only with miRNA-18a mimics.

Conclusions: miRNA-18a negatively affects HIF-1α expression and inhibits the metastasis of OSCC, thereby suggesting its potential as a therapeutic target for antimetastatic strategies in OSCC.

Keywords: HIF-1α; Hypoxia; Invasion; MicroRNA; Migration; Oral squamous cell carcinoma.

Fig. 1

Identification of miRNA-18a associated with HIF-1α in OSCC. A Predictive analysis of HIF-1α and miRNAs according to three miRNA databases (Targetscan, MiRDB, and PICTAR). B Schematic drawing showing the specific binding sites between HIF-1α and the miR-17-92 cluster according to the results of a sequence alignment assay. C mRNA expression of miR-17, -18a, -19a, and -20a in OSCC relative to that in adjacent normal tissues, as assessed using qRT-PCR. U6 was used as the control gene. D mRNA expression of HIF-1α and p53 in OSCC tissues relative to that in adjacent normal tissues according to qRT-PCR analysis. GAPDH was used as the control gene. *Significant difference in expression between the nontumor and tumor conditions (p < 0.05). Tissue samples were pooled. All experiments were performed at least in triplicate. miR-17, microRNA-17; miR-18a, microRNA-18a; miR-20, microRNA-20a; miRs, microRNAs; Nor, normal tissue; OSCC, oral squamous cell carcinoma

Fig. 2

Expression of miR-18a in OSCC cell lines under hypoxic conditions. A Protein expression of HIF-1α in YD-10B and HSC-2 cells under hypoxic conditions according to western blot analysis. B Intensity graph showing the expression levels of proteins under hypoxic conditions in OSCC cell lines. GAPDH was used as the control gene. C mRNA expression of miR-18a and D miR-20a in OSCC cell lines under hypoxic conditions. U6 was used as the control gene. *Significant difference in expression between the normoxic and hypoxic conditions (p < 0.05). miR-18a, microRNA-18a; miR-20, microRNA-20a; Normoxic, normoxic conditions; Hypoxic, hypoxic conditions. The original picture of western blots of A was shown in Additional file 2: Fig. S1

Fig. 3

Expression of HIF-1α and PCNA induced by miRNA-18a in OSCC under hypoxic conditions. mRNA expression of A miRNA-18a and B HIF-1α induced by miRNA-18a mimics in OSCC cell lines according to qRT-PCR analysis. U6 was used as the control gene. C Protein expression of HIF-1α and PCNA induced by C miR-18a mimics or D miRNA-18a and CoCl₂ in OSCC cell lines according to western blot analysis. Intensity graph showing the expression level of proteins after treatment with C miR-18a mimics or D miRNA-18a and CoCl₂ in OSCC cell lines, as determined with western blotting. *Significant difference in expression between control- and miRNA-18a-treated or miRNA-18a and CoCl₂-treated OSCC cell lines (p < 0.05). GAPDH was used as the control gene. All experiments were performed at least in triplicate. miR-18a, microRNA-18a; CoCl₂, cobalt chloride. The original pictures of western blots of C and D were shown in Additional file 2: Fig. S2, S3 and Fig. S4, S5.

Fig. 4

Cell viability, migration, and invasion abilities in OSCC cell lines according to miRNA-18a treatment. A Representative images acquired after an EdU assay in YD-10B and HSC-2 cells with and without miRNA-18a treatment (scale bar: 200 μm; original magnification: × 10). B Graphs showing the mean positive cell number for proliferation in five random regions in each well in the two tested OSCC cell lines. C Graphs showing cell viability, as measured using an MTT assay in YD-10B and HSC-2 cells. Representative images of D migration ability (scale bar: 200 μm; original magnification: × 10) and E invasion ability (scale bar: 200 μm; original magnification: × 20) in YD-10B and HSC-2 cells transfected with miRNA-18a. Graphs represent the mean migrated and invaded cells induced by miRNA-18a treatment in YD-10B and HSC-2 cells in five random regions in each well (bottom panels). *Significant difference between the control- and miRNA-18a-treated conditions (p < 0.05). miR-18a, microRNA-18a

Fig. 5

Cell viability, migration, and invasion abilities of OSCC cell lines under hypoxic conditions with miRNA-18a treatment. A Representative images from the EdU assay in which YD-10B and HSC-2 cells were treated with CoCl₂ and miR-18a or miRNA-18a alone (scale bar: 200 μm; original magnification: × 10). B Graphs showing the mean positive cell number for proliferation in five random regions in each well in the two tested OSCC cell lines. C Graphs showing cell viability, as measured by an MTT assay in YD-10B and HSC-2 cells. Representative images of D migration ability (scale bar: 200 μm; original magnification: × 10) and E invasion ability (scale bar: 200 μm; original magnification: × 20) in YD-10B and HSC-2 cells under CoCl₂ treatment induced with miRNA-18a treatment. Graphs showing the mean number of migrated and invaded cells induced by miRNA-18a treatment in YD-10B and HSC-2 cells under hypoxic conditions (bottom panels). These values were obtained from five random regions in each well. *Significant difference between cells treated with miRNA-18a alone and those treated with miRNA-18a under hypoxic conditions (p < 0.05). miR-18a, microRNA 18a; CoCl₂, cobalt chloride