Effects of arecoline on proliferation of oral squamous cell carcinoma cells by dysregulating c-Myc and miR-22, directly targeting oncostatin M

<div><p>Arecoline, the major alkaloid of areca nut, is known to induce oral carcinogenesis, however, its mechanism is still needed to elucidate. This study investigated the effects of arecoline on cell viability and cell-cycle progression of oral squamous cell carcinoma (OSCC) cells as well as a relevant cellular gene expression. The results showed that a low concentration of arecoline (0.025 μg/ml) increased OSCC cell viability, proportion of cells in G2/M phase and cell proliferation. Simultaneously, it induced IL-6, STAT3 and c-Myc expression. Interestingly, c-<i>myc</i> promoter activity was also induced by arecoline. MiR-22 expression in arecoline-treated OSCC cells was suppressed and comparable to an upregulated c-Myc expression. In arecoline-treated OSCC cells, oncostatin M (OSM) expression was significantly upregulated and inversely correlated with miR-22 expression. Likewise, OSM expression and its post-transcriptional activity were significantly decreased in miR-22-transfected OSCC and 293FT cells. This result demonstrated that miR-22 directly targeted OSM. Interestingly, miR-22 played an important role as a tumor suppresser on suppressing cell proliferation, migration and cell-cycle progression of OSCC cells. This result suggested the effect of arecoline to promote cell proliferation and cell-cycle progression of OSCC cells might be involved in induction of c-Myc expression and reduction of miR-22 resulting in OSM upregulation.</p></div

The effects of arecoline on cell viability and cell-cycle progression.

<p>Cytotoxicity (A and B) and cell proliferation (C and D) were determined in arecoline-untreated or treated OSCC cell lines at various concentrations for 24 hours using the MTT assay. Statistical significance of the differences of cell viability (%) was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001). Cell-cycle phase distribution (E and F) in ORL-48(T) cells treated with 0 and 0.025 μg/ml of arecoline in synchronized condition was analyzed by flow cytometry. The percentages of G0/G1, S and G2/M population (G) of arecoline-treated cells were compared to untreated ORL-48(T) cells as control. Statistical significance of the differences of G2/M population was analyzed using Paired <i>t</i>-test (*<i>P</i> < 0.05).</p

Relative expression levels of miR-22 and OSM.

<p>100 and 500 ng/well of mock control (pIRES2-EGFP vector) and pIRES-miR-22 vectors were transfected into ORL-48(T) and ORL-136(T) cells. At 24 and 48 hours post-transfection, pri-miR-22 (A-B) and OSM (C-D) expression was determined by RT-PCR. Protein levels of OSM (E) were determined in ORL-48(T) and ORL-136(T) cells at 48 hours after transfection with 100 ng of mock control and pIRES-miR-22 vectors. Relative intensity of OSM protein band (F-G) was calculated using ImageJ 1.49v software. Statistical significance of the differences was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001).</p

Effect of arecoline on IL-6 and STAT3 in ORL-48(T) and ORL-136(T) cells.

<p>ORL-48(T) and ORL-136(T) cells were treated with 0, 0.025 and 25 μg/ml arecoline for 24 hours. Expression of IL-6 (A and D) and STAT3 (B and E) were investigated by RT-PCR and their amplicons were visualized by 2% agarose gel electrophoresis (C and F). Statistical significance of the differences of relative expression was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05 and **<i>P</i> < 0.01).</p

MiR-22 targets OSM and miR-22 functions in cell proliferation, migration and cell-cycle assay.

<p>The construct of the miR-22 targets sequence within the OSM 3′UTR WT and Mut in pGL3-Control vector. The luciferase gene was linked to the 3′UTR WT and Mut of OSM. 293FT cells were co-transfected with 250 ng pIRES-miR-22 and 100 ng pGL3-OSM 3′UTR WT or Mut vectors (A). The normalized luciferase activity in pIRES-miR-22 and pGL3-OSM 3′UTR WT or Mut co-transfected cells was relative to normalized luciferase activity of pIRES2-EGFP and OSM 3′UTR WT or Mut co-transfected cells (B). A green fluorescence expression vector (pEGFP-N3) was transfected for monitoring transfection efficiency. Statistical significance of the differences of luciferase activity was analyzed using Two-way ANOVA (*<i>P</i> < 0.05). Cell proliferation and migration in pIRES-miR-22-transfected ORL-48(T) cells were measured by a hemocytometer and wound healing assay at different incubation time points (C-E). The photograph was taken under 4X objective lens NIS-Elements Advanced Research Imaging Software version 3.0. Statistical significance of the differences of cell viability and wound closure was analyzed using Student's <i>t</i>-test (*<i>P</i> < 0.05 and ***<i>P</i> < 0.001). Cell-cycle assay in miR-22 or mock-transfected ORL-48(T) for 48 hours post-transfection was performed by flow cytometry (F). Statistical significance of the differences of G2/M and G0/G1 population was analyzed using Paired <i>t</i>-test (*<i>P</i> < 0.05 and (**<i>P</i> < 0.01, respectively).</p

Primer sequences.

<p>Primer sequences.</p