Table 1.
Primer sequences.
Fig 1.
The effects of arecoline on cell viability and cell-cycle progression.
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 (*P < 0.05, **P < 0.01 and ***P < 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 t-test (*P < 0.05).
Fig 2.
The effects of arecoline on c-Myc expression and transcriptional activity.
ORL-48(T) cells treated with 0, 0.025 and 25 μg/ml of arecoline for 24 hours were assayed to determine levels of c-Myc expression in mRNA (RT-PCR) (A) and protein (western blot) (B). Relative c-Myc expression and relative intensity of c-Myc protein band were investigated in RT-PCR and western blot result (C-D). Statistical significance of the differences was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*P < 0.05, **P < 0.01 and ***P < 0.001). Mock or pGL3-cMYCP vector-untransfected or transfected ORL-48(T) cells were treated with 0, 0.025, 0.25 and 25 μg/ml of arecoline for 24 and 48 hours (E). The transcriptional activity of the c-myc promoter was determined by luciferase activity. Statistical significance of the differences of luciferase activity was analyzed using Two-way ANOVA (*P < 0.05, **P < 0.01 and ***P < 0.001).
Fig 3.
Effect of arecoline on IL-6 and STAT3 in ORL-48(T) and ORL-136(T) cells.
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 (*P < 0.05 and **P < 0.01).
Fig 4.
The effects of arecoline on miR-22 and OSM expression.
Both ORL-48(T) and ORL-136(T) cells were exposed to 0, 0.025 and 25 μg/ml of arecoline for 24 hours. Pri-miR-22 expression was detected by RT-PCR. β-actin was used as internal controls (A-B). ORL-48(T) cells were treated with various concentrations of arecoline for 24 hours; then OSM mRNA expression in these cells was analyzed using RT-PCR (C-D). OSM protein levels in ORL-48(T) (E) and ORL-136(T) (F) cells were examined by western blot and relative intensities were analyzed by ImageJ 1.49v software (G-H). Statistical significance of the differences of relative expression was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*P < 0.05, **P < 0.01 and ***P < 0.001).
Fig 5.
Relative expression levels of miR-22 and OSM.
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 (*P < 0.05, **P < 0.01 and ***P < 0.001).
Fig 6.
MiR-22 targets OSM and miR-22 functions in cell proliferation, migration and cell-cycle assay.
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 (*P < 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 t-test (*P < 0.05 and ***P < 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 t-test (*P < 0.05 and (**P < 0.01, respectively).