Additional file 1: of Epidermal growth factor receptor pathway mutation and expression profiles in cervical squamous cell carcinoma: therapeutic implications

Table S1. KRAS and PIK3CA mutations detected

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

STING and IFN-κ transcripts were down-regulated by HPV E2s.

<p>(A) STING transcripts level analyzed by RT-PCR was significantly down-regulated by HPV16 E2, HPV18 E2 and the HPV18 E2 TAD in HPK and B) IFN-κ transcription level was significantly down-regulated by HPV16 E2, HPV18 E2 and HPV18 TAD in HPK. C) STING transcripts level was modulated by E2 in NUH49 as shown in (A). D) IFN-κ transcripts level was modulated by E2 in ITB3 and NUH49 as in (B). (A–D) HPV18 E2TAD significantly down-regulated STING and IFN-κ genes transcription when compared to HPV18 E2DBD. (*<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001).</p

Transcriptional reduction of IFN-κ in STING-silenced HPK and poly I:C stimulated HPK.

<p>(A) STING and IFN-κ transcription level in HPK cells after transfection with siSTING and siIFNK for 48 h. IFN-κ transcription level in STING-knockdown HPK was reduced by ∼20% (<i>P</i> = 0.06). STING transcription level in IFN-κ-knockdown HPK was not changed. (B) ISGs transcription levels were down-modulated in IFN-κ-knockdown HPK. (C) TAD of HPV18 E2 abrogated STING transcription in Poly I:C induced HPK cells. Cells transduced with GFP as control and GFP-E218TAD were stimulated with poly I:C (10 µg/ml) for 3, 6, and 12 h. STING transcription level was measured at each time point. (D) IFN-κ transcription level was abrogated by HPV18 E2TAD when examined at each time point of IFN-κ induction by poly I:C (E) ISGs were determined after stimulation of HPK expressing HPV18 E2TAD with poly I:C (10 µg/ml) for 3 h. (*<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001).</p

HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes.

<p>(A) GFP signals of recombinant adenoviruses GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD under fluorescence microscope on live cells (10X). (B) GFP protein expression was detected by western blotting analysis. GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD showed the expected protein sizes of ∼32 kD, 62 kD, 64 kD, 50 kD, and 45 kD, respectively and are indicated by asterisks. The β-actin protein was used as loading control. (C) The mRNA expression levels of recombinant adenoviruses GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD are shown as CT values (mean±SD). (D) The suppressive effect of HPV E2 on HPV18 E6/E7 transcription in HeLa cells is shown as relative transcripts levels compared to cells not expressing E2 using real-time PCR. (*<i>P</i>≤0.05).</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

STING and IFN-κ down-regulation in HPV positive clinical specimens.

<p>(A) HPV E2s transcription level was significantly increased in HPV positive normal and LSIL when compared to HPV negative normal cases (<i>P</i><0.05). (B) STING transcription level was significantly decreased in LSIL with HPV positive and HPV E2 positive cases (<i>P</i><0.05). Normal cases with HPV positive also showed low STING transcription level when compared to normal HPV negative cases (<i>P</i><0.05). (C) IFN-κ transcription level was significantly down-regulated in normal HPV positive cases and HPV positive LSIL cases when compared to normal HPV negative cases (<i>P</i><0.05). (*<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001).</p

Transcription network- associated genes modulated by HPV16E2 in HPK.

<p>Transcription network- associated genes modulated by HPV16E2 in HPK.</p

Top 10 modulated pathways in HPK expressing HPV16 E2 protein.

<p>Top 10 modulated pathways in HPK expressing HPV16 E2 protein.</p