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

Up-Regulation of miR-21 Is Associated with Cervicitis and Human Papillomavirus Infection in Cervical Tissues.

MicroRNA-21 (miR-21) is recognized as an oncomir and shows up-regulation in many types of human malignancy. The aim of this study was to investigate the association of miR-21 expression associated with HPV infection in normal and abnormal cervical tissues. Cervical tissue samples with different cytological or histopathological grades were investigated for HPV by PCR and for miR-21 and programmed cell death, protein 4 (PDCD4) expression using quantitative real-time PCR (qRT-PCR). Laser capture microdissection (LCM) of stromal and epithelial tissues and in situ hybridization (ISH) using locked nucleic acid (LNA) probes were performed on a subset of fixed specimens. Cell line experiments were conducted on fibroblasts stimulated in culture media from HeLa cells, which were then assessed for miR-21, PDCD4, IL-6 and α-SMA expression by qRT-PCR. Twenty normal cervical cell, 12 cervicitis, 14 cervical intraepithelial neoplastic I (CIN I), 22 CIN II-III and 43 cervical squamous cell carcinoma (SCC) specimens were investigated. miR-21 levels were significantly lower in normal than in abnormal tissues. The expression of miR-21 in HPV negative normal cytology was significantly lower than in HPV positive samples in abnormal tissue and SCC. The miR-21 expression was significantly higher in HPV negative cervicitis than HPV negative normal cells. LCM and ISH data showed that miR-21 is primarily expressed in the tumor-associated stromal cell microenvironment. Fibroblasts treated with HeLa cell culture media showed up-regulated expression of miR-21, which correlated with increased expression of α-SMA and IL-6 and with down-regulation of PDCD4. These results demonstrate that miR-21 is associated with HPV infection and involved in cervical lesions as well as cervicitis and its up-regulation in tumor-stroma might be involved in the inflammation process and cervical cancer progression

E2 Proteins of High Risk Human Papillomaviruses Down-Modulate STING and IFN-κ Transcription in Keratinocytes

<div><p>In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-κ that is involved in immune evasion and HPV persistence<b>.</b> To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-κ and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-κ and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-κ were further determined in clinical specimens by real time PCR. STING and IFN-κ were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-κ transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.</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

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