Supplementary Materials and Methods, Supplementary Tables 1 through 10, and Supplementary Figures 1 through 20 from MicroRNA-211 Enhances the Oncogenicity of Carcinogen-Induced Oral Carcinoma by Repressing TCF12 and Increasing Antioxidant Activity
Supplementary Materials and Methods. Table S1. Cell cultivation conditions. Table S2. siRNAs used in the present study. Table S3. TaqMan assay probes used in the present study. Table S4. Primers used in the present study. Table S5. shRNA clones used in the present study. Table S6. Paired OSCC samples for qRT-PCR and Western blot analysis. Table S7. OSCC samples in TMA for IHC and ISH analysis. Table S8. Primary antibodies or associated reagents used in the present study. Table S9. The complimentarity between miR-211 and the 3’UTR of TCF12 gene. Table S10. Jaspar prediction scores. Fig S1. Genotyping of K14-EGFP-miR-211 transgenic mouse model. Fig S2. GFP immunoreactivity and thickness in squamous epithelium. Fig S3. Immunohistochemistry of the cell proliferation and survival proteins in tongue epithelium. Fig S4. Induction of mouse esophageal tumorigenesis by 4NQO. Fig. S5. Representative histopathological sections of orthotopic xenografts and neck lymph nodes. Fig. S6. Analysis of pri-miR-211 expression in OSCC cells. Fig. S7. miR-211 staining and TCF12 immunoreactivity in mouse tongue tissues. Fig. S8. Analysis of TCF12 protein in subcellular fractions in FaDu cells. Fig. S9. Phenotypic analysis of OECM1 cell with TCF12 expression. Fig. S10. Correlation between miR-211 expression and TCF12 mRNA expression in OSCC tumors. Fig. S11. miR-211 staining and TCF12 immunoreactivity in OSCC. Fig. S12. TCF12 immunoreactivity in mouse multistep carcinogenesis. Fig. S13. Color diagram of Fig. 4A, a. Fig S14. Analysis of TCF12 associated biological processes and gene interaction networks. Fig. S15. Prediction of the potential regulation of TCF12 on the promoter activity of downstream genes using Jasper software. Fig. S16. qRT-PCR analysis to confirm the regulation of TCF12 on downstream genes across different OSCC cells. Fig. S17. Correlation between the expression of TCF12 and FAM213A in database. Fig. S18. FAM213A immunoreactivity. Fig. S19. Color diagram of Fig. 6D. Fig. S20. Kaplan-Meier survival analysis.
Funding
Ministry of Science and Technology
Taipei Veterans General Hospital
Department of Education
Health and Welfare Surcharge of tobacco
History
ARTICLE ABSTRACT
miR-211 expression in human oral squamous cell carcinoma (OSCC) has been implicated in poor patient survival. To investigate the oncogenic roles of miR-211, we generated K14-EGFP-miR-211 transgenic mice tagged with GFP. Induction of oral carcinogenesis in transgenic mice using 4-nitroquinoline 1-oxide (4NQO) resulted in more extensive and severe tongue tumorigenesis compared with control animals. We found that 4NQO and arecoline upregulated miR-211 expression in OSCC cells. In silico and experimental evidence further revealed that miR-211 directly targeted transcription factor 12 (TCF12), which mediated suppressor activities in OSCC cells and was drastically downregulated in tumor tissues. We used GeneChip analysis and bioinformatic algorithms to identify transcriptional targets of TCF12 and confirmed through reporter and ChIP assays that family with sequence similarity 213, member A (FAM213A), a peroxiredoxin-like antioxidative protein, was repressed transcriptionally by TCF12. FAM213A silencing in OSCC cells diminished oncogenic activity, reduced the ALDH1-positive cell population, and increased reactive oxygen species. TCF12 and FAM213A expression was correlated inversely in head and neck carcinoma samples according to The Cancer Genome Atlas. OSCC patients bearing tumors with high FAM213A expression tended to have worse survival. Furthermore, 4NQO treatment downregulated TCF12 and upregulated FAM213A by modulating miR-211 both in vitro and in vivo. Overall, our findings develop a mouse model that recapitulates the molecular and histopathologic alterations of human OSCC pathogenesis and highlight a new miRNA-mediated oncogenic mechanism. Cancer Res; 76(16); 4872–86. ©2016 AACR.Usage metrics
