Additional file 1 of Histone lysine methyltransferase SMYD3 promotes oral squamous cell carcinoma tumorigenesis via H3K4me3-mediated HMGA2 transcription

Additional file 1: Fig. S1. Identification of SMYD3 for diagnosis of OSCC. A–E ROC curve analyses of SMYD3 in TCGA, meta-GEO, TCGA, GSE37991, and GSE30784 datasets. AUC values are shown. Fig. S2. The DNA methylation and genomic mutation profile in the TCGA-OSCC dataset. A The correlation of SMYD3 expression and DNA methylation level in TCGA-OSCC cohort. B Value differences of DNA methylation probes in normaland tumortissues from TCGA-OSCC cohort. C The lollipop plot illustrates the differential distribution of somatic mutation in the TCGA-OSCC dataset for SMYD3. D, E ROC curve analyses of SMYD3 in qRT-PCR and IHC staining of collected samples, respectively. Ns, not significant, *P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Fig. S3. High expression of SMYD3 indicates increased H3K4me3 modification and HMGA2 expression. A–F IHC images of high and low protein expression of SMYD3, H3K4me3 and HMGA2. Scale bars: 100 μm. Fig. S4. Biological function and pathway enrichment analysis. A The results of GO analysis of RNA-seq on two groups of CAL-27 transfected with NC and SMYD3 siRNA. B The results of KEGG analysis of RNA-seq on two groups of CAL-27 transfected with NC and SMYD3 siRNA. Fig. S5. SMYD3 facilitates OSCC cell stemness maintenance and proliferation in vitro and tumorigenesis in vivo. A, B SMYD3 mRNA and protein levels in CAL-27 and UM-SCC-1 cell lines. C SMYD3 mRNA levels in OSCC cells transfected with NC and SMYD3 siRNAs. D–G Quantitative statistical results of SMYD3 knockdown in vitro experiments. H SMYD3 mRNA levels in OSCC cells transfected with vector and SMYD3 plasmid. I–K Quantitative statistical results of SMYD3 overexpression in vitro experiments. L The protein expressions of SMYD3 and H3K4me3 were detected after transfection of CAL-27 cell line with SMYD3 plasmids. M, N SMYD3 mRNA and protein levels in CAL-27 transfected with shNC and shSMYD3. *P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Fig. S6. BCI-121 suppresses OSCC cells stemness maintenance and proliferation. A H3K4me3 levels in OSCC cells under different concentrations of BCI-121. B, C The effect of different concentrations of BCI-121 on cell viability determined by CCK8 assays in CAL-27 and UM-SCC-1 cell lines. D SMYD3 mRNA levels in OSCC cells following BCI-121 treatment. E–H Quantitative statistical results of BCI-121 treatment in vitro experiments. Ns, not significant, *P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Fig. S7. BCI-121 impedes the chemical-induced primary OSCC formation. A, B Representative H&E images of tongue lesions. Scale bars, 200 μm. C The number of clusters was determined using SOM clustering. Fig. S8. HMGA2 is upregulated in OSCC, and high expression of HMGA2 predicts a poor prognosis. A HMGA2 was overexpressed in OSCC samples from the meta-GEO dataset. Numbers in parentheses indicate the sample size. B Patients with high HMGA2 expression have a worse prognosis than those with low HMGA2 expression in the meta-GEO dataset. Numbers in parentheses indicate the sample size. C Quantitative result of qRT-PCR of HMGA2 in 20 paired adjacent normal and OSCC tissues. D The protein levels of HMGA2 in 12 pairs of OSCC tissuesand adjacent normal tissuesmeasured by Western blotting. E Images of IHC staining for HMGA2 in normal tissues and different histologic grades of OSCC tissues. Scale bars: 50 μm. Fig. S9. HMGA2 is a downstream target gene of SMYD3. A The SMYD3 binding sitein human HMGA2 promoter and the corresponding base mutation. B, C HMGA2 mRNA and protein levels in CAL-27 transfected with NC and HMGA2 siRNA. D Upon the completion of the ten-day tumorsphere formation assay, the proteins were extracted from the spheroid cells on the 10th day. The expression of SOX2 in SMYD3-upregulated cells was nullified following HMGA2 knockdown. E–H The tumorsphere formation and proliferation capacities of SMYD3-upregulated cells were abrogated by HMGA2 knockdown. Scale bars: 50 μm. *P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Fig. S10. SMYD3 affects OSCC cell stemness maintenance and proliferation via HMGA2. A, B HMGA2 mRNA and protein levels in CAL-27 transfected with vector and HMGA2 plasmid. C–G The SOX2 expression, tumorsphere formation and proliferation capacities of HMGA2-upregulated cells were abrogated by SMYD3 knockdown. Scale bars: 50 μm. *P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001