In vitro and in silico validation of CA3 and FHL1 downregulation in oral cancer

Abstract

Background: Aberrant methylation is a frequent event in oral cancer.

Methods: In order to better characterize these alterations, a search for genes downregulated by aberrant methylation in oral squamous cell carcinoma (OSCC) was conducted through the mining of ORESTES dataset. Findings were further validated in OSCC cell lines and patients' samples and confirmed using TCGA data. Differentially expressed genes were identified in ORESTES libraries and validated in vitro using RT-PCR in HNSCC cell-lines and OSCC tumor samples. Further confirmation of these results was performed using mRNA expression and methylation data from The Cancer Genome Atlas (TCGA) data.

Results: From the set of genes selected for validation, CA3 and FHL1 were downregulated in 60% (12/20) and 75% (15/20) of OSCC samples, respectively, and in HNSCC cell lines. The treatment of cell lines JHU-13 and FaDu with the demethylating agent 5'-aza-dC was efficient in restoring CA3 and FHL1 expression. TCGA expression and methylation data on OSCC confirms the downregulation of these genes in OSCC samples and also suggests that expression of CA3 and FHL1 is probably regulated by methylation. The downregulation of CA3 and FHL1 observed in silico was validated in HNSCC cell lines and OSCC samples, showing the feasibility of integrating different datasets to select differentially expressed genes in silico.

Conclusions: These results showed that the downregulation of CA3 and FHL1 data observed in the ORESTES libraries was validated in HNSCC cell lines and OSCC samples and in a large cohort of samples from the TCGA database. Moreover, it suggests that expression of CA3 and FHL1 could probably be regulated by methylation having an important role the oral carcinogenesis.

Keywords: CA3; FHL1; Gene expression; Methylation; OSCC.

Fig. 1

RT-PCR analysis of CA3, FHL1, ANXA6, WDR26, HMGN4, C9orf64, FSTL1, CCN1, SAR1B and NFE2L1 expression in five HNSCC cell lines (represented above). Note, the expression of CA3 is not detectable in JHU-13 (O13) cell line and FHL1 is downregulated in FaDu cell line. Legend: (Ladder) 100 bp DNA Ladder (Invitrogen). + positive control (SW 480 tumor cell line) and NTC (no template control). ACTB mRNA was used to evaluate quantity in each RT-PCR reaction

Fig. 2

Gene expression profile of FHL1 and CA3 in 20 OSCC samples and 10 histologically normal oral mucosa samples. The Y-axis shows the log2 fold-change downregulation of the relative expression (2^-ΔΔCt). The dotted line indicates the cut-off adopted (2-fold downregulation)

Fig. 3

In silico TCGA validation of mRNA expression and methylation data. Heatmap analysis from TCGA data for methylation status (β-values) and mRNA expression of CA3 and FHL1 genes in normal and OSCC samples. The black vertical line divides normal from tumor samples

Fig. 4

In silico TCGA evaluation of methylation status and mRNA expression of CA3 and FHL1. a and b boxplots show mean methylation β-values in normal and tumor tissues, for CA3 and FHL1 genes, respectively. c and d boxplots show mRNA expression distributions, in normal and tumor tissues, for CA3 and FHL1 genes, respectively. Statistical p values denote Student’s ttest between normal and OSCC samples. e and f show Pearson’s correlation of mean methylation β-values and mRNA expression, for CA3 and FHL1 genes, respectively

Fig. 5

RT-PCR assay for CA3 and FHL1 expression analysis after 5'-aza-dC treatment with 1 μM for 7 days. a Expression levels of CA3 in JHU-13 cell line. Note the gradative increase of CA3 expression from 3 to 7 days; b Expression levels of FHL1 in FaDu cell line. Note that FHL1 expression was restored at the third day of treatment. ACTB expression was used to evaluate the load quantity in each well. + positive control (HCT tumor cell line) and – negative control (without DNA)