Study the gene expression of E-cadherin, syndecan1, matrix metalloproteinases-2, -7, -9, -14 and tissue inhibitors of metalloproteinases-1 and -2 in canine oral melanoma

Abstract

The objectives of this study were to 1.) select the suitable reference genes for quantitative real-time polymerse chain reaction in the most common canine oral cancers: oral melanoma (OM) and oral squamous cell carcinoma (OSCC), 2.) study the gene expression of E-cadherin (CDH1), syndecan 1 (SDC1), matrix metalloproteinases-2, -7, -9, -14 (MMP2, MMP7, MMP9, MMP14) and tissue inhibitors of metalloproteinases-1 and -2 (TIMP1, TIMP2) in canine OM at the mRNA level and study the CDH1, SDC1 and Ki-67 protein expression by immunohistochemistry, and 3.) study the association of gene expression and the tumor, node, metastasis (TNM) stage. Twelve OM tissues with different TNM stages together with 7 OSCC and 8 normal gingival tissues were collected for reference gene selection. Five algorithms were used to evaluate 6 candidate reference genes, including beta actin (ACTB), beta-2-microglobulin (B2M), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13a), ribosomal protein S5 (RPS5), and ribosomal protein S19 (RPS19). The result showed that the cohort of the most suitable reference genes for canine OM and OSCC were ACTB, RPS5 and RPS19 and they were then used to normalize target genes in qRT-PCR. In OM, CDH1 and SDC1, encoding tumor suppressor proteins, were found to be down-regulated, corresponding to the IHC results. MMP7 gene was also found to be down-regulated whereas MMP2 and MMP14, encoding the proMMP2 activator, were upregulated, indicating cancer progression. TIMP1, encoding an inhibitor of tumor progression and metastasis, and TIMP2, encoding an inhibitor of MMP2, were upregulated in the early stage (stages 1 and 2) and late stage (stages 3-4) of the disease, respectively. Ki-67 expression, an indicator of proliferating cells, was also found to be upregulated in OM. In conclusion, the present study reported the suitable reference genes for canine OM and OSCC. The association of tumor suppressing gene and some MMP gene expression with OM was presented, regardless of the disease stages. Upregulation of TIMPs in the different stages of OM should be further studied for the possibility to be used as pathological and diagnostic markers of the disease stages.