Establishment and miRNA expression characterization of a canine mammary gland tumor cell line

Canine mammary gland tumors (CMGTs) are the most common neoplasms in sexually intact female dogs. CMGTs have been suggested as a model for studying human breast cancer because of several similarities, including the relative age of onset, risk factors, incidence, histological and molecular features, biological behavior, metastatic pattern, and responses to therapy. In the present study, we established a new cell line, the SNP cell line, from a CMGT. A tumor formed in each NOD.CB17-Prkdcscid/J mouse at the site of subcutaneous SNP cell injection, with an average size at 7 days after inoculation of 49.9 mm3. SNP cells are characterized by proliferation in a tubulopapillary pattern. Moreover, we examined miRNA expression in the cultured cells and found that the expression values of miRNA-143 and miRNA-138a showed the greatest increase and decrease, respectively, of all miRNAs observed. These miRNAs might therefore play a significant role in the malignancy of SNP cells. SNP cells might serve as a model for future genetic analysis and clinical treatments of human breast tumors. SNP cells or normal mammary gland tissue were freshly frozen within 30 minutes of harvesting or surgical excision and stored at -80 °C until further use for an Affymetrix® GeneChip® miRNA array. An Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) was used to assess the quality of total RNA (1,000 ng). The biotin-labeled sample was prepared with a Flash TagTM Biotin HSR RNA Labeling Kit (Affymetrix, Inc.) according to the manufacturer’s protocol, and 21.5 µl of biotin-labeled sample was combined with 110.5 μl of Hybridization Master Mix. The array was incubated at 48 °C and 60 rpm for 18 hours in a GeneChip® Hybridization Oven 645 (Affymetrix, Inc.) according to the manufacturer’s protocol. A GeneChip® Fluidics Station 450 (Affymetrix, Inc.) was used to wash the array according to the manufacturer’s protocol before scanning with a GeneChip® Scanner 3000 7G (Affymetrix, Inc.) following the protocol in the Affymetrix® GeneChip® Command Console User Manual.