Detection on the expression of the transcription factors: oct-4, nanog, and sox-2 genes in canine cutaneous mast cell tumors

Abstract

The objectives of this study were to determine the expression of Oct-4, Nanog, and Sox-2 in both mRNA and proteins levels. The 30 biopsied MCT cases, from the Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University, were used in this study. The samples were divided into two parts; part I, the biopsied samples were processed for paraffin section and stained with H&E stain, for grading based on Patnaik’s system and Kiupel’s system and the other part was detected the expression of Oct-4, Nanog, and Sox-2 genes. The MCT samples were classified into three grades; grade I, II, and III by Patnaik system; grade I, 40% (12/30), grade II, 26% (8/30) and grade III, 33.33% (10/30). And, Kiupel’s system which were low grade, 66.66% (20/30) and high grade, 33.33% (10/30) respectively. The RT-PCR and qRT-PCR were used for detecting Oct-4, Nanog, and Sox-2 mRNA in MCT cases. The results showed that all of MCT samples expressed both Oct-4 and Sox-2 at mRNA level (100 %; 30/30). While, Nanog could not be detected in all MCT samples (0%). For qRT-PCR, the Oct-4 gene in high grade tended to be higher than low grade parallelly in grade III also tended to be higher than grade I and II respectively . On the contrary, Sox-2 expression, in grade III and high grade tended to be lower than grade I and low grade respectively, when compared to internal control. However, the results had no statistically significant for the experiments among the grading system. The immunohistochemical expression of Oct-4, Nanog, and Sox-2 in MCT samples were conducted. There were positive expression of Oct-4, Nanog, and Sox-2 in all the samples at 90% (27/30), 80 % (24/30) and 80% (24/30 ) respectively. Regards to the positive cells and the grade of MCT, it was demonstrated that there were no statistically difference of Oct-4 and Nanog positive cells in all grades (p≥0.05) . On the contrary, the positive cells of Sox-2 in high grade were higher than low grade MCT samples with statistically difference (p≤0.05). The results of Western blot technique showed the expression of Oct-4, Nanog, and Sox-2 protein with low reaction intensity. Which were; Oct-4, 1 band at 43 kDa, Nanog, 3 bands, at 45,35 and 25 kDa, approximately, Sox-2, 4 bands, at 77,65,35 and 26 kDa approximately in all MCT samples respectively when compared to positive control.. In addition, the demonstration of Intranuclear Immunocytofluorescence was conducted on the expression of Oct-4, Nanog, and Sox-2 proteins which the positively localizations were shown in intranuclear and intracytoplasmic of MCT cells. Additionally, the expression of the Oct-4, Nanog, and Sox-2 proteins could be potentially detected by Surface Plasmon Resonance, SPR technique in MCT. However, there were some limitations of the technique, due to their high sensitivity and nonspecific appearances. By the obtained results, it is revealed that the major embryonic transcription factors, Oct-4, Nanog, and Sox-2 mRNA and proteins in the MCT tissue and could be detected. Which could be possibly beneficial on the development of the prognostic marker of this tumor in the future.