Mutation and expression study of p21 and c-Myc genes in different tumors

Abstract

Companion animals such as dogs are considered the best preclinical model for cancer and many other complex human diseases. Many features make the dog an attractive model for human cancer biology research. Firstly, the great genetic homology between the human and dog genome as compared to other animals. Secondly, dogs live in the same environment and share the similar risk factors or disease characteristics with humans. Dogs and cats are the most beloved creatures among the pet species. Therefore, animal passionate are more anxious about their companion animal's health and husbandry techniques. The cancer incidence rate is increasing due to less rigorous managerial efforts, fewer animal health care facilities and economic constraints. The first part of the study was conducted at PG Lab, IBBT UVAS Lahore Pakistan, Forty three canines and twelve feline tumor and normal samples were collected from Pet Center UVAS from 2012 to 2015. The genomic DNA was extracted by using the Qiagen DNA extraction kit and specific set of primers were designed for the amplification of p21 and C-MYC gene. The amplified products were cleaned and sequenced for the detection of SNP/mutation. To determine the differential expression of p21 and C-MYC gene, total RNA was extracted and converted into cDNA using the Thermo Scientific RNA purification kit and cDNA synthesis kit respectively. Real time q-PCR ABI-7500 was used and a TaqMan primer probe for the studied genes and GAPDH housekeeping gene were specifically designed to detect and target the gene sequence. Exon 2 and exon 3 of both genes were sequenced but no polymorphism was detected in available samples. However, the expression of p21 gene was increased in 53% and 38% of all the studied tumors in canine and feline respectively, out of which 75% in canine mammary adenocarcinoma, 50% in canine OSCC, 33% in canine peri-anal adenocarcinoma, 60 % in canine mast cell tumors and 40% in canine soft tissue sarcomas, 40 % in feline mammary adenocarcinoma and 33 % in feline soft tissue sarcoma. . Similarly, C-MYC gene expression was increased in 80% and 75 % of all the studied tumors in canine and feline respectively, out of which 62% in canine mammary adenocarcinoma, 60 % in feline mammary adenocarcinoma, 75% in canine oral squamous cell carcinoma, 66 % in canine peri-anal adenocarcinoma, 80 % in canine mast cell tumors and 100% in canine and feline soft tissue sarcoma samples. The second part of the study was conduct at Veterinary Medicine Lab, University of Cambridge UK, Twelve canine soft tissue sarcomas were collected from Queens Veterinary School, University of Cambridge and thirteen soft tissue sarcomas sample from UVAS Lahore Pakistan for the expression profiling of microRNAs (miR-9, miR-23a, miR-92a, miR-125b, miR-451, miR-499) in correlation with structural genes (MIF, MDR, CDKN1A, C-MYC). MicroRNA expression experiments were performed using Qiagen Rotter Gene 6000 RT-qPCR and SYBER Green chemistry. The HPRT housekeeping gene, three SNORDS & RUN6 were used to normalize the structural genes and microRNs respectively. Expression data was analyzed by the Genepattern software. miR- 9a, miR- 92a, miR-499a showed the high expression while miR-23a, miR-125b and miR-451a showed low expression. Further more significant positive and negative correlations were observed among the genes investigated. Similarly, the above methodology was used on twenty human oral dysplasia and OSSC samples to determine the differential microRNA expression of 15 microRNAs (17-92 cluster family of microRNA, miR-21a, miR106b, miR-125a, miR-145, mir146a, miR146b, miR-182, miR451 and miR-let7a), Deregulation of microRNAs was mainly observed in 17-92 cluster family (miR-17a. miR-18a, miR-19a. miR-19b, miR-20a, miR-92a) in oral squamous cell carcinoma when compared to oral dysplasia. Significant positive and negative correlations were also observed among the microRNAs. In conclusion, p21 and C-MYC showed an altered expression but no polymorphism was detected in the limited canine and feline tumor samples analyzed in this study, suggesting the need of more tumors samples to better understand the gene role in specific tumors pathogenesis Moreover, the panel of miRNAs used for STS and human OSCC, may be a useful addition to available data and helpful for a better understanding of tumor biology.