Characterizing binding sites of heat responsive microRNAs and their expression pattern in heat stressed PBMCs of native cattle, exotic cattle and riverine buffaloes.

Abstract

It is generally believed that due to evolutionary differences and adaptation to tropical conditions, Indian native cattle has superior heat tolerant ability than Bos taurus cattle. In the present study, 3'-UTR of two most important heat responsive genes i.e., heat shock protein 70.1 (HSP70.1) and heat shock factor- 1 (HSF-1) were sequence characterized in different breeds of Indian native cattle to identify the variations and miRNA binding sites. In addition, the impact of heat stress was assessed in a total of 57 PBMCs samples of native Sahiwal cows (Bos indicus), exotic Holstein cows (Bos taurus) and Murrah buffaloes (Bubalus bubalis) using various cellular parameters like cell viability, cytotoxicity and apoptosis. Further, expression profile of 12 heat responsive miRNAs were also evaluated in unstressed and stressed PBMCs to understand post transcriptional changes in native cows, exotic cows and Murrah buffaloes. The sequence data showed 3'-UTR of HSP70.1 gene of Indian cattle to be exactly similar to Bos taurus with no miRNA binding site. Whereas, sequencing of 3'-UTR of HSF-1 gene revealed 3 SNPs at positions G1762T; C1811T and C1983T with 7 well conserved miRNA binding sites. The impact of heat stress on various cellular parameters in terms of cell viability, cytotoxicity and apoptosis was highest in PBMCs of Holstein cows followed by Murrah buffaloes and Sahiwal cows. Further, in contrast to Holstein Frisian cows and Murrah buffaloes, the expression pattern of 12 heat responsive miRNAs, in heat stressed PBMCs of Sahiwal cows were quite distinct. There was a significant (p < 0.05) induction in expression of most of the miRNAs after heat stress in PBMCs of Sahiwal cows followed by a rapid decline. The distinct cellular response and pattern of miRNA expression across cattle types and buffaloes might be influencing their PBMCs tolerance level to heat stress.