The HSP90AA1 gene is involved in heat stress responses and its functional genetic polymorphisms are associated with heat tolerance in Holstein cows.

As the stress-inducible isoform of the heat-shock protein 90 (HSP90), the HSP90AA1 gene encodes HSP90α and plays an important role in heat stress (HS) response. Therefore, this study aimed to investigate the role of the HSP90AA1 gene in cellular responses during HS and to identify functional SNPs associated with thermotolerance in Holstein cattle. For the in vitro validation experiment of acute HS, cells from the Madin-Darby bovine kidney cell line were exposed to 42°C for 1 h, and various parameters were assessed, including cell apoptosis, cell autophagy, and the cellular functions of HSP90α by using its inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Furthermore, the polymorphisms identified in the HSP90AA1 gene and their functions related to HS were validated in vitro. Acute HS exposure induced cell apoptosis, cell autophagy, and upregulated expression of the HSP90AA1 gene. Inhibition of HSP90α by 17-AAG treatment had a significant effect on the expression of the HSP90α protein and increased cell apoptosis. However, autophagy decreased in comparison to the control treatment when cells were exposed to 42°C for 1 h. Five SNPs identified in the HSP90AA1 gene were significantly associated with rectal temperature and respiration score in Holstein cows, in which the rs109256957 SNP is located in the 3' untranslated region (3' UTR). Furthermore, we demonstrated that the 3' UTR of HSP90AA1 is a direct target of bta-miR-1224 by cell transfection with exogenous microRNA (miRNA) mimic and inhibitor. The luciferase assays revealed that the SNP rs109256957 affects the regulation of bta-miR-1224 binding activity and alters the expression of the HSP90AA1 gene. Heat stress-induced HSP90AA1 expression maintains cell survival by inhibiting cell apoptosis and increasing cell autophagy. The rs109256957 located in the 3' UTR region is a functional variation and it affects the HSP90AA1 expression by altering its binding activity with bta-miR-1224, thereby associating with the physiological parameters of Holstein cows.