miR-27a-3p relieves heat stress-induced mitochondrial damage and aberrant milk protein synthesis through MEK/ERK pathway in BMECs

Abstract

With global warming, heat stress has become a primary factor that compromises the health and milk quality of dairy cows. Here, we investigated the function and underlying regulatory mechanism of miR-27a-3p in bovine mammary epithelial cells (BMECs) under heat-stress conditions. The current study showed that miR-27a-3p could prevent heat stress-induced BMEC oxidative stress and mitochondrial damage by regulating the balance between mitochondrial fission and fusion processes. Importantly, we found that miR-27a-3p could increase cell proliferation under heat stress conditions by regulating the MEK/ERK pathway and cyclin D1/E1. Interestingly, miR-27a-3p is also involved in the regulation of milk protein synthesis-related protein expression, such as CSN2 and ELF5. Inhibition of the MEK/ERK signaling pathway by AZD6244 blocked the regulatory function of miR-27a-3p in cell proliferation and milk protein synthesis in BMECs under heat stress conditions. Our findings demonstrated that miR-27a-3p protects BMECs from heat stress-induced oxidative stress and mitochondrial damage through the MEK/ERK pathway, thereby promoting BMECs proliferation and lactation in dairy cows. The potential regulatory mechanism of miR-27a-3p in attenuating heat stress-induced apoptosis and lactation defect in BMECs.

Keywords: Cell proliferation; Lactation; MEK/ERK signaling pathway; Mitochondrial damage; miR-27a-3p.

Fig. 1

miR-27a-3p is involved in heat stress-induced apoptosis in BMECs. (A) The mRNA levels of Hsp70, Bax, Bcl2, CSN2, and ELF5 after the expression of miR-27a-3p in heat stress-treated BMECs. (B-C) The protein levels of Hsp70, Bax, and Bcl2 were determined by western blotting in BMECs after expression of miR-27a-3p. **p < 0.01; ***p < 0.001. Data are presented as mean ± SEM from at least three independent experiments

Fig. 2

miR-27a-3p attenuates heat stress-induced oxidative stress in BMECs. (A) Representative images of DCFH-DA staining in control and different treatment groups. (B) Fluorescence intensity of the ROS level was quantified in control and different treatment groups. (C) The anti-oxidative enzyme activity of SOD was measured in control and different treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM from at least three independent experiments

Fig. 3

miR-27a-3p attenuates heat stress-induced mitochondrial function defects in BMECs. (A) The ATP level was measured in control and different treatment groups. (B-C) The levels of mitochondrial fusion and fission-related proteins were determined in control and different treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM from at least three independent experiments

Fig. 4

miR-27a-3p is critical for the proliferation and activation of the MEK/ERK signaling pathway in BMECs. (A) Representative images of EdU staining in control and different treatment groups. (B) Fluorescence intensity of EdU was quantified in control and different treatment groups. (C) The mRNA level of cyclin D1 and cyclin E1 was examined in control and different treatment groups. (E–F) The phosphorylation level of MEK1/2 and ERK1/2, and MEK1/2, ERK1/2, Cyclin D1, and Cyclin E1 protein levels were examined by western blot in control and different treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM from at least three independent experiments

Fig. 5

Inhibition of MEK/ERK signaling pathway blocks the positive effect of miR-27a-3p on BMECs. (A) Representative images of EdU staining after inhibition of MEK/ERK signaling pathway under heat stress condition. (B) Fluorescence intensity of EdU was quantified in heat stress group and AZD6244 treatment group. (C-D) The phosphorylation level of MEK1/2 and ERK1/2, and MEK1/2, ERK1/2, Cyclin D1, Cyclin E1, CSN2, and ELF5 protein levels were examined by western blot in heat stress group and AZD6244 treatment group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM from at least three independent experiments