MiR- 223 alleviates the heat-stress-induced inhibition of cell proliferation by targeting PRDM1.

BACKGROUND

Heat stress, exacerbated by global warming, has emerged as a significant concern for both the health of dairy cattle and the quality of milk production. In vitro investigations suggest that primary bovine mammary epithelial cells exhibit enhanced levels of programmed cell death when subjected to elevated ambient temperatures, potentially resulting in a reduction in the total number of mammary epithelial cells within the mammary gland, thereby partially elucidating the diminished milk yield in lactating cows under heat stress. In vivo, heat stress affects both milk synthesis and secretion by directly acting on mammary epithelial cells and by altering hormonal levels and metabolic pathways, which can lead to long-term effects on mammary growth. Future research should focus on elucidating the molecular mechanisms by which heat stress regulates mammary development. Previous studies have demonstrated that heat stress induction results in a significant downregulation of miR- 223 in MAC-T cells; therefore, miR- 223 may play a crucial role in the response to heat stress. Nevertheless, the mechanism by which miR- 223 confers resistance to heat stress in MAC-T remains unclear.

METHODS

Here, to investigate how miR- 223 regulates the proliferation of MAC-T cells, we performed a combination of miRNA- 223 overexpression and inhibition strategies. We transfected MAC-T cells with miR- 223 mimics or inhibitors and evaluated the impact on cell proliferation using CCK- 8 assay, EdU assay, and RT-qPCR. Additionally, MAC-T cells subjected to heat stress were used to investigate how miR- 223 and its target gene regulate cell proliferation under heat stress, either by promoting or alleviating the inhibition of cell proliferation, as assessed by EdU assay, CCK- 8 assay, and RT-qPCR.

RESULTS

In this study, we investigated the effects of heat stress on MAC-T cell proliferation and gene expression. Bioinformatics analysis identified PRDM1 as a key regulator of proliferation, and it was selected for further investigation. RT-qPCR validated the upregulation of PRDM1 under heat stress, confirming its role in regulating cell proliferation. The results revealed that miR- 223 mimic promoted cell proliferation, with PRDM1 identified as its target gene. Importantly, after heat stress, the miR- 223 mimic or the knockdown of PRDM1 in MAC-T was proven to partially reverse the inhibition of proliferation.

CONCLUSION

Consequently, the miR- 223 targeting PRDM1 might be important in alleviating heat-stress-induced inhibition of cell proliferation. This would potentially alleviate heat stress-induced damage to the mammary gland, thereby improving milk production in dairy cows.