Xia Siqi, Fan Huimei, Xiao Jianghai, Shen Ci, Yan Yongping, Wang Meigui, Tang Tao, Sun Wenqiang, Wang Jie, Jia Xianbo, Lai Songjia
State Key Laboratory of Swine and Poultry Breeding Industrycollege of Animal Science and Technology, Sichuan Agricultural University, Ya'an, People's Republic of China.
Key Laboratory of Livestock and Poultry Multi-Omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, People's Republic of China.
BMC Genomics. 2025 May 12;26(1):470. doi: 10.1186/s12864-025-11567-0.
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.
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.
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.
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.
全球变暖加剧了热应激，这已成为奶牛健康和牛奶生产质量的重大问题。体外研究表明，原代牛乳腺上皮细胞在环境温度升高时程序性细胞死亡水平增强，这可能导致乳腺内乳腺上皮细胞总数减少，从而部分解释了热应激下泌乳奶牛产奶量下降的原因。在体内，热应激通过直接作用于乳腺上皮细胞以及改变激素水平和代谢途径来影响乳汁的合成和分泌，这可能对乳腺生长产生长期影响。未来的研究应侧重于阐明热应激调节乳腺发育的分子机制。先前的研究表明，热应激诱导导致MAC-T细胞中miR-223显著下调；因此，miR-223可能在热应激反应中起关键作用。然而，miR-223赋予MAC-T细胞耐热性的机制仍不清楚。
在此，为了研究miR-223如何调节MAC-T细胞的增殖，我们结合了miRNA-223过表达和抑制策略。我们用miR-223模拟物或抑制剂转染MAC-T细胞，并使用CCK-8测定、EdU测定和RT-qPCR评估对细胞增殖的影响。此外，使用遭受热应激的MAC-T细胞来研究miR-223及其靶基因如何在热应激下调节细胞增殖，通过EdU测定、CCK-8测定和RT-qPCR评估，是促进还是减轻对细胞增殖的抑制。
在本研究中，我们研究了热应激对MAC-T细胞增殖和基因表达的影响。生物信息学分析确定PRDM1是增殖的关键调节因子，并选择其进行进一步研究。RT-qPCR验证了热应激下PRDM1的上调，证实了其在调节细胞增殖中的作用。结果显示，miR-223模拟物促进细胞增殖，PRDM1被确定为其靶基因。重要的是，热应激后，MAC-T中miR-223模拟物或PRDM1的敲低被证明可部分逆转对增殖的抑制。
因此，靶向PRDM1的miR-223可能在减轻热应激诱导的细胞增殖抑制方面很重要。这可能会减轻热应激对乳腺的损伤，从而提高奶牛的产奶量。
