Chen Zhi, Xu Xin, Tan Taoling, Chen Daijie, Liang Huanjie, Sun Kaidi, Li Mingxun, Zhang Huimin, Mao Yongjiang, Yang Zhangping
College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
Joint International Research Laboratory of Agriculture & Agri-Product Safety, Ministry of Education, Yangzhou University, Yangzhou, China.
Reprod Domest Anim. 2019 Jun;54(6):882-891. doi: 10.1111/rda.13438. Epub 2019 May 3.
Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus-induced mastitis. In the present study, we overexpressed and suppressed miR-145 to investigate the function of miR-145 in Mac-T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac-T cytokines and in cell proliferation. We found that overexpression of miR-145 in Mac-T cells significantly reduced the secretion of IL-12 and TNF-α, but increased the secretion of IFN-γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real-time PCR (qRT-PCR), Western blotting and luciferase multiplex verification techniques, we found that miR-145 targeted and regulated FSCN1. Knock-down of FSCN1 significantly increased the secretion of IL-12, while the secretion of TNF-α was significantly downregulated in Mac-T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta-miR-145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.
奶牛乳腺炎是影响牛奶质量和食品安全的一个有害因素。乳腺炎通常是指由致病微生物感染引起的炎症。我们近年来的研究揭示了miRNA调控在金黄色葡萄球菌诱导的乳腺炎中的作用。在本研究中,我们过表达和抑制miR-145以研究miR-145在Mac-T细胞中的功能。采用流式细胞术、酶联免疫吸附测定(ELISA)和EdU染色来检测几种Mac-T细胞因子分泌的变化以及细胞增殖情况。我们发现,在Mac-T细胞中过表达miR-145可显著降低白细胞介素-12(IL-12)和肿瘤坏死因子-α(TNF-α)的分泌,但增加γ-干扰素(IFN-γ)的分泌;同时牛乳腺上皮细胞的增殖也受到抑制。通过定量实时聚合酶链反应(qRT-PCR)、蛋白质免疫印迹法和荧光素酶多重验证技术,我们发现miR-145靶向并调控丝状肌动蛋白1(FSCN1)。敲低FSCN1可显著增加IL-12的分泌,而在Mac-T细胞中TNF-α的分泌则显著下调。乳腺组织受到金黄色葡萄球菌感染后,机体启动炎症反应;牛miR-145(Bta-miR-145)表达下调,从而降低了对FSCN1基因的抑制作用;FSCN1表达上调促进乳腺上皮细胞增殖,以使受损组织得以恢复。本研究结果将有助于理解奶牛抵抗金黄色葡萄球菌感染的免疫机制,并为乳腺炎的防治提供实验室研究依据。