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| METTL3基因在奶牛乳腺上皮细胞炎症反应中的表达及功能分析 |
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王正兴1,2, 李彦龙1,2, 邢明旭1,2, 秦铭1,2, 王玉梅1,2, 王兴平1,2, 罗仍卓么1,2
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1.宁夏大学 动物科技学院，宁夏 银川 750021;2.宁夏回族自治区反刍动物分子细胞育种重点实验室，宁夏 银川 750021
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| 摘要: |
| 【 目的】探讨甲基转移酶样蛋白3（methyltransferase-like 3，METTL3）基因在奶牛乳腺上皮细胞（bovin mammary epithelial cells，bMECs）炎症反应中的表达及功能，为解析奶牛乳房炎分子调控机制奠定基础。【 方法】采用生物信息学方法分析奶牛METTL3蛋白质的结构特征；采用50 μg/mL的脂多糖（lipopolysaccharide，LPS）构建bMECs炎症模型，在此基础上通过实时荧光定量PCR（RT-qPCR）检测LPS诱导不同时间点（0，3，6，12和24 h）炎症 因子基因（IL-6和IL-8）及METTL3基因的表达水平，并利用CCK-8和RT-qPCR检测干扰METTL3基因后bMECs的细胞活力、炎症因子、细胞增殖及凋亡相关基因表达的变化。【 结果】生物信息学分析结果表明，METTL3基因的开放阅读框（ORF）长度为1 743 bp，编码一个由580个氨基酸组成的蛋白质，该蛋白存在59个潜在的翻译后修饰位点，可能通过表观转录调控机制参与细胞炎症进程。在LPS诱导bMECs的不同时间点，IL-6和IL-8表达量随诱导时间延长而升高，于24 h达到表达峰值（P<0.01），表明成功建立了bMECs炎症模型。与0 h相比，METTL3在LPS刺激3，6，12和24 h的表达水平均极显著上调（P<0.01），其中6 h表达量最高，呈现炎症早期响应特征。在干扰METTL3表达后，bMECs内促炎因子IL-6的表达水平极显著下调（P<0.01），IL-8显著下调（P<0.05），IL-1β亦呈现下降趋势，细 胞增殖基因CDK2、CDK4和PCNA的表达水平均极显著升高（P<0. 01），而细胞凋亡基因CASP3、CASP9和BAX的表达水平均极显著降低（P<0.01）。干扰METTL3后，bMECs细胞活力显著升高（P<0.05）。【 结论】METTL3通过加剧炎症反应、促进细胞凋亡并抑制细胞增殖而加重LPS诱导的bMECs炎症损伤，提示METTL3是调控奶牛乳腺炎 进程的关键分子。 |
| 关键词: 奶牛 奶牛乳腺上皮细胞 炎症反应 METTL3 |
| DOI：10. 13207/j. jnwafu. 2026. 08. 002 |
| 分类号: |
| 基金项目:国家自然科学基金项目（32460826）；国家现代农业产业技术体系项目（CARS-36）；宁夏自然科学基金项目（2024AAC03116） |
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| Analysis on the expression and function of METTL3 gene in the inflammatory response of bovine mammary epithelial cells |
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WANG Zhengxing1,2, LI Yanlong1,2, XING Mingxu1,2, QIN Ming1,2, WANG Yumei1,2, WANG Xingping1,2, LUORENG Zhuoma1,2
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1.School of Animal Science and Technology，Ningxia University，Yinchuan，Ningxia 750021，China;2.Key Laboratory of Molecular Cell Breeding of Ruminants，Ningxia Hui Autonomous Region，Yinchuan，Ningxia 750021，China
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| Abstract: |
| 【 Objective】This study aimed to investigate the expression and function of methyltransferase-like 3（METTL3） in the inflammatory response of bovine mammary epithelial cells （bMECs），so as to lay a foundation for elucidating the molecular regulatory mechanisms of bovine mastitis.【Method】The structure cha-racteristics of the bovine METTL3 protein were analyzed by using bioinformatics methods. An inflammatory model of bMECs was established based on 50 μg/mL LPS treatment. On this basis，RT-qPCR was used to de?tect the expression levels of inflammatory factors （IL-6 and IL-8） and the METTL3 gene at different time points （0，3，6，12，and 24 h） after LPS induction. CCK-8 assay and RT-qPCR were employed to examine cell viability，the expression of inflammatory factors，and the expression of cell proliferation and apoptosis-related genes after METTL3 knockdown.【Result】Bioinformatics analysis results indicated that the open reading frame （ORF） of the METTL3 gene was 1 743 bp in length，encoding a protein of 580 amino acids. This protein con?tained 59 potential post-translational modification sites and might be involved in cellular inflammatory processes through epitranscriptional regulatory mechanisms. At different time points after LPS induction，the expression levels of IL-6 and IL-8 continued to increase over time，peaking at 24 h （P<0. 01），indicating the successful es-tablishment of an inflammatory model of bMECs. The expression level of METTL3 was highly significantly up-regulated at 3，6，12，and 24 h after LPS stimulation （P<0.01） compared with that at 0 h，with the highest ex-pression level observed at 6 h，demonstrating the characteristic of early inflammatory response. After METTL3 knockdown，the expression level of the pro-inflammatory factor IL-6 was highly significantly downregulated （P<0.01），IL-8 was significantly downregulated （P<0. 05），and IL-1β also showed a downward trend. The expression levels of cell proliferation-related genes CDK2，CDK4，and PCNA were highly significantly in?creased （P<0. 01），while the expression levels of apoptosis-related genes CASP3，CASP9，and BAX were highly significantly decreased （P<0. 01）. In addition，cell viability was significantly increased after METTL3 knockdown （P<0.05）.【Conclusion】METTL3 aggravates LPS-induced inflammatory injury in bMECs by ex?acerbating the inflammatory response，promoting cellular apoptosis，and inhibiting cell proliferation. This finding identifies METTL3 as a key regulator in the progression of bovine mastitis. |
| Key words: dairy cows bovin mammary epithelial cells inflammatory response METTL3 |
