gga-miR-138-5p靶向SelM调控鸡缺硒性肝程序性坏死机制的研究

The liver is the main target organ of selenium deficiency. Our group found that selenium deficiency could induce oxidative stress and cell necrosis in liver of chickens, while the expression of SelM was decreased and the expression of miR-138-5p was increased. SelM can regulate the antioxidant capacity and calcium homeostasis of cells. MiR-138-5p regulates cell development and differentiation, and miR-138-5p has a potential targeting relationship with SelM. To confirm the target relationship between miR-138-5p and SelM, and the role of miR-138-5p/SelM in programmed necrosis in chickens induced by selenium deficiency. We aimed to replicate the model of selenium-deficiency hepatic programmed necrosis in vivo and in vitro culture of chickens. Based on this model, qPCR, immunoblotting, luciferase reporter system, flow cytometry, and transmission electron microscopy were used to identify morphological and programmed necrosis signaling pathway genes (RIP3, MLKL, etc.), antioxidant markers, inflammatory factors (NF-κB, PTGE,etc.), mitochondrial homeostasis, and energy metabolism (HK, PK, etc.) related indicators, aiming to elucidate the mechanism of selenium-deficient hepatic programmed necrosis from the perspective of miR-138-5p/SelM. It provides basis for preventing selenium deficiency and a reference for comparative medicine.

肝脏是硒缺乏的主要靶器官，课题组研究发现，鸡硒缺乏时肝组织发生氧化应激和细胞坏死，同时SelM表达降低和miR-138-5p表达升高。SelM能够调控细胞的抗氧化和钙稳态，miR-138-5p具有调节细胞发育和分化功能，miR-138-5p与SelM具有潜在靶向关系。为证实miR-138-5p与SelM的靶向关系及miR-138-5p/SelM在鸡缺硒性肝程序性坏死中的作用，本课题拟在复制鸡缺硒性肝细胞程序性坏死模型和体外培养肝细胞的基础上，采用qPCR、免疫印迹、荧光素酶报告系统、流式细胞术、透射电镜等方法，通过形态学观察、程序性坏死信号通路基因（RIP3、MLKL等）、抗氧化指标、炎症因子（NF-κB、PTGE等）、线粒体稳态和能量代谢（HK、PK等）相关指标的检测等，从miR-138-5p/SelM角度阐明鸡缺硒性肝细胞程序性坏死的发生机制，为防治硒缺乏提供依据，为比较医学提供借鉴。

课题组研究发现，鸡硒缺乏时肝组织发生氧化应激和细胞坏死，同时SelM表达降低和miR-138-5p表达升高。SelM能够调控细胞的抗氧化和钙稳态，miR-138-5p具有调节细胞发育和分化功能，miR-138-5p与SelM具有潜在靶向关系。基于此，提出miR-138-5p靶向SelM调控鸡缺硒性肝程序性坏死的科学假设。本试验在人工复制鸡缺硒性肝脏损伤病理模型、建立miR-138-5p敲低和过表达模型以及SelM敲低LMH细胞模型的基础上，对抗氧化水平、Ca2+通路相关指标、能量代谢相关指标、程序性坏死相关指标、炎症相关指标等进行检测，结果表明：1）硒缺乏引起miR-138-5p的表达上调和SelM的表达下调，二者具有靶向调控关系。2）过表达miR-138-5p和敲低SELM降低肝脏抗氧化酶活性，自由基及氧化酶活性增加，引起氧化应激，可被NAC抑制。3）过表达miR-138-5p和敲低SelM增加RIPK1、RIPK3和MLKL的表达水平，NAC和BAPTA-AM可有效缓解SelM敲低引起的细胞程序性坏死，表明氧化应激和钙离子参与miR-138-5p/SelM导致的细胞程序性坏死。4）过表达miR-138-5p和敲低SelM降低SERCA1和增加CAMKⅡα的表达水平，引起钙离子过载，NAC和BAPTA-AM可有效缓解SelM敲低引起的钙离子失衡，表明miR-138-5p/SelM引起的细胞钙离子失衡是由氧化应激导致的。5）过表达miR-138-5p和敲低SelM上调SDHB，LDHB，HK1，HK2，PK和PFK的表达，下调PDHX的表达，引起能量代谢障碍，NAC和BAPTA-AM可有效缓解SelM敲低引起的能量代谢失衡，表明miR-138-5p/SelM引起的细胞能量代谢障碍是由氧化应激和钙离子失衡导致的。6）过表达miR-138-5p和敲低SelM增加NLRP3、IL1β、TNFα和NFκB等炎症因子的表达水平，NAC和BAPTA-AM可有效缓解SelM敲低引起的炎症因子表达上调，表明氧化应激和钙离子参与miR-138-5p/SelM导致的肝脏炎症。表明miR-138-5p/SelM通过ROS/钙超载诱导调节程序性坏死诱发肝炎，这一结果阐明了缺硒性肝炎发生的具体生物学机制，为缺硒性肝脏损伤的研究提供了理论基础，为比较医学的研究提拱了借鉴。