【作者】 吴昊；

【导师】 徐世文；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2023， 硕士

【摘要】 microRNA（miRNA）在维持骨骼肌稳态等方面发挥重要的生物学作用。miRNA通过抑制靶m RNA的转录后翻译,从而影响许多基因的表达,参与对机体器官发育以及功能的调控。硒（Selenium,Se）是人和动物所必需的微量元素,主要以硒蛋白的形式发挥作用,充足的硒能够保障畜禽骨骼肌的健康发育。硒蛋白T（SelT）是硒蛋白家族的重要成员之一,具有很强的氧化还原调节功能,miR-365-3p具有靶向调控SelT的功能。在肌纤维的形成过程中,成肌细胞首先进入细胞周期进行快速的增殖。但SelT在维护成肌细胞氧化还原平衡,促进细胞增殖的作用和调控机制仍缺乏研究。基于此,本研究在建立了缺硒肉鸡模型,SelT敲低/过表达和miR-365-3p敲低/过表达成肌细胞与鸡胚模型,以及H₂O₂或线粒体氧化应激抑制剂Mito-TEMPO处理成肌细胞等模型的基础上,应用H&E染色、免疫组化染色、免疫荧光染色、TUNEL染色、流式细胞术、Ed U染色、实时定量PCR、蛋白免疫印迹和试剂盒等试验方法,通过检测成肌细胞氧化应激水平、线粒体功能、线粒体生物发生能力、ATP含量、细胞周期、细胞凋亡、增殖潜力、MHC含量等,旨在阐明缺硒通过miR-365-3p/SelT轴诱导线粒体氧化应激,抑制线粒体生物发生,进而导致成肌细胞增殖障碍的作用机制。结果表明:（1）缺硒显著上调肉鸡骨骼肌组织中miR-365-3p的表达并下调SelT的水平（p<0.05）,导致鸡骨骼肌肌纤维出现断裂,纤维排列无序松散,间质多脂肪细胞,并出现炎性细胞浸润和纤维坏死,肌纤维的横截面积缩小,骨骼肌萎缩和肉鸡整体体重的显著降低（p<0.05）。miR-365-3p过表达或SelT敲低鸡胚的骨骼肌变化与体内变化相似。相反敲低miR-365-3p或过表达SelT的鸡胚骨骼肌肌纤维横截面积显著增加,肌纤维饱满充盈（p<0.05）。上述结果表明硒缺乏会通过miR-365-3p/SelT轴诱导鸡骨骼肌的损伤。（2）氧化应激、DNA氧化损伤以及凋亡水平的检测结果显示,缺硒显著增加肌肉组织中T-NOS的活性以及H₂O₂和丙二醛（MDA）的含量并降低了抗氧化酶CAT、Gpx、GSH、SOD和T-AOC的活性,上调γH2AX和8-OH DG的水平以及TUNEL阳性细胞数（p<0.05）。miR-365-3p过表达或SelT敲低的鸡胚组织和成肌细胞中得到与体内试验趋势一致的结果。使用Mito-TEMPO靶向抑制Mito SOX可有效缓解成肌细胞因SelT水平降低所引起的上述指标的变化。此外,过表达SelT有效抑制了H₂O₂处理诱导的氧化应激（p<0.05）。但当miR-365-3p mimic与SelT质粒共转染时,SelT对细胞的保护作用消失。以上结果表明缺硒通过miR-365-3p抑制SelT,诱导线粒体氧化应激、DNA损伤,促进细胞凋亡。（3）线粒体动力学基因的检测结果显示,缺硒引起骨骼肌线粒体融合分裂基因MFN1、MFN2、OPA1、DRP1、CLPP、PGC-1a、TRAM和PPAR-γ的表达下调。过表达miR-365-3p或敲低SelT引起成肌细胞中上述指标相似的变化。使用Mito-TEMPO靶向抑制Mito SOX可以显著减轻因SelT水平降低所引起的线粒体稳态紊乱（p<0.05）。H₂O₂处理后,与SelT质粒单独转染的成肌细胞相比,miR-365-3p mimic与SelT质粒共转染显著下调了上述动力学基因的表达（p<0.05）。以上结果说明,缺硒通过miR-365-3p抑制SelT,诱导成肌细胞线粒体稳态失调。（4）线粒体氧化磷酸化功能检测结果显示,缺硒显著下调骨骼肌组织中氧化磷酸化（OXPHOS）酶NDFB8、SDHB、VQCRC2、MTCO1和ATP5A1的水平以及ATP的生成（p<0.05）。过表达miR-365-3p或敲低SelT也造成了成肌细胞线粒体JC-1膜电位的丧失,并阻断细胞的氧化磷酸化进程,最终导致鸡胚肌肉组织和成肌细胞中ATP的合成显著减少（p<0.05）。Mito-TEMPO处理可有效缓解因SelT水平减少所引起的氧化磷酸化功能失调。此外,过表达SelT可显著抑制H₂O₂处理引起的上述指标的变化。但H₂O₂处理同样导致miR-365-3p mimic与SelT质粒共转染细胞中氧化磷酸化功能的失调（p<0.05）。上述结果表明,缺硒通过miR-365-3p抑制SelT,引起成肌细胞氧化磷酸化功能障碍。（5）通过对组织和细胞中MHC水平和成肌细胞细胞周期、增殖相关指标的检测,评估了成肌细胞的损伤以及增殖能力。结果显示,缺硒显著下调骨骼肌组织中MHC的水平。过表达miR-365-3p或敲低SelT也引起鸡胚肌肉组织和成肌细胞中ki67和MHC水平的显著下调,G0/G1期与G2/M期成肌细胞百分比增加,S期成肌细胞百分比减少,Ed U阳性细胞数减少,伴随着p53/p21信号通路的激活与细胞周期蛋白Cyclin A,Cyclin B,Cyclin D和Cyclin E水平的显著降低（p<0.05）。使用Mito-TEMPO靶向抑制Mito SOX可显著缓解因SelT水平降低所造成的成肌细胞周期阻滞（p<0.05）。同样,H₂O₂处理miR-365-3p mimic与SelT质粒共转染细胞得到了上述相似的结果,而H₂O₂处理SelT过表达细胞则不会引起上述指标的显著变化。上述结果表明缺硒通过miR-365-3p抑制SelT,诱导成肌细胞周期阻滞,抑制其增殖。综上所述,缺硒通过miR-365-3p靶向抑制SelT,破坏线粒体内氧化还原稳态,阻碍ATP的生成,诱导成肌细胞周期阻滞并促进细胞凋亡,最终导致肉鸡骨骼肌的损伤。缺硒通过miR-365-3p/SelT轴调控线粒体氧化应激诱导成肌细胞增殖障碍。这项研究为进一步探索SelT的功能提供了一个新的方向,也为比较医学提供参考。更多还原

【Abstract】 MicroRNA（miRNA）plays an important biological role in maintaining skeletal muscle homeostasis and other aspects.Mi RNA affects the expression of many genes and participates in the regulation of organ development and function by inhibiting the post transcriptional translation of target m RNA.Selenium（Se）is an essential trace element for humans and animals,which mainly functions in the form of selenoprotein.Adequate Se can ensure the healthy development of animal skeletal muscles.Selenoprotein T（SelT）is an important member of the selenoprotein family,which has a strong redox regulation function.miR-365-3p has the function of targeted regulation of SelT.During the formation of muscle fibers,myoblasts first enter the cell cycle for rapid proliferation.However,there is still a lack of research on the role and regulatory mechanism of SelT in maintaining the redox balance of myoblasts and promoting cell proliferation.Based on this,here,we established Se deficient broiler models,SelT knockdown/overexpression and miR-365-3p knockdown/overexpression myoblasts and chicken embryo models,as well as H₂O₂ or mitochondrial oxidative stress inhibitor Mito-TEMPO treated myoblast models.We applied H&E staining,immunohistochemistry staining,immunofluorescence staining,TUNEL staining,flow cytometry,Ed U staining,real-time quantitative PCR,protein immunoblotting,and kit testing methods,By detecting the levels of oxidative stress,mitochondrial function,mitochondrial biogenic ability,ATP content,cell cycle,apoptosis,proliferation potential,MHC content,etc.in myoblasts,the aim is to elucidate the mechanism of Se deficiency inducing mitochondrial oxidative stress through the miR-365-3p/SelT axis,inhibiting mitochondrial biogenesis,and ultimately leading to dysproliferation of myoblasts.The results indicate that:（1）Se deficiency significantly upregulated the expression of miR-365-3p in broiler skeletal muscle tissue and downregulated the level of SelT（p<0.05）.Se deficiency led to fracture of skeletal muscle fibers in chickens,disordered and loose fiber arrangement,abundant adipocytes in the stroma,and inflammatory cell infiltration and fiber necrosis.The cross-sectional area of muscle fibers decreases,skeletal muscle atrophy,and a significant decrease in overall body weight of broilers（p<0.05）.The skeletal muscle changes in miR-365-3p overexpression or SelT knockdown chicken embryos were similar to those in vivo.On the contrary,knocking down miR-365-3p or overexpressing SelT significantly increased the cross-sectional area of skeletal muscle fibers in chicken embryos,and the muscle fibers were plump and filled（p<0.05）.The above results indicate that Se deficiency can induce damage to chicken skeletal muscles through the miR-365-3p/SelT axis.（2）The detection results of oxidative stress,DNA oxidative damage,and apoptosis levels showed that Se deficiency significantly increased the content of H₂O₂ and malondialdehyde（MDA）and activity of T-NOS in muscle tissue and decreased the activity of antioxidant enzymes CAT,Gpx,GSH,SOD and T-AOC,increased the levels ofγH2AX and 8-OH DG and the number of TUNEL positive cells（p<0.05）.Results consistent with in vivo experimental trends were obtained in chicken embryo tissues and myoblasts overexpressing miR-365-3p or knocking down SelT.Targeted inhibition of Mito SOX using Mito-TEMPO could effectively alleviate the changes in the aforementioned indicators caused by the decrease in SelT levels in myoblasts.In addition,overexpression of SelT effectively inhibited oxidative stress induced by H₂O₂ treatment（p<0.05）.However,when miR-365-3p mimic was co transfected with the SelT plasmid,the protective effect of SelT on cells disappeared.The above results indicate that Se deficiency inhibits SelT through miR-365-3p,induces mitochondrial oxidative stress,DNA damage,and promotes cell apoptosis.（3）The detection results of mitochondrial dynamics genes showed that Se deficiency caused the expression of fusion and division genes MFN1,MFN2,OPA1,DRP1,CLPP,PGC-1a,TRAM,and PPAR-γin skeletal muscle mitochondria were downregulated.Overexpression of miR-365-3p or knockdown of SelT resulted in similar changes in the indicators in myoblasts.Targeted inhibition of Mito SOX using Mito-TEMPO could significantly alleviate mitochondrial homeostasis disorders caused by decreased SelT levels（p<0.05）.H₂O₂ treatment did not cause significant changes in the expression of kinetic genes,but when miR-365-3p mimic was co transfected with the SelT plasmid,H₂O₂ treatment significantly downregulated the expression of the genes（p<0.05）.The above results indicate that Se deficiency inhibits SelT through miR-365-3p and induces mitochondrial homeostasis disorders in myoblasts.（4）The results of mitochondrial oxidative phosphorylation function test showed that Se deficiency significantly reduced the levels of oxidative phosphorylation（OXPHOS）enzymes NDFB8,SDHB,VQCRC2,MTCO1 and ATP5A1,and ATP production in skeletal muscle tissues（p<0.05）.Overexpression of miR-365-3p or knockdown of SelT also caused the loss of mitochondrial JC-1 membrane potential of myoblasts,blocked the process of oxidative phosphorylation of cells,and finally led to a significant reduction of ATP synthesis in chicken embryo muscle tissue and myoblasts（p<0.05）.Mito-TEMPO treatment could effectively alleviate the oxidative phosphorylation dysfunction caused by the reduction of SelT level.After H₂O₂treatment,compared with myoblast cells transfected with SelT plasmid alone,co transfection of miR-365-3p mimic with SelT plasmid significantly downregulated the expression of the kinetic genes（p<0.05）.The above results indicate that Se deficiency inhibits SelT through miR-365-3p,resulting in dysfunction of oxidative phosphorylation in myoblasts.（5）By detecting MHC levels in tissues and cells,as well as indicators related to myoblast cell cycle and proliferation,the damage and proliferation ability of myoblasts were evaluated.The results showed that Se deficiency significantly downregulated the level of MHC in skeletal muscle tissue.Overexpression of miR-365-3p or knockdown of SelT also caused significant downregulation of ki67 and MHC levels in chicken embryo muscle tissue and myoblasts.The percentage of myoblasts in G0/G1 and G2/M phases increased,the percentage of myoblasts in S phase decreased,and the number of Ed U positive cells decreased,accompanied by activation of p53/p21 signaling pathway and significant reduction of Cyclin A,Cyclin B,Cyclin D,and Cyclin E levels（p<0.05）.Targeted inhibition of Mito SOX using Mito-TEMPO could significantly alleviate the myoblast cell cycle arrest caused by decreased SelT levels（p<0.05）.Similarly,H₂O₂ treatment of miR-365-3p mimic and SelT plasmids co transfected cells yielded similar results,while H₂O₂treatment of SelT overexpressing cells did not cause significant changes in these indicators.The above results indicate that Se deficiency inhibits SelT,induces myoblast cell cycle arrest,and inhibits its proliferation through miR-365-3p.In summary,Se deficiency inhibits SelT through miR-365-3p targeting,disrupts mitochondrial redox homeostasis,hinders ATP generation,induces myoblast cell cycle arrest,promotes cell apoptosis,and ultimately leads to damage to broiler skeletal muscles.Selenium deficiency induces mitochondrial redox oxidative stress through miR-365-3p/SelT axis,causing myoblasts proliferation disorders.This study provides a new direction for further exploring the function of SelT and also provides reference for comparative medicine.更多还原