miRNA-1和miRNA-133在鸭骨骼肌发育中的表达及功能初步研究

发布时间：2018-07-03 16:42

本文选题：鸭 + miRNA-1　；参考：《扬州大学》2017年硕士论文

【摘要】：microRNAs(miRNAs)是一类非编码RNA,短单链,长度约22 bp,且在进化过程中高度保守。miRNAs在单细胞和多细胞生物中广泛存在,在转录后水平对基因表达进行调控。现有研究表明miRNA-1和miRNA-133来源于同一对双顺反子(Bicistronic pairs),并对骨骼肌的发生发育有重要的调控作用,而关于鸭miRNA-1和miRNA-133对骨骼肌发育的研究还尚未见有报道。本实验以体型大小不同的樱桃谷鸭和莆田黑鸭(白羽系)作为实验对象,通过实时荧光定量技术构建鸭miRNA-1和miRNA-133的组织表达谱和发育性表达谱;并通过转染miRNA的模拟物或抑制物,采用双荧光素酶报告基因系统对鸭miRNA-1和miRNA-133的功能进行初步探讨,以了解其在鸭骨骼肌发育中的调控作用。主要研究结果如下:1.为探明樱桃谷鸭和莆田黑鸭(白羽系)的肌肉生长发育与肌纤维发育规律,分别对38、42、45、49、56等不同日龄胸肌重、腿肌重、肌纤维面积等表型进行了测定。结果显示,樱桃谷鸭42日龄胸肌重达205 g,腿肌重达238 g,其肌纤维面积分别是5835 μm2和12406μm2,而莆田黑鸭(白羽系)42日龄胸肌重仅为129g,腿肌重175g,其肌纤维面积分别是926 μm2和4089 μm2,表明樱桃谷鸭与莆田黑鸭(白羽系)在胸肌、腿肌发育和肌纤维发育等方面差异显著。2.为构建鸭miRNA-1和miRNA-133的组织表达谱和发育性表达谱,本文通过实时荧光定量检测樱桃谷鸭和莆田黑鸭(白羽系)的miRNA-1和miRNA-133表达量。检测结果显示,miRNA-1和miRNA-133在心肌、胸肌和腿肌等肌肉中呈特异性表达。同时检测胚胎期和早期生长发育过程中的肌肉组织miRNA的表达量,发现鸭胸肌和腿肌miRNA-1和miRNA-133的表达呈现出类似的变化趋势,即在胚胎期后期miRNA表达急剧上升,而在整个生长发育期miRNA的表达基本恒定。樱桃谷鸭中表达量分别在胚胎期28天和生长期42天达到峰值,且在这两个时间点,樱桃谷鸭的表达量显著高于莆田黑鸭(P0.05)。3.为探明鸭miRNA-1和miRNA-133对骨骼肌发育的作用,将miRNA-1 mimic或inhibitor和miRNA-133 mimic或inhibitor转染至鸭成肌细胞,实时荧光定量检测结果显示,miRNA mimic或inhibitor可有效促进或抑制其表达;且过表达miRNA-1可促进相邻成肌细胞相互融合,而过表达miRNA-133细胞融合现象很少;CCK-8细胞增殖检测结果表明,降低miRNA-1表达可促进成肌细胞增殖,而降低miRNA-133则可抑制成肌细胞增殖;成肌细胞分化标记标志基因表达检测结果显示,转染miRNA-1 mimic,分化标记标志基因MEF2d、Myod表达量显著地上升,而转染miRNA-133 inhibitor,MEF2d、Myod基因的表达量显著地上升。以上结果充分说明miRNA-1可促进鸭成肌细胞分化,miRNA-133可促进成肌细胞增殖。4.为进一步阐明miRNA-1和miRNA-133在鸭骨骼肌发育中的作用机制,通过文献检索和生物信息学预测,确定miRNA-1和miRNA-133的候选靶基因。实时荧光定量检测转染miRNA的模拟物和抑制物之后候选靶基因的表达量,结果显示miRNA-1可显著降低其候选靶基因HDAC4的表达(P0.01),miRNA-133也可降低其候选靶基因SRF、TGFBR1的表达(P0.01)。双荧光素酶报告系统进一步检测结果表明,miRNA-1可抑制pGL-Basic-HDAC4荧光素酶报告基因活性;而miRNA-133并未降低pGL-Basic-SRF,pGL-Basic-TGFBR1荧光表达强度。因此,鸭miRNA-1可通过靶向HDAC4促进鸭成肌细胞分化;而miRNA-133可影响SRF、TGFBR1的表达,并促进鸭成肌细胞增殖。
[Abstract]:MicroRNAs (miRNAs) is a class of non coded RNA, short single chain, length about 22 BP, and highly conservative.MiRNAs is widely distributed in single and multicellular organisms during evolution and regulates gene expression at post transcriptional level. Existing studies show that miRNA-1 and miRNA-133 originate from the same pair of CIS counter (Bicistronic pairs) and to skeletal muscle. The development of miRNA-1 and miRNA-133 on skeletal muscle development has not yet been reported. In this experiment, the tissue expression profiles and development of duck miRNA-1 and miRNA-133 were constructed by real time fluorescence quantitative technique with different sizes of Cherry Valley ducks and Putian black ducks (white feather system). The function of duck miRNA-1 and miRNA-133 was preliminarily discussed by the double luciferase reporter gene system by transfection of miRNA mimics or inhibitors. The main results were as follows: 1. the development and development of the muscle and development of Cherry Valley Duck and Putian black duck (white feather) were the main results. The development regularity of muscle fiber was measured on the chest muscle weight, leg muscle weight and muscle fiber area of 38,42,45,49,56, respectively. The results showed that the 42 day old breast muscle of Cherry Valley ducks was 205 g and the leg muscles weighed 238 g, and the muscle fiber area was 5835 m2 and 12406 Mu M2 respectively, while the 42 day old chest muscle weight of Putian black duck (white feather system) was only 129G, leg muscles. The muscle fiber area of 175g was 926 Mu m2 and 4089 M2 respectively, indicating that cherry valley duck and Putian black duck (white feather system) were significantly different in the breast muscle, leg muscle development and muscle fiber development..2. was the tissue expression spectrum and developmental expression profile of miRNA-1 and miRNA-133 in ducks, and the real time fluorescence quantitative detection of Cherry Valley Duck and Putian black duck ( The expression of miRNA-1 and miRNA-133 in the white feather system. The results showed that miRNA-1 and miRNA-133 were specifically expressed in the muscles of the myocardium, the chest muscle and the leg muscles. Meanwhile, the expression of miRNA in the muscle tissue of the embryo and early growth and development was detected, and the expression of miRNA-1 and miRNA-133 in the breast and leg muscles of the duck showed a similar change. The expression of miRNA increased rapidly at the end of the embryo period, while the expression of miRNA in the whole growth period was basically constant. The expression of Cherry Valley ducks reached a peak at 28 days in the embryo period and 42 days in the growth period, and the expression of Cherry Valley ducks was significantly higher than that of Putian black duck (P0.05).3. as the miRNA-1 and miRNA-133 of the ducks. The role of skeletal muscle development is to transfect miRNA-1 mimic or inhibitor and miRNA-133 mimic or inhibitor into duck myoblasts. Real-time fluorescence quantitative detection results show that miRNA mimic or inhibitor can effectively promote or inhibit its expression; and overexpression miRNA-1 can promote adjacent myoblasts fusion, and overexpression miRNA-133 cell fusion now The results of CCK-8 cell proliferation detection showed that the reduction of miRNA-1 expression could promote the proliferation of myoblast, while the reduction of miRNA-133 could inhibit the proliferation of myoblast. The detection results of the marker gene expression of myoblast differentiation showed that miRNA-1 mimic, differentiation marker gene MEF2d, Myod expression increased significantly, and miRNA-13 was transfected to miRNA-13. The expression of 3 inhibitor, MEF2d and Myod genes increased significantly. The above results fully indicate that miRNA-1 can promote the differentiation of duck adult myoblast. MiRNA-133 can promote the proliferation of myoblast by.4. to further clarify the mechanism of miRNA-1 and miRNA-133 in the development of skeletal muscle in ducks. By literature retrieval and bioinformatics prediction, miRNA-1 and miRNA- are determined. 133 candidate target gene. Real-time fluorescence quantitative detection of the expression of candidate target gene after transfection of miRNA mimics and inhibitors. The results show that miRNA-1 can significantly reduce the expression of the candidate target gene HDAC4 (P0.01), miRNA-133 also reduces the candidate target gene SRF, TGFBR1's apparent (P0.01). Double luciferase reporter system is further detected. The results showed that miRNA-1 could inhibit the activity of pGL-Basic-HDAC4 luciferase reporter gene, while miRNA-133 did not reduce pGL-Basic-SRF and pGL-Basic-TGFBR1 fluorescence intensity. Therefore, ducks miRNA-1 can promote the differentiation of duck myoblasts by targeting HDAC4, and miRNA-133 can affect the expression of SRF, TGFBR1, and promote the proliferation of duck myoblasts.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S834

【参考文献】