microRNA在饲粮蛋白水平调控母猪情期启动中的作用及机制

MicroRNA is a short single-stranded RNA which are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs) resulting in target degradation or translational repression. Studies showed that the protein levels affecting progeny miRNA expression thereby affecting its physiological function. The level of protein and amino acids had effects on the progeny puberty onset in mice, but its precise mechanism is still unclear. We have a hypothesis that dietary protein levels affect miRNA expression in the reproductive axis thereby regulates puberty onset in gilts. Using gilts as model, this project will investigate the effects of protein level on the expression of miRNAs, employ high-throughput genome-wide transcriptome analytic techniques to explore the dynamic expression of miRNAs in the hypothalamus and pituitary, further utilize cell culture, plasmid transfection, dual luciferase assay and site directed mutagenesis to study the mechanism of special miRNAs regulate the Kisspeptin/GPR54/GnRH signaling pathways affect gilts' puberty onset. These would have a important theoretical significance at deeply understand the relationship between protein level and gilt puberty onset and clarify the mechanism of puberty onset, and have a great practical significance at establish nutrition programs for puberty onset and reproductive capacity of gilt lifespan.

MicroRNA是一种单链小分子RNA，在基因表达的转录后水平通过与靶基因mRNA特异性碱基配对引起mRNA降解或抑制其翻译，从而对基因表达进行调控。鼠上研究发现，蛋白水平影响后代miRNA表达进而影响其发情表现，但其精确机制不清楚。饲粮蛋白水平能否通过调节生殖轴miRNA表达进而调控情期启动，在猪上未见报道。本项目以后备母猪为对象，研究蛋白水平对后备母猪发情相关miRNA表达的影响，采用高通量测序检测miRNA在下丘脑、垂体中的表达，并进一步通过细胞培养、质粒转染、双荧光素酶分析和基因定点突变考察特定miRNA调控Kisspeptin/GPR54/GnRH信号途径影响后备母猪发情启动的机制，这对深入认识蛋白水平与后备母猪发情启动的关系及阐明发情启动的机制将具有重大的理论意义，同时对建立后备母猪的营养方案，调控母猪适时发情和终生繁殖力具有实践指导意义。

MicroRNA是一种单链小分子RNA，在基因表达的转录后水平通过与靶基因mRNA特异性碱基配对引起mRNA降解或抑制其翻译，从而对基因表达进行调控。鼠上研究发现，蛋白水平影响后代miRNA表达进而影响其发情表现，但其精确机制不清楚。饲粮蛋白水平能否通过调节生殖轴miRNA表达进而调控情期启动尚不清楚。CircRNAs是非编码RNA的一种，因为它不具有5'末端帽子和3'末端尾巴结构、并能以共价键形成闭合环，所以不易受核酸外切酶的酶切作用而表达稳定。已有研究证实，miR-17-5p具有抑制细胞及组织生长的作用，而circRNAs则可与miRNAs结合后抑制其活性，进而调控其靶基因的表达。但是circRNAs调控miR-17-5p对细胞生长发育的影响尚不清楚。本项目以鼠为对象，研究蛋白水平对雌性大鼠初情启动和情期循环的影响，并进一步通过细胞培养、质粒转染、双荧光素酶分析考察miR-17-5p与circRNAs调控细胞生长的机制。结果表明，蛋白质限饲可损伤性腺发育、卵泡成熟和E2合成，通过下丘脑ERα通路改变kisspeptin和GnRH的活化影响初情启动；4%蛋白限饲影响发情周期、激素、基因表达，造成此现象的原因主要为饲粮蛋白水平影响肝脏IGF-1合成，通过作用于下丘脑IGF-1R影响KISS-1基因表达，从而对下丘脑-垂体-性腺轴进行调控；miR-17-5p过表达降低了细胞增殖和存活，而circ-Amotl1过表达却增加了细胞增殖、存活和迁移的能力，促进细胞生长；通过RT-PCR、Western blotting、荧光素酶报告分析发现，circ-Amotl1通过提高Stat3的核转位能力以及降低miR-17-5p的表达来实现增强细胞活力。本试验的研究对深入认识蛋白水平与雌性动物发情启动的关系及阐明发情启动的机制将具有重大的理论意义。