CircRNA作为ceRNA参与miR-423-5p调控猪骨骼肌纤维发育的机制研究

猪肉品质与肌纤维类型密切相关，瘦肉型长白猪与脂肪型蓝塘猪肌纤维类型存在明显差异。前期结合长白猪和蓝塘猪背最长肌mRNAome、circRNAome及miRNAome测序数据，通过整合基因组学的方法初步构建猪肌发育调控网络，发现存在5个候选circRNAs可能作为竞争性内源RNA参与miR-423-5p上游调控。因此，本项目拟首先通过克隆测序、FISH、核质分离RT-qPCR技术明确候选circRNA是否真实成环、亚细胞定位及出核规律；利用荧光素酶报告基因、ChiRP及RIP技术验证miR-423-5p与候选circRNA的靶向关系；以猪骨骼肌卫星细胞为材料，过表达或干扰候选circRNA并设计miR-423-5p拯救实验，确定候选circRNA作为ceRNA所调控的网络构成及影响猪肌纤维发育的分子机制，这不仅对阐明肌肉发育这一生命现象具有重要意义，还可以为优质肉猪选育提供理论依据。

The pork quality traits are closely related to muscle fiber characteristics, and significant differences of myofiber types have been observed between Landrace that represented for the lean-type western pig breed and a typical Chinese indigenous obese-type breed termed as Lantang. In the past, integration analysis of mRNAome, circRNAome and miRNAome data of porcine longissimus muscle between Landrace and Lantang piglets showed 5 circRNA candidates can be as competitive endogenous RNAs to mediate miR-423-5p expression in myogenesis. Based on the previous researches, we will validate the reality, intracellular location and expression of circRNA candidates using gene cloning by T vector, RT-qPCR and Fluorescence in situ hybridization (FISH). In addition, the target relationships between miR-423-5p and circRNA candidates will be further validated by luciferase reporter gene assays, Chromatin Isolation by RNA Purification (ChiRP) and RNA Binding Protein Immunoprecipitation (RIP), respectively. Next, the ceRNA network and mechanism of muscle growth and development regulated by circRNA candidates will be determined by transfection of over-expression or siRNA recombinant vector. Overall, our study will clarify the regulatory mechanisms of muscle growth and development, and also lead to a new theoretical basis for pig breeding and pork quality improvement.