基于ceRNA网络研究miR-143-3p调控奶山羊乳腺上皮细胞凋亡的分子机制

Our previous studies found that miR-143-3p was high expressed in dairy goat mammary gland tissues at late lactation, and which can promote the apoptosis of mammary epithelial cells cultured in vitro, but the direct target genes of miR-143-3p and the signal pathways involved have not yet been determined. lncRNA can interact with miRNA as a competitive endogenous RNA (ceRNA), and involved in the expression regulation of target genes, but the lncRNA in the regulation of mammary epithelial cell apoptosis mediated by miR-143-3p remains unclear.. Based on the model of mammary epithelial cells cultured in vitro, this project will design an over-expression group of miR-143-3p and a control group for lncRNA-sequencing and RIP-sequencing, to screen and identify the lncRNA and target gene regulated by miR-143-3p via dual luciferase reporter gene assay system, Co-IP, qRT-PCR and western blot combining with bioinformatics analysis, to specify the ceRNA network of miR-143-3p regulating apoptosis of mammary epithelial cells, and to explore the molecular signaling pathway involved by miR-143-3p in mammary epithelial cell apoptosis further through GO annotation and KEGG pathway analysis combining with RNAi, qRT-PCR, western blot, MTT and flow cytometry. Finally, the ceRNA network and signaling pathway involved by miR-143-3p in mammary epithelial cell apoptosis in dairy goat are elucidated.

申请人前期研究发现，miR-143-3p在奶山羊泌乳末期乳腺组织中高表达，且能促进原代乳腺上皮细胞凋亡，但其直接作用的靶基因及参与的信号通路尚未确定。lncRNA能作为一种竞争性内源RNA (ceRNA) 与miRNA相互作用，参与对靶基因的表达调控，但在miR-143-3p介导的奶山羊乳腺上皮细胞凋亡中可能参与调控的lncRNA尚不清楚。. 本项目拟以奶山羊原代乳腺上皮细胞为模型，设计miR-143-3p过表达组和对照组，基于lncRNA-Seq和RIP-Seq，利用双荧光素酶报告基因、免疫共沉淀、qRT-PCR、western blot等方法，鉴定受miR-143-3p介导调控的lncRNA及靶基因，通过GO和KEGG分析，结合RNAi、western blot、流式细胞术等试验验证，阐明miR-143-3p调控奶山羊乳腺上皮细胞凋亡的ceRNA网络及分子机制。

本研究基于前期发现，miR-143-3p在奶山羊泌乳末期乳腺组织中高表达，且能促进体外培养的乳腺上皮细胞凋亡。但miR-143-3p直接作用的靶基因及参与的信号通路尚未明确。lncRNA能作为一种竞争性内源RNA (ceRNA) 与miRNA相互作用，参与对靶基因的表达调控，而miR-143-3p介导的参与调控奶山羊乳腺上皮细胞活性的lncRNA尚不清楚。. 本课题以体外培养的奶山羊原代乳腺上皮细胞为实验材料，设计miR-143-3p过表达组和对照组，基于lncRNA-Seq和mRNA-Seq，获得受miR-143-3p调控的差异表达lncRAs和mRNAs，结合生物信息学分析，构建了miR-143-3p参与的ceRNAs调控网络。同时，利用双荧光素酶报告基因系统、qRT-PCR、western blot等方法，鉴定ceRNAs网络中受miR-143-3p介导调控的lncRNA及靶向mRNAs。对获得的目的关键基因，通过MTT、FCM、qRT-PCR、western blot等方法，进一步分析了其对乳腺上皮细胞的效应。. 本研究通过构建miR-143参与的ceRNAs调控网络，从lncRNA、mRNA和miRNA层面解析了miR-143-3p调控奶山羊乳腺上皮细胞活性的作用，其对进一步深入研究miR-143-3p对奶山羊乳腺发育与泌乳的调控提供理论支持，对非编码RNA在奶山羊乳腺发育与泌乳的调控功能研究提供一定理论参考。