摘要
实验室前期通过RNA测序发现1个新的环状RNA分子circINHBA,并确定其在猪健康和早期闭锁卵泡中呈现显著差异表达。本研究旨在鉴定并分析circINHBA的表达模式,探索其在猪卵泡颗粒细胞(GCs)凋亡中的作用,为揭示调控猪卵泡闭锁的机制、筛选调控猪卵泡闭锁和GCs凋亡的靶标调节物提供依据。根据circINHBA,设计跨反向剪切位点的发散引物,通过PCR、琼脂糖凝胶电泳、Sanger测序鉴定circINHBA的环状结构,并通过使用或不使用Rnase R消化的实时荧光定量PCR(RT-qPCR)进行稳定性检验;利用核质分离技术和荧光原位杂交技术(FISH)分析猪卵泡GCs中circINHBA的亚细胞定位;RT-qPCR检测circINHBA在猪健康和早期闭锁卵泡中的表达情况,通过circINHBA干扰试验分析其对凋亡标志基因BCL2和BAX表达的影响;利用流式细胞术分析circINHBA对猪卵泡GCs凋亡的影响。结果:成功验证circINHBA为一种新的环状RNA分子,具有稳定的环状结构;定量分析发现,circINHBA在健康卵泡中的表达量显著高于早期闭锁卵泡,且亚细胞定位结果表明其主要分布于细胞质;特异性si-RNA干扰试验及凋亡标志基因检测发现circINHBA具有抑制猪卵泡GCs凋亡的作用。综上,本研究证实了1个全新circINHBA分子对猪卵泡GCs凋亡的影响,揭示了其在猪卵泡闭锁过程中的潜在作用,为进一步探究环状RNA分子在猪卵泡闭锁过程中的调控作用及分子机制奠定了基础。
A new circular RNA,circINHBA,was identified by RNA sequencing in a previous study of our laboratory,and it presented significant differential expression between healthy and early atretic follicles in pigs.The aim of this study was to identify and analyze the expression pattern of circINHBA and to determine its role in apoptosis of porcine follicular granulosa cells(GCs),so as to provide a basis for unravelling the regulatory mechanism of porcine follicular atresia,and for screening the targets to regulate porcine follicular atresia and apoptosis of GCs.The divergent primers across the reverse shear site were designed,and the ring structure of circINHBA was identified by PCR,agarose gel electrophoresis,and Sanger sequencing.The stability of circINHBA was tested by qPCR with or without RNase R digestion.The subcellular localisation of circINHBA in porcine follicular GCs was analyzed using nucleoplasmic separation and fluorescence in situ hybridisation(FISH).The expression of circINHBA in healthy and early atretic follicles of pigs was detected by qPCR,and its effect on the expression of apoptosis marker genes BCL2 and BAX was analyzed by circINHBA interference assay.Finally,the effect of circINHBA on the apoptosis of porcine follicular GCs was analyzed by flow cytometry.The result was that we identified a new circular RNA molecule,circINHBA,and successfully validated its circular structure and confirmed its stability.The quantitative analysis revealed that the expression of circINHBA was significantly higher in healthy follicles than that in early atretic follicles,and that subcellular localization showed that it was mainly distributed in the cytoplasm.The specific si-RNA interference assay and apoptosis marker gene detection showed that circINHBA inhibited apoptosis in porcine follicular GCs.In conclusion,our study confirmed the effect of a novel circINHBA molecule on the apoptosis of porcine follicular GCs,revealed its potential role in porcine follicular atresia,and laid the foundation for further investigation
作者
王彩霞
秦鑫鑫
李文洁
程晓龙
张瑀格
张金璧
潘增祥
WANG Caixia;QIN Xinxin;LI Wenjie;CHENG Xiaolong;ZHANG Yuge;ZHANG Jinbi;PAN Zengxiang(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)
出处
《畜牧与兽医》
CAS
北大核心
2024年第10期14-20,共7页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金项目(31672421,32002175)。
