摘要
【目的】本研究旨在揭示miR-145-4在蛋鸭卵泡发育中的作用及调控机制。【方法】分离培养蛋鸭卵泡颗粒细胞,转染miR-145-4相似物(mimic)、抑制物(inhibitor)后,利用实时荧光定量PCR法检测细胞增殖凋亡相关基因的表达情况,采用RNAhybrid和Targetscan程序预测miR-145-4的靶基因及其与靶基因磷脂酰肌醇-3-激酶催化亚单位α(phosphatidylinositol-3-kinase catalytic subunitα,PIK3CA)3′-UTR的结合位点,分别构建PIK3CA基因3′-UTR的野生型和突变型双荧光素酶载体,与miR-145-4 mimic和mimic-NC共转染蛋鸭卵泡颗粒细胞后,采用双荧光素酶报告系统验证miR-145-4与靶基因PIK3CA的结合关系,最后采用实时荧光定量PCR法检测miR-145-4对蛋鸭卵泡颗粒细胞中PIK3CA基因表达的影响。【结果】实时荧光定量PCR结果显示,与对照组相比,过表达miR-145-4后CyclinB2基因的表达量极显著降低(P<0.01),BCL2基因的表达量极显著增加(P<0.01);抑制miR-145-4后,CyclinB2基因表达量极显著升高(P<0.01),BCL2基因表达量极显著降低(P<0.01)。预测结果显示,miR-145-4与PIK3CA基因3′-UTR存在结合位点。PCR扩增和测序结果显示,PIK3CA基因3′-UTR的野生型和突变型载体构建成功。双荧光素酶检测结果显示,与突变型及空载体共转染组相比,野生型组双荧光素酶活性显著降低(P<0.05),说明miR-145-4能够与PIK3CA基因3′-UTR结合。实时荧光定量PCR结果表明,在蛋鸭卵泡颗粒细胞中过表达miR-145-4后,PIK3CA基因表达量显著降低(P<0.05),而抑制miR-145-4后,PIK3CA基因表达量极显著升高(P<0.01)。【结论】miR-145-4通过正向调控靶基因PIK3CA促进蛋鸭卵泡颗粒细胞的凋亡,进而调控蛋鸭的卵泡发育。
【Objective】The purpose of this study was to reveal the role and regulatory mechanism of miR-145-4 in follicular development for egg duck.【Method】The egg duck follicular granulosa cells were isolated and cultured.After miR-145-4 mimic and inhibitor were transfected,the expression of cell proliferation and apoptosis related genes were detected by Real-time quantitative PCR.RNAhybrid and Targetscan programs were used to predict the target gene and predict the potential binding sites of miR-145-4 to the 3′-UTR of phosphatidylinositol-3-kinase catalytic subunitα(PIK3CA)gene.The wild type and mutant type dual-luciferase reporter vector of PIK3CA gene 3′-UTR were constructed and co-transfeacted with miR-145-4 mimic and mimic-NC into duck granulose cells.The binding relationship between miR-145-4 and target gene PIK3CA was verified by double luciferase reporting system.And the effect of miR-145-4 on the expression of PIK3CA in granulosa cells of egg ducks was detected by Real-time quantitative PCR.【Result】The results of Real-time quantitative PCR showed that after over expression of miR-145-4,compared with control group,the expression of CyclinB2 gene was extremely significantly decreased(P<0.01),and the expression of BCL2 gene was extremely significantly increased(P<0.01).After inhibiting miR-145-4,compared with control group,the expression of CyclinB2 gene was extremely significantly increased(P<0.01),and the expression of BCL2 was extremely significantly decreased(P<0.01).The predict results showed that miR-145-4 could be binding to PIK3CA gene 3′-UTR.PCR amplification and sequencing results indicated that the wild type and mutant type vectors of PIK3CA gene 3′-UTR were successfully constructed.The results of double luciferase assay showed that the double luciferase activity of wild-type group was significantly lower than that of mutant and empty vector co-transfection groups(P<0.05),indicating that miR-145-4 could bind to PIK3CA gene 3′-UTR.The results of Real-time quantitative PCR showed that the expression of PIK3CA gene decreased significantly after overexpression of miR-145-4 in egg duck follicular granulosa cells(P<0.05),while the expression of PIK3CA gene increased extremely significantly after inhibition of miR-145-4(P<0.01).【Conclusion】miR-145-4 promoted the apoptosis of follicle granulosa cells by positively regulating the target gene PIK3CA,and then regulated the follicular development of egg ducks.
作者
吴艳
皮劲松
张昊
梁振华
杜金平
潘爱銮
申杰
蒲跃进
WU Yan;PI Jinsong;ZHANG Hao;LIANG Zhenhua;DU Jinping;PAN Ailuan;SHEN Jie;PU Yuejin(Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Science,Wuhan 430064,China;Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Wuhan 430064,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第3期809-816,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(32072709)
湖北省自然科学基金(2020CFB655)
湖北省重点研发计划(2020BBA034)
湖北省技术创新专项(2019ABA084)
财政部和农业农村部:国家现代农业产业技术体系资助(CARS-42)
湖北省农业科学院领军人才项目(L2018017)
湖北省农业科技创新中心项目(2016-620-000-001-023)。