miR-145-4调控蛋鸭卵泡发育机制的研究

Study on the Mechanism of miR-145-4 Regulating Follicle Development in Egg Ducks

【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.