摘要
【目的】探明miRNA-18b和HIF-1α之间的关系。【方法】以NCSU-23胚胎培养液为对照组,Ber加入NCSU-23培养液中为Ber组,在对照组的原核胚细胞质中显微注射10pLmiRNA-18b过表达剂(agomir)、低表达剂(antagomir)和阴性片段分别为miRNA-18bagomir组、miRNA-18bantagomir组和阴性片段对照(NC)组。观察IVF胚胎2-、4-、8-细胞和桑椹胚、囊胚发育率;通过qRT-PCR、Western-blot和双荧光素酶报告基因的方法验证miRNA-18b的靶基因HIF-1α。【结果】Ber组和miRNA-18bantagomir组,可极显著提高猪IVF囊胚发育率(P<0.01)。Ber组和miRNA-18bantagomir组能上调猪IVF胚胎中HIF-1α表达水平,miRNA-18b agomir组能下调HIF-1α表达水平;HIF-1α的表达水平随着胚胎发育呈上升趋势,4-~8-细胞阶段更为明显;在HIF-1α的3’UTR wt、mut重建载体转染产生的双萤光素酶活性上,miRNA-18b与NC组均无差异(P>0.05)。【结论】Ber和抑制miRNA-18b的表达能够促进4-、8-细胞和桑椹胚时期HIF-1α的表达从而促进猪IVF胚胎体外发育,其中4-~8-细胞时期作用更为明显。
【Objective】To investigate the effects of berberine(Ber)and microRNA(miRNA)-18 bon hypoxia-inducible factor-1α(HIF-1α)in embryosof in vitro fertilization(IVF).【Methods】NCSU-23 medium was used as a control group,with Ber added to NCSU-23 for the Ber group.The miRNA-18 bover expression agent(agomir),miRNA-18 blow expression agent(antagomir)and negative fragments were microinjected(10 pL)into the cytoplasm of the zygotes of control group as the miRNA-18 bagomir group,miRNA-18 bantagomir group and negative control(NC)group.The rates 2-cell,4-cell,8-cell,morula and blastocyst were observed.qRT-PCR and Western-blot and double luciferase reporter gene methods were used to verify the target gene HIF-1αof miRNA-18 b.【Results】In the Ber and miRNA-18 bantagomir groups,the rate of blastocysts were highly significantly increased(P<0.01)comparing with the control group.Ber and low miRNA-18 b expressionincreased significantlythe expressions of HIF-1αin IVF embryos,high miRNA-18 bexpression reducedsignificantly the expressions HIF-1αin IVF embryosand the expression of HIF-1αincreased with the development of embryos,especially from the 4-cell to 8-cell stage.On the double luciferase activity produced by transfection of the 3’UTR wt and mutreconstitution vector of HIF-1α,there was no difference between miRNA-18 band NC group(P>0.05).【Conclusion】Ber and inhibiting expression of miRNA-18 b promoted the expression of HIF-1αin 4-cell,8-cell and morula embryos thus the development of IVF embryoes.The expression of HIF-1αincreased with the development of embryosfrom 4-cell to 8-cell stage was more obvious.
作者
王俊丽
刘兵
张超
戴佳格
张紫薇
于博
高建明
WANG Junli;LIU Bing;ZHANG Chao;DAI Jiage;ZHANG Ziwei;YU Bo;GAO Jianming(Animal Science and Technology College,Ueijing University of Agriculture,Beijing 102206,China;College of Animal Science and Technology,China Agriculture University,Beijing 100193,China)
出处
《北京农学院学报》
2019年第4期89-94,共6页
Journal of Beijing University of Agriculture
基金
北京市教委科学研究重点项目（KZ201610020017）
国家自然基金项目（31572402）
