gga-miR-218 通过靶向 Stra8 在精原干细胞减数分裂中的调控功能。

Regulatory functions of gga-miR-218 in spermatogonial stem cells meiosis by targeting Stra8.

机构信息

College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu province, PR China; College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu province, PR China; Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou 225009, Jiangsu province, PR China.

Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, Jiangsu province, PR China.

出版信息

Mech Dev. 2020 Dec;164:103636. doi: 10.1016/j.mod.2020.103636. Epub 2020 Aug 13.

DOI:10.1016/j.mod.2020.103636

PMID:32798699

Abstract

MicroRNAs play a crucial role in sperm formation, but its specific function remains unknown. Here, we found that gga-miR-218 regulates chicken sperm formation through in/ex vivo experiments. We constructed over-expression/interference carrier to overexpress and inhibit gga-miR-218 in chicken spermatogonial stem cells, separately, the detection of haploid and QRT-PCR of meiosis related genes revealed that gga-miR-218 inhibits meiosis. After injection of miR-218 in vivo, semen concentration and HE (Hematoxylin and Eosin staining) revealed that gga-miR-218 inhibits meiosis. Meanwhile, we discovered that gga-miR-218 could target Stra8 by prediction software which can inhibit the wild-type fluorescence activity by co-transfection of gga-miR-218 with the Stra8 3' untranslated regions fluorescent reporter vector (wild-type/mutant), QRT-PCR and Western blot showed that gga-miR-218 inhibits the expression level of Stra8 by targeting its 3' untranslated regions directly. Finally, we suggest that gga-miR-218 could target to srta8 directly and inhibit spermatogenesis.

摘要

微小 RNA 在精子形成中发挥着关键作用，但具体功能尚不清楚。在这里，我们通过体内外实验发现 gga-miR-218 通过调控鸡精原干细胞中 gga-miR-218 的表达来调节鸡精子的形成。我们构建了过表达/干扰载体，分别在鸡精原干细胞中过表达和抑制 gga-miR-218，通过检测单倍体和减数分裂相关基因的 QRT-PCR 发现 gga-miR-218 抑制减数分裂。体内注射 miR-218 后，精液浓度和 HE（苏木精和伊红染色）染色结果表明 gga-miR-218 抑制减数分裂。同时，我们通过预测软件发现 gga-miR-218 可以靶向 Stra8，共转染 gga-miR-218 和 Stra8 3'UTR 荧光报告载体（野生型/突变型）可以抑制野生型荧光活性，QRT-PCR 和 Western blot 结果显示 gga-miR-218 通过直接靶向其 3'UTR 抑制 Stra8 的表达水平。最后，我们认为 gga-miR-218 可以直接靶向 srta8 并抑制精子发生。