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Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1
Journal of Animal Science and Biotechnology ( IF 6.3 ) Pub Date : 2020-10-14 , DOI: 10.1186/s40104-020-00506-6
Xiaopeng An 1 , Haidong Ma 1, 2 , Yuhan Liu 1 , Fu Li 1 , Yuxuan Song 1 , Guang Li 1 , Yueyu Bai 3 , Binyun Cao 1
Affiliation  

MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes, including proliferation, development and apoptosis. Our previous study has showed that miR-101-3p is differentially expressed in dairy goat ovaries compared single with multiple litters. The objective of this research was to explore the potential function and molecular mechanism of miR-101-3p via its target STC1 in goat ovarian growth and development. cDNA libraries were constructed using goat granulosa cells transfected with miR-101-3p mimics and negative control by RNA-sequencing. In total, 142 differentially expressed unigenes (DEGs) were detected between two libraries, including 78 down-regulated and 64 up-regulated genes. GO and KEGG enrichment analysis showed the potential impacts of DEGs on ovarian development. STC1 was singled out from DEGs for further research owing to it regulates reproductive-related processes. In vitro, bioinformatics analysis and 3′-UTR assays confirmed that STC1 was a target of miR-101-3p. ELISA was performed to detect the estrogen (E2) and progesterone (P4) levels. CCK8, EdU and flow cytometry assays were performed to detect the proliferation and apoptosis of granulosa cells. Results showed that miR-101-3p regulated STAR, CYP19A1, CYP11A1 and 3β-HSD steroid hormone synthesis-associated genes by STC1 depletion, thus promoted E2 and P4 secretions. MiR-101-3p also affected the key protein PI3K, PTEN, AKT and mTOR in PI3K-AKT pathway by STC1, thereby suppressing proliferation and promoting apoptosis of granulosa cells. In vivo, the distribution and expression levels of miR-101-3p in mouse ovaries were determined through fluorescence in situ hybridisation (FISH). Immunohistochemistry results showed that STC1 expression was suppressed in mouse ovaries in miR-101-3p-agonist and siRNA-STC1 groups. Small and stunted ovarian fragments, decreased numbers of follicles at diverse stages were observed using Hematoxylin-eosin (HE) staining, thereby showing unusual ovarian development after miR-101-3p overexpression or STC1 depletion. Inhibition of miR-101-3p manifested opposite results. Taken together, our results demonstrated a regulatory mechanism of miR-101-3p via STC1 in goat granulosa cells, and offered the first in vivo example of miR-101-3p and STC1 functions required for ovarian development.

中文翻译:

miR-101-3p通过STC1对体外山羊颗粒细胞和体内卵巢发育的影响

miRNA 在调节多种生物过程(包括增殖、发育和细胞凋亡)中充当关键的转录后基因介质。我们之前的研究表明,与单窝和多窝相比,miR-101-3p 在奶山羊卵巢中的表达差异很大。本研究旨在探讨miR-101-3p通过其靶标STC1在山羊卵巢生长发育中的潜在功能和分子机制。使用转染 miR-101-3p 模拟物的山羊颗粒细胞和通过 RNA 测序的阴性对照构建 cDNA 文库。总共在两个文库之间检测到 142 个差异表达的 unigenes (DEG),包括 78 个下调基因和 64 个上调基因。GO和KEGG富集分析显示了DEGs对卵巢发育的潜在影响。STC1 被从 DEG 中挑选出来进行进一步研究,因为它调节与生殖相关的过程。在体外,生物信息学分析和 3'-UTR 测定证实 STC1 是 miR-101-3p 的靶标。进行ELISA以检测雌激素(E2)和孕酮(P4)水平。进行CCK8、EdU和流式细胞仪检测以检测颗粒细胞的增殖和凋亡。结果表明,miR-101-3p通过STC1耗竭调节STAR、CYP19A1、CYP11A1和3β-HSD类固醇激素合成相关基因,从而促进E2和P4的分泌。MiR-101-3p还通过STC1影响PI3K-AKT通路中的关键蛋白PI3K、PTEN、AKT和mTOR,从而抑制增殖,促进颗粒细胞凋亡。体内,通过荧光原位杂交(FISH)测定小鼠卵巢中miR-101-3p的分布和表达水平。免疫组织化学结果显示,miR-101-3p-激动剂和 siRNA-STC1 组小鼠卵巢中 STC1 的表达受到抑制。使用苏木精-伊红 (HE) 染色观察到小的和发育不良的卵巢碎片,不同阶段的卵泡数量减少,从而在 miR-101-3p 过表达或 STC1 耗尽后显示出异常的卵巢发育。抑制 miR-101-3p 表现出相反的结果。总之,我们的结果证明了 miR-101-3p 通过 STC1 在山羊颗粒细胞中的调节机制,并提供了卵巢发育所需的 miR-101-3p 和 STC1 功能的第一个体内例子。免疫组织化学结果显示,miR-101-3p-激动剂和 siRNA-STC1 组小鼠卵巢中 STC1 的表达受到抑制。使用苏木精-伊红 (HE) 染色观察到小的和发育不良的卵巢碎片,不同阶段的卵泡数量减少,从而在 miR-101-3p 过表达或 STC1 耗尽后显示出异常的卵巢发育。抑制 miR-101-3p 表现出相反的结果。总之,我们的结果证明了 miR-101-3p 通过 STC1 在山羊颗粒细胞中的调节机制,并提供了卵巢发育所需的 miR-101-3p 和 STC1 功能的第一个体内例子。免疫组织化学结果显示,miR-101-3p-激动剂和 siRNA-STC1 组小鼠卵巢中 STC1 的表达受到抑制。使用苏木精-伊红 (HE) 染色观察到小的和发育不良的卵巢碎片,不同阶段的卵泡数量减少,从而在 miR-101-3p 过表达或 STC1 耗竭后显示出异常的卵巢发育。抑制 miR-101-3p 表现出相反的结果。总之,我们的结果证明了 miR-101-3p 通过 STC1 在山羊颗粒细胞中的调节机制,并提供了卵巢发育所需的 miR-101-3p 和 STC1 功能的第一个体内例子。从而在 miR-101-3p 过表达或 STC1 耗尽后显示出异常的卵巢发育。抑制 miR-101-3p 表现出相反的结果。总之,我们的结果证明了 miR-101-3p 通过 STC1 在山羊颗粒细胞中的调节机制,并提供了卵巢发育所需的 miR-101-3p 和 STC1 功能的第一个体内例子。从而在 miR-101-3p 过表达或 STC1 耗尽后显示出异常的卵巢发育。抑制 miR-101-3p 表现出相反的结果。总之,我们的结果证明了 miR-101-3p 通过 STC1 在山羊颗粒细胞中的调节机制,并提供了卵巢发育所需的 miR-101-3p 和 STC1 功能的第一个体内例子。
更新日期:2020-10-15
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