Bta-miR-101对牛睾丸支持细胞增殖、凋亡及分泌的影响

doi:10.11843/j.issn.0366-6964.2024.03.017

摘要/Abstract

摘要： 旨在探究bta-miR-101对牛睾丸支持细胞增殖、凋亡及分泌的影响。本研究采集3日龄（n=3）与13月龄（n=3）健康公牛的睾丸组织，构建安格斯牛组织表达谱，验证bta-miR-101在两个时期的差异表达。利用在线工具（TargetScan、miRTarBase、miRDB及miRWalk）预测miR-101的靶基因，并利用KOBAS对其进行GO和KEGG通路富集分析。将miR-101的模拟物与抑制物分别转染至牛睾丸支持细胞中，利用RT-qPCR、Western Blotting以及CCK-8等技术检测细胞增殖、凋亡及分泌相关基因表达量以及细胞增殖系数。通过4种在线软件预测到26个bta-miR-101的共同靶基因，GO和KEGG富集分析发现这些基因主要富集在核质、蛋白质结合、JAK-STAT 的受体信号通路上，这些信号通路与细胞分化、细胞周期、细胞凋亡等生物过程有着不同程度的关联。RT-qPCR结果显示，bta-miR-101在牛初生期睾丸组织中的表达量显著高于性成熟时期（P<0.05），与实验室前期测序结果一致。转染miR-101的模拟物与抑制物后，发现相较于对照组，miR-101 mimics可以促进增殖相关基因在mRNA和蛋白水平的表达量，并显著升高支持细胞的增殖系数，同时抑制凋亡基因在mRNA和蛋白水平的表达量（P<0.05）；miR-101 inhibitor可以抑制增殖相关基因在mRNA和蛋白水平的表达量，同时促进凋亡基因在mRNA和蛋白水平的表达量（P<0.05）。在细胞分泌方面，miR-101对分泌标志基因GDNF、BMP4的转录有影响，但对雄激素结合蛋白的分泌影响不显著。综上所述，miR-101促进牛睾丸未成熟支持细胞的增殖并抑制其凋亡，对雄激素结合蛋白的分泌无显著影响。

关键词: 牛睾丸支持细胞, miR-101, 增殖, 凋亡

Abstract: The study aimed to explore the effects of bta-miR-101 on bovine testicular sertoli cells' proliferation, apoptosis and secretion.In this study, testicular tissues of healthy bulls (n=3) aged 3 days and 13 months were collected to construct the expression profile of Angus cattle tissues. The differences in bta-miR-101 expression were verified between the two periods. Online tools (TargetScan, miRTarBase, miRDB, and miRWalk) were used to predict the target gene of miR-101, and KOBAS was used for enrichment analysis of GO and KEGG pathways. The mimics and inhibitors of miR-101 were transfected into bovine testicular sertoli cells. Cell proliferation, apoptosis, secretion-related gene expression, and cell proliferation coefficient were detected by RT-qPCR, Western Blotting, and CCK-8 techniques.There were 26 common target genes for bta-miR-101 predicted by 4 online tools. GO and KEGG enrichment analysis showed that these genes were mainly enriched in the nucleoplasm, protein-binding, and JAK-STAT receptor signaling pathways, which were associated with cell differentiation, cell cycle, apoptosis, and other biological processes to varying degrees. The results of RT-qPCR showed that bta-miR-101 expression was significantly higher in testis tissue at birth than at sexual maturity (P<0.05), consistent with previous laboratory sequencing results. After transfection of miR-101 mimics and inhibitors, it was found that miR-101 mimics could promote the expression of proliferation-related genes at mRNA and protein levels, significantly increase the proliferation coefficient of sertoli cells, and inhibit the expression of apoptosis genes at mRNA and protein levels (P<0.05); miR-101 inhibitors inhibited the expression of proliferation-related genes at mRNA and protein levels and promoted the expression of apoptotic genes at mRNA and protein levels (P<0.05). In terms of cell secretion, miR-101 had an effect on the transcription of secretion marker genes GDNF and BMP4, but had no significant effect on the secretion of androgen-binding proteins. In conclusion, miR-101 promoted proliferation and inhibited apoptosis of immature sertoli cells of the bovine testis, and had no significant effect on the secretion of androgen-binding proteins.

Key words: bovine testicular sertoli cell, miR-101, proliferation, apoptosis

中图分类号:

S823.3

虎巧燕, 翟相钦, 李一丹, 韩家乐, 雷初朝, 党瑞华. Bta-miR-101对牛睾丸支持细胞增殖、凋亡及分泌的影响[J]. 畜牧兽医学报, 2024, 55(3): 1040-1051.

HU Qiaoyan, ZHAI Xiangqin, LI Yidan, HAN Jiale, LEI Chuzhao, DANG Ruihua. Effects of bta-miR-101 on Proliferation, Apoptosis and Secretion of Bovine Testicular Sertoli Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1040-1051.

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