Abstract

Immature Sertoli cell (SC) proliferation determines the final number of mature SCs and further regulates spermatogenesis. Accumulating evidence demonstrated that microRNAs (miRNAs) play an important role in SC proliferation, differentiation, and apoptosis. However, the effect and molecular mechanism of miRNA on bovine immature SC remain to be poorly understood. In this study, miRNA sequencing of testes collected in mature (24-mo old) and immature (neonatal) bulls was conducted to determine the miRNA expression profiles. MicroRNA-34b was one of the differentially expressed miRNAs and was selected for in-depth functional studies pertaining to SC growth. The results showed that miR-34b mimic transfection in primary Sertoli cells (PSC) inhibited cell proliferation and induced cell cycle arrested at G2 phase and decreased the expression of cell cycle-related genes such as CCNB1, CDK1, CDC25C, and C-MYC. MicroRNA-34b overexpression also leads to increased cell apoptosis, with proapoptotic genes P53 and BAX upregulated, while antiapoptotic gene BCL2 decreased. However, miR-34b knockdown had the opposite effects. Through a combination of transcriptome sequencing, bioinformatics analysis, dual-luciferase reporter assay, and Western blotting, mitogen-activated protein kinase kinase1 (MAP2K1), also known as MEK1, was identified as a target of miR-34b. In addition, PSC proliferation inhibition was mediated by cell cycle arrest and apoptosis with MAP2K1 interference. Overexpression of MAP2K1 effectively reversed the miR-34b-repressed PSC cell growth. Moreover, both miR-34b overexpression and MAP2K1 knockdown decreased the protein levels of P-ERK1/2, while MAP2K1 overexpression showed opposite effects. In summary, data suggest that miR-34b regulates PSC proliferation and apoptosis through the MEK/ERK signaling pathway. These data provide a theoretical and experimental framework for further clarifying the regulation of cell growth in PSC of bovine.

中文翻译：

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Bta-miR-34b 通过靶向牛原代支持细胞中的 MAP2K1，通过 MEK/ERK 通路抑制增殖并促进细胞凋亡。

摘要

未成熟的支持细胞 ( SC ) 增殖决定了成熟 SCs 的最终数量并进一步调节精子发生。越来越多的证据表明，微小RNA（miRNA）在SC增殖、分化和凋亡中起重要作用。然而，miRNA对牛未成熟SC的作用和分子机制仍然知之甚少。在本研究中，对成熟（24 个月大）和未成熟（新生）公牛的睾丸进行 miRNA 测序以确定 miRNA 表达谱。MicroRNA-34b 是差异表达的 miRNA 之一，被选中用于与 SC 生长有关的深入功能研究。结果表明 miR-34b 在原代支持细胞 ( PSC) 中模拟转染) 抑制细胞增殖并诱导细胞周期停滞在 G2 期，并降低细胞周期相关基因如CCNB1、CDK1、CDC25C和C-MYC 的表达。MicroRNA-34b 过表达也导致细胞凋亡增加，促凋亡基因P53和BAX上调，而抗凋亡基因BCL2减少。然而，miR-34b 敲低具有相反的效果。通过转录组测序、生物信息学分析、双荧光素酶报告基因检测和蛋白质印迹法的组合，丝裂原活化蛋白激酶激酶 1 ( MAP2K1)，也称为 MEK1，被确定为 miR-34b 的靶标。此外，PSC 增殖抑制是由细胞周期停滞和细胞凋亡与 MAP2K1 干扰介导的。MAP2K1 的过表达有效地逆转了 miR-34b 抑制的 PSC 细胞生长。此外，miR-34b 过表达和 MAP2K1 敲低都降低了 P-ERK1/2 的蛋白质水平，而 MAP2K1 过表达则显示出相反的效果。总之，数据表明 miR-34b 通过 MEK/ERK 信号通路调节 PSC 增殖和凋亡。这些数据为进一步阐明牛 PSC 中细胞生长的调节提供了理论和实验框架。