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Bta-miR-34b inhibits proliferation and promotes apoptosis via the MEK/ERK pathway by targeting MAP2K1 in bovine primary Sertoli cells.

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.

SUBMITTER: Zhang L

PROVIDER: S-EPMC7598574 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMicroRNAs act as post-transcriptional regulators that repress translation or degrade mRNA transcripts. Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically bta-miR-365-3p, in muscle biology, we investigated its functions in regulating primary bovine myoblast proliferation and differentiation.ResultsFirstly, we found that bta-miR-365-3p was predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting results showed that overexpression of bta-miR-365-3p significantly reduced the expression levels of cyclin D1 (CCND1), cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) but stimulated the expression levels of muscle differentiation markers, i.e., MYOD1, MYOG at both mRNA and protein level. Moreover, downregulation of bta-miR-365-3p increased the expression of CCND1, CDK2 and PCNA but decreased the expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining results showed that increased levels of bta-miR-365-3p suppressed cell proliferation but promoted myotube formation, whereas decreased levels of bta-miR-365-3p resulted in the opposite consequences. Finally, we identified that activin A receptor type I (ACVR1) could be a direct target of bta-miR-365-3p. It was demonstrated that bta-miR-365-3p can bind to the 3'UTR of ACVR1 gene to regulate its expression based on dual luciferase gene reporter assays. Consistently, knock-down of ACVR1 was associated with decreased expressions of CDK2, CCND1 and PCNA but increased expression of MYOG and MYOD1 both at mRNA and protein level.ConclusionCollectively, these data suggested that bta-miR-365-3p represses proliferation but promotes differentiation of bovine myoblasts through several biological mechanisms involving downregulation of ACVR1.

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Bta-miR-34b inhibits proliferation and promotes apoptosis via the MEK/ERK pathway by targeting MAP2K1 in bovine primary Sertoli cells.

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