Unraveling the hnRNPK/miR-133a-3p/UCP2 axis: a novel regulatory circuit governing porcine skeletal muscle satellite cell fate

Abstract

Objective

MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by targeting mRNA 3’UTRs, critically influencing skeletal muscle development. While miR-133a-3p is implicated in myogenesis, its regulatory interplay with RNA-binding proteins (RBPs) in porcine skeletal muscle satellite cells (PSCs) remains unexplored. This study elucidates the functional role and molecular mechanism of miR-133a-3p through a novel hnRNPK/miR-133a-3p/UCP2 axis in PSCs.

Methods

RT-qPCR and western blot were used for gene expression analysis. miR-133a-3p mimic and miR-133a-3p inhibitor were conducted to overexpression and knockdown the expression of miR-133a-3p in PSCs, respectively. The hnRNPK overexpression vector was designed using the pcDNA3.1(+) vector. 5-ethynyl-2′-deoxyuridine (EdU) assays were conducted for cell proliferation. Immunofluorescence detection was employed for cell differentiation analysis. The dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and biotin-labeled miRNA pull-down assays were utilized for regulatory mechanism analysis of hnRNPK/miR-133a-3p/ UCP2.

Results

Transcriptional profiling revealed miR-133a-3p upregulation during PSCs differentiation. Gain- and loss-of-function assays demonstrated that miR-133a-3p suppresses proliferation but promotes differentiation; its inhibition yielded opposite effects. Comprehensive mechanistic studies (dual-luciferase, RIP, and biotin pull-down assays) identified hnRNPK as a direct binding partner of miR-133a-3p. Subsequent validation confirmed UCP2 as a downstream target, with hnRNPK attenuating miR-133a-3p-mediated UCP2 repression. Notably, hnRNPK antagonized miR-133a-3p's pro-differentiation effects, revealing its role as a negative myogenesis regulator.

Conclusion

Collectively, we report a novel hnRNPK/miR-133a-3p/UCP2 axis governing mitochondrial gene expression and PSCs differentiation. This work advances understanding of RBP-miRNA interaction in post-transcriptional myogenic regulation and provides new targets for muscle biology and regenerative medicine.