miR-F4-C12 Functions on the Regulation of Adipose Accumulation by Targeting PIK3R1 in Castrated Male Pigs

SIMPLE SUMMARY: MicroRNAs play crucial roles in regulating adipogenesis and fat storage; their role in regulating castrated male pig adipose growth is worth elucidating. Four nine-fold differentially expressed miRNAs were selected to investigate their functions on the regulation of adipose development based on our previous study. In 3T3-L1 cells and backfat tissues of castrated and intact male pigs, miR-F4-C12 was identified as involved in the adipose development using qRT-PCR and oil O staining. PIK3R1 was proposed by the TargetScan, miRDB and starBase as a target of miR-F4-C12 and verified through a dual-luciferase reporter assay and Western blot. These results revealed that miR-F4-C12 may regulate adipose accumulation in castrated male pigs by targeting PIK3R1. Our data provide a valuable foundation to understand the molecular mechanisms involved in adipose tissue metabolism to castration-induced sex hormone deficiency. ABSTRACT: MicroRNAs (miRNAs) constitute small regulatory molecules for a wide array of biological activities (18~24 nucleotides in length), including adipogenesis and adipose deposition. Their effect is, however, incompletely defined in inducing fat accumulation in castrated male pigs. Based on our study, four nine-times miRNAs were selected to examine their functions in adipose formation activities. In 3T3-L1 cells and backfat tissues of castrated and intact male pigs, miR-F4-C12 was identified as a factor in adipose development utilizing quantitative real-time PCR (qRT-PCR). Further, miR-F4-C12 was identified to promote fat development, suggesting that miR-F4-C12 was involved in adipogenesis. Moreover, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was proposed by the TargetScan, miRDB and starBase as a target of miR-F4-C12 and verified through a two-luciferase reporter assay. The over-expression of miR-F4-C12 dramatically decreases the PIK3R1 protein level in 3T3-L1 cells. The mRNA and protein levels of PIK3R1 in castrated pigs are reduced relative to intact pigs, providing further evidence that PIK3R1 is involved in regulating adipose accumulation. These results suggest that miR-F4-C12 involves adipose development and may regulate subcutaneous adipose tissue accumulation by targeting PIK3R1 in castrated male pigs.