The Positive Role Of PU.1as LncRNA In Porcine Preadipocyte Adipogenesis And Its Molecular Mechanism

Antisense long non-coding RNA (AS lncRNA) plays an important role in refinedregulation of animal gene expression. However, to date, its functions and molecularmechanisms on domestic animal adipogenesis are largely unknown. Previously, we foundinconsequent expression between PU.1mRNA and its protein in porcine adipose component:in preadipocytes, mRNA was of high abundance, but protein level is very low; while inmature adipocytes, the situation was just the opposite. We concluded PU.1AS lncRNAmight be involved in this process.Therefore, in this study, we first identified a novel AS lncRNA transcribed fromporcine PU.1gene by strand-specific RT-PCR. Second, we analyzed the PU.1mRNA, ASlncRNA and protein expression profiles in various porcine tissues and during preadipocytedifferentiation. Then, pLentiHI-PU.1shRNA and pLentiHI-PU.1AS shRNA were appliedto infect porcine primary preadipocytes induced to adipogenesis. Oil red O staining wasused to we examined the lipid accumulation and real-time qPCR and western blotting wereconducted to detected the adipogenetic and lipolytic markers and PU.1expression afterinfection. Finally, we explored the mechanisms between zebrafish and pig.The mainfindings are as follows:Strand-specific primer R3, R4, R5were respectively used in the RT reactions addingthe RNA samples from the adipose tissues of Guanzhong Black pigs, and the cDNAproducts were applied in the following PCR reaction as the templates. As expected, we gotseveral purpose bands after amplification using different forward and reverse primers,sizing321bp,349bp,391bp,420bp and449bp. The results demonstrated that PU.1ASlncRNA was transcribed in pig.(2) PU.1AS lncRNA was widely expressed and generally lower than the mRNA levelsin various porcine tissues. In the tissues of which PU.1mRNA levels were about and about,AS lncRNA increased as protein reduced, and vice versa. It suggested that PU.1AS lncRNAcould regulate PU.1protein level.(3) During the preadipocyte differentiation, PU.1mRNA raised at day2and thendecreased gradually, while the PU.1AS lncRNA exhibited a similar trend with that of PU.1 mRNA but decreased sharper. Surprisingly, PU.1protein level was rising duringdifferentiation, hinting a negative role in adipogenesis.(4) Knockdown of endogenous PU.1promoted preadipocyte adipogenesis, whereasknockdown of endogenous PU.1AS lncRNA had an opposite effect. What is more, in thePU.1shRNA1treatment group, adipogenic genes (PPARγ and FAS) were significantlyup-regulated (P <0.05), while downregulated in the PU.1AS shRNA1treatment group (P <0.05). The lipolytic genes (ATGL and HSL) were contrast to that of the adipogenic genes.(5) Knockdown of PU.1AS lncRNA did not influence the mRNA levels, butupregulated the protein levels. Thus, we predicted that PU.1AS lncRNA participated in thePU.1translation regulation. Consequently, an RNA duplex was detected by endogenousribonuclease protection assay combined with RT-PCR, indicating that PU.1AS lncRNAexerted its positive function through forming a sense-antisense RNA duplex with PU.1mRNA to block translation.(6) Knockdown of zebrafish PU.1AS lncRNA upregulated mRNA levels of PU.1andthe early adaptive immune related genes in vivo, but we did not detected an RNA duplex,prompting that the mechanism of AS lncRNAs varied in species.Collectively, PU.1AS lncRNA had a positive effect on adipogenesis through forming asense-antisense RNA duplex with PU.1mRNA to block PU.1mRNA translation. However,AS lncRNA the mechanism of AS lncRNAs varied in species. These results furtherprovided a target for lncRNA regulating animal adipogenesis and improvement forregulatory mechanism of lncRNAs.