MiR-9-5p promotes rabbit preadipocyte differentiation by suppressing leptin gene expression

Abstract Background: MicroRNAs are a class of small non-coding RNAs and participate in the regulation of cell differentiation programs. Previous studies have demonstrated that miR-9-5p play a key role during cancer cell research, but the mechanisms regulating of miR-9-5p in adipogenesis remain poorly understood. This study intended to investigate its significance in rabbits with high quality meat by observing the regulating effect of miR-9-5p in pre-adipocytes and finding related targets. Methods: In this study, a dual luciferase reporter assay was employed to validate the targeting relationship between miR-9-5p and leptin gene. We also utilized quantitative reverse transcription PCR (qRT-PCR), western blot, determination of triglyceride and oil red O staining assay to analyze the regulation of miR-9-5p and leptin gene during adipocyte differentiation. Results: The analysis demonstrated that during pre-adipocyte differentiation, miR-9-5p was up-regulated and the fat formation related biomarkers fatty acid-binding protein 4(FABP4), CCAAT-enhancer binding protein α(C/EBPα), and peroxisome proliferator activated receptorγ (PPARγ) were also up-regulated. Meanwhile, the oil red O staining assay revealed that the accumulation of lipid droplets increased. We explored the expression pattern and role of miR-9-5p in adipogenesis using white pre-adipocytes. The results showed that miR-9-5p was up-regulated during pre-adipocyte differentiation and overexpression of miR-9-5P enhanced lipid accumulation. Furthermore, we found overexpression of miR-9-5p significantly up regulated the mRNA levels of marker gene PPARγ, C/EBPα and FABP4, as well as the protein levels of PPARγ and content of triglyceride, the results suggested that miR-9-5p might be involved in the regulation of rabbit pre-adipocyte differentiation. We predicted leptin is a target of miR-9-5p by using bioinformatics tools and the conclusion was validated in a luciferase reporter assay. Finally, we verified inhibition of leptin by si-leptin promoted rabbits pre-adipocyte differentiation. Conclusion: In a word, these results indicate that miR-9-5p promotes rabbits white pre-adipocyte differentiation by targeting leptin .