Construction of lentiviral RNAi vector of PPAR gamma gene in cashmere goats and comparison with the transcriptome analysis of adipose cells treatments

摘要

As an important trait influencing meat quality, the deposition of Intramuscular fat (IMF) is affected by preadipocyte differentiation. Nuclear receptor peroxisome proliferator-activated receptor gamma(PPAR gamma) plays an important role in the regulation of glucose homeostasis and lipid metabolism. In this study, lentivirus-mediated RNA interference (RNAi) was applied to inhibit PPAR gamma gene expression, which can explore the effects of PPAR gamma silencing on the preadipose of cashmere goats in vitro. The roles of PPAR gamma gene in proliferation and differentiation of adipocytes were investigated by MTT assay and Oil Red O staining extraction assay. Using Real-time PCR and Western blotting analyses, the gene expression levels in goat adipocytes were detected at 24, 36, 48 and 72 h after transfection. By the high-throughput sequencing, the comparative transcriptome analysis of four different samples including preadipocyte, adipocyte, shRNA-PPAR gamma intramuscular fat cell and control group in Cashmere goats was performed. The results showed that PPAR gamma gene could increase proliferation of goat adipocyte, but decrease their differentiation. The novel genes screened with 152, 450, 456, 412, 39 and 445 different expressions by high-throughput sequencing. The genes, such as PREF-1, PREF-1, FGF, TGF, TNF, RA, IL-21 and EGF had higher expression in shRNA-PPAR gamma intramuscular fat cells. The 10 pathways that related to adipocyte differentiation were discovered by GO and KEGG analysis. The most significant pathways involved in PPAR gamma, WNT, mTOR, and MAPK signaling. Some key gens including LPL, FABP, FATP, AP2, RXRG and CD36 were enriched in PPAR gamma signal pathway. All of these findings will provide a new reference for understanding adipose transcriptome and identifying novel pathways regulating adipose physiology.