关键词: 牛SPARC基因, 可选择性多聚腺苷酸化, 细胞增殖, 成脂分化

Abstract: To investigate the regulation of alternative polyadenylation（APA） of the bovine secreted protein acidic and rich in cysteine（SPARC）gene in the proliferation and differentiation of preadipocytes，the existence of APA phenomenon of bovine SPARC gene was verified by 3' rapid amplification of cDNA ends（3' RACE）. Using dual luciferase reporter gene assay，qPCR，Western blotting and other molecular biology techniques，the differential expression characteristics of SPARC gene 3'untranslated regions（3'UTR）‑APA isoforms were analyzed.The reporter gene vector was set as wild‑type short 3'UTR group（WT‑3'UTRS） ， wild‑type long 3'UTR group （WT‑3'UTRL） ， mutant long 3'UTR group（Mut‑3'UTRL‑P1），respectively. The protein expression vector was set as wild‑type short 3'UTR fusion group WT‑SPARCS），wild‑type long 3'UTR fusion group（WT‑SPARCL），and mutant long 3'UTR fusion group Mut‑SPARCL‑P1）. Through functional gain and loss test，CCK‑8 proliferation test，oil red O staining and qRT‑PCR technology，SPARC overexpression group（pCMV‑SPARC），empty vector control group（pCMV），SPARC interference group（siSPARC）and negative control group（NC）were set up to systematically explore the effects of SPARC gene and its 3'UTR‑APA isoforms on the proliferation and adipogenic differentiation of bovine preadipocytes. The results showed that there were two 3'UTR‑APA isoforms in bovine SPARC gene. Compared with the WT‑3'UTRS group，the luciferase activities of the WT‑3'UTRL and Mut‑3'UTRL‑P1 groups were significantly or extremely significantly decreased.Compared with the WT‑SPARCS group，the mRNA and protein expression levels of the SPARC gene in the WT‑SPARCL and Mut‑SPARCL‑P1 groups were both highly significantly downregulated. Compared with the pCMV group，the mRNA expression levels of proliferation marker genes in the pCMV‑SPARC group were extremely significantly upregulated，which significantly promoted cell proliferation，while the mRNA expression levels of adipogenic marker genes were extremely significantly downregulated，and lipid droplet accumulation was extremely significantly reduced. Compared with the NC group，the mRNA expression level of proliferation marker genes in the siSPARC group showed the opposite trend. Compared with the WT‑SPARCL group，the mRNA expression levels of proliferation marker genes in the WT‑SPARCS group were generally significantly or highly significantly upregulated，and cell proliferation was significantly promoted；meanwhile，the mRNA expression levels of adipogenic marker genes were highly significantly down‑regulated，and lipid droplet accumulation was significantly reduced.In conclusion，the functional differences of the APA regulation mechanism of bovine SPARC gene in the proliferation and differentiation of its preadipocytes were revealed，which not only provided a theoretical reference for analyzing the molecular mechanism of its participation in the post‑transcriptional regulation of bovine fat deposition，but also laid a theoretical foundation for molecular breeding of high‑quality beef cattle.

Key words: Bovine SPARC gene, Alternative polyadenylation, Cell proliferation, Adipogenesis differentiation