ABSTRACT

Preadipocyte proliferation is a critical and precisely orchestrated procedure in adipogenesis, which is highly regulated by microRNAs (miRNAs). A previous study identified that the expression of miR-33a is different in intramuscular fat (IMF) tissues from steers and bulls. In the present study, miR-33a was overexpressed in bovine preadipocytes, and a total of 781 differentialy expressed genes were found, including 348 upregulated and 433 downregulated genes. Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analyses of the differentially expressed genes enriched cell division and cell cycle respectively. MiR-33a overexpression decreased the rate of preadipocyte proliferation. Synchronously, the mRNA and protein expression levels of proliferation-related marker genes, including cyclin B1 (CCNB1) and proliferating cell nuclear antigen (PCNA), were decreased. In contrast, inhibiting miR-33a increased the rate of preadipocyte proliferation, and expression levels of CCNB1 and PCNA. Furthermore, based on luciferase reporter assays, miR-33a targeted directly cyclin-dependent kinase 6 (CDK6)-3ʹUTR and inhibited CDK6 protein expression. Interestingly, the silencing of CDK6 inhibited bovine preadipocyte proliferation and proliferation-related genes. Therefore, miR-33a inhibits the proliferation of bovine preadipocytes. CDK6 is the target gene of miR-33a and may be involved in the effects of miR-33a on bovine preadipocyte proliferation.

 抽象的

前脂肪细胞增殖是脂肪生成过程中一个关键且精确协调的过程，受到 microRNA (miRNA) 的高度调控。先前的一项研究发现，在阉牛和公牛的肌内脂肪（IMF）组织中，miR-33a 的表达是不同的。本研究中，miR-33a在牛前脂肪细胞中过表达，共发现781个差异表达基因，其中上调基因348个，下调基因433个。基因本体论和京都基因和基因组百科全书（KEGG）对差异表达基因的通路分析分别丰富了细胞分裂和细胞周期。 MiR-33a 过度表达降低了前脂肪细胞增殖率。同时，包括细胞周期蛋白B1（CCNB1）和增殖细胞核抗原（PCNA）在内的增殖相关标记基因的mRNA和蛋白表达水平降低。相反，抑制 miR-33a 会增加前脂肪细胞增殖率以及 CCNB1 和 PCNA 的表达水平。此外，基于荧光素酶报告基因检测，miR-33a 直接靶向细胞周期蛋白依赖性激酶 6 (CDK6)-3ʹUTR 并抑制 CDK6 蛋白表达。有趣的是，CDK6 的沉默抑制了牛前脂肪细胞增殖和增殖相关基因。因此，miR-33a抑制牛前脂肪细胞的增殖。 CDK6是miR-33a的靶基因，可能参与miR-33a对牛前脂肪细胞增殖的影响。