Expression profile of CCND2 gene and the overexpression effects on proliferation of skeletal muscle cells in Luchuan pigs

【Objective】This study aimed to investigate the expression pattern of cell cycle protein （CCND2） gene in skeletal muscle tissues of Luchuan pigs and its regulatory mechanism of proliferation of skeletal muscle cells in Luchuan pigs， so as to provide theoretical basis for the genetic improvement of meat production traits in Luchuan pigs.【Method】Spatiotemporal expression characteristics of CCND2 gene during development of porcine skeletal muscle cells were investigated. CCND gene was overexpressed and knocked down， and the relative expression of porcine skeletal muscle cell proliferation marker genes was detected by real-time fluorescence quantitative PCR. Porcine skeletal muscle cell proliferation activity was detected using CCK-8 and EdU cell proliferation activity assay kits. Differentially expressed genes （DEGs） were screened by transcriptome sequencing， and GO functional annotation and KEGG signaling pathway enrichment analysis were performed.【Result】The relative expression of CCND2 gene in the skeletal muscle of Landrace pigs during the embryonic period was significantly higher than that in Tongcheng pigs （P<0.01， the same below）. Knockdown of CCND2 gene significantly decreased the relative expression of CDK4 gene （P<0.05， the same below）. After overexpression of the CCND2 gene， the relative expression of CDKN1B gene significantly decreased ， the relative expression of KI67 gene extremely significantly decreased， and the relative expression of PCNA gene extremely significantly increased. CCK-8 and EdU cell proliferation activity assays showed that the knockdown of CCND2 gene slowed cell proliferation， while the overexpression of CCND2 gene accelerated cell proliferation. A total of 120 DEGs were screened； 80 DEGs were up-regulated and 40 DEGs were down-regulated in overexpressed CCND2 gene. Up-regulated DEGs were significantly annotated in GO functional terms related to cell proliferation， such as the mitotic spindle midzone， JAK-STAT signaling pathway， midbody， and signal transduction， and were significantly enriched in Wnt signaling pathway， cell adhesion molecules， FoxO signaling pathway， and cAMP signaling pathway. Down-regulated DEGs were significantly annotated in GO functional terms related to biological processes， such as positive regulation of the ERK1/ERK2 cascade， negative regulation of NF-κB activity， and negative regulation of IκB kinase/NF-κB signaling， and were significantly enriched in signaling pathways such as the calcium signaling pathway， fatty acid metabolism， signaling pathways regulating pluripotency of stemceus， metabolic pathways， and cholesterol metabolism.【Conclusion】The CCND2 gene promotes the proliferation of skeletal muscle cells in Luchuan pigs， a process that may be related to the transcriptional regulation of genes involved in signaling pathways such as Wnt， FoxO， and cAMP. The CCND2 gene may be a key candidate gene contributing to the differences in skeletal muscle development between Chinese local pigs and Western lean-type pigs， and can serve as a potential target for elucidating skeletal muscle development in pigs.