The Study Of The Mechanism Of Regulating PK15 Cell Proliferation About Porcine NR2F2 Gene

Chicken ovalbumin upstream promoter transcription factor 2(COUP-TFⅡ,also known as NR2F2),an important member of the nuclear receptor family,is belongs to the steroid hormone receptor superfamily.As a transcription factor,NR2F2 gene is widely expressed in tissues.It can directly activate or inhibit the transcriptional activity of target genes,regulate multiple signaling pathways,participate in cell differentiation,neurogenesis,organ formation processes,and play a regulatory role in the process of bone,muscle and fat formation and metabolic balance.At present,the research of NR2F2 gene mainly focuses on its mechanism of action on human cancer and other diseases,as well as the muscle and fat tissues of mice,but the research of NR2F2 gene in pigs has not been reported.The purpose of this study was to explore the mechanism of NR2F2 gene and its effect on cell proliferation,apoptosis and migration by regulating downstream genes,so as to provide a theoretical basis for further research on the molecular mechanism of NR2F2 gene affecting pig muscle growth and development through TGF-βsignaling pathway.The CDS sequence of NR2F2 gene was obtained by cloning and sequencing in this study.The protein structure,function and localization of the NR2F2 gene were predicted by bioinformatics analysis.The temporal and spatial expression of NR2F2 gene in pigs was detected by qRT-PCR.The lentivirus vector of NR2F2 gene was constructed and transfected into PK15 cell.The changes in gene expression related to cell proliferation were detected by qRT-PCR and WB methods.The cell proliferation was detected by cck-8 and EdU methods.The effects on cell cycle,apoptosis and migration were detected by scratch test and flow cytometry.NR2F2 and Smad4 protein binding verified by CO-IP.Further through the detection of TGF-βsignaling pathways and save the test to explore the influence of the NR2F2 gene regulatory mechanisms of cell proliferation.The results showed that the CDS region sequence of NR2F2 gene of Mashen pig was successfully obtained,and it has a total length of 846 bp and code of 281 amino acids.The protein is highly conserved in different species.The NR2F2 gene of Mashen pig was widely expressed in all tissues,and the expression level was the highest in kidney and spleen,moderate in lung,and low in other tissues.Three transfection methods were used to transfer the empty vector and the overexpressed NR2F2 gene vector into the PK15 cells,among which the lentivirus transfection method had high transfection efficiency,high cell survival rate and stable inheritance.PK15 cell lines with stable expression of NR2F2 gene were successfully obtained,which significantly increased the expression of NR2F2 gene.The overexpression of NR2F2 gene significantly increased the expression of PCNA and Ki67 genes,promoted cell transformation from G1 phase to S phase,inhibited cell apoptosis,and promoted cell migration.NR2F2 and Smad4 protein binding was verified by CO-IP.After overexpression of NR2F2 gene,the expression of Smad4 gene was increased,and the expression of downstream target genes cyclin-related factors Cyclin B,CDK1,CDK4 and Cyclin D1 significantly increased in the TGF-β signaling pathway.After knock out of NR2F2 gene,the Smad4 gene expression was decreased,and the expression of downstream target genes cyclin-related factors Cyclin B,CDK1,CDK4 and Cyclin D1 were significantly decreased in the TGF-β signaling pathway.And overexpression of NR2F2 gene interfered with the expression of Smad4,the expression levels of Cyclin B,CDK1,CDK4 and Cyclin D1 were detected which were decreased,indicating that the NR2F2 gene affected cell proliferation by regulating the Smad4 gene.To sum up,this study successfully cloned pigs NR2F2 gene CDS region,and though the overexpression and knockout experiments,preliminary discussed NR2F2 gene regulation through Smad4 gene expression influence TGF-β signaling pathway,in turn,affects the mechanism of cell proliferation.This study supplemented the TGF-β signaling pathway that regulates cell proliferation networks,provided a theoretical basis for further research on the molecular mechanism of NR2F2 gene affecting pig growth development through TGF-β signaling pathway,and was of great significance for further research on Mashen pig.