NR2F2基因调控猪PK15细胞增殖和凋亡的研究

Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells

旨在探究NR2F2基因对猪PK15细胞增殖和凋亡的影响。本研究在猪PK15细胞中过表达NR2F2基因,通过qRT-PCR、Western blot、CCK-8、EdU、流式细胞术、划痕试验等方法检测了过表达NR2F2基因对细胞增殖的影响;采用CO-IP技术验证NR2F2与Smad4蛋白结合能力;用qRT-PCR方法检测了NR2F2基因对Smad 4基因表达以及下游基因Cyclin B、CDK1、CDK4、Cyclin D 1表达的影响;挽救试验过表达NR2F2基因且干扰Smad 4基因检测下游基因及增殖基因Ki67、PCNA的表达。结果发现,过表达NR2F2基因极显著上调了Ki 67基因的表达(P<0.01),显著上调了PCNA的表达(P<0.05),促进了细胞周期的进程,抑制了细胞凋亡,促进了细胞的增殖。CO-IP结果证明了NR2F2与Smad4蛋白物理性结合,且发现过表达NR2F2基因后,Smad 4基因表达显著上升(P<0.01),Smad 4基因调控的下游基因Cyclin B、CDK1、CDK4、Cyclin D 1表达显著升高(P<0.01);干扰NR2F2基因后,Smad 4基因表达显著下降(P<0.01),Smad 4基因调控的下游基因表达显著降低(P<0.01)。挽救试验发现,过表达NR2F2基因后,干扰Smad 4基因的表达,能显著降低Cyclin D1、Cyclin B、CDK 1以及Ki67、PCNA的表达(P<0.01,P<0.05)。本研究结果表明,过表达NR2F2基因后促进了细胞的增殖,抑制凋亡,且NR2F2蛋白能与Smad4蛋白结合并影响Smad 4基因的表达,进而影响下游基因的表达。

This study aimed to explore the effect of NR2F2 gene on the proliferation and apoptosis of porcine PK15 cells.In this study,the effect of overexpression of NR2F2 gene on cell proliferation was detected by qRT-PCR,Western blot,CCK-8,EdU,flow cytometry,scratch assay and other methods in porcine PK15 cells.CO-IP technology was used to verify the binding ability of NR2F2 to Smad4 protein.The effect of NR2F2 gene on Smad 4 gene expression and the expression of downstream gene Cyclin B,CDK1,CDK4,Cyclin D 1 were detected by qRT-PCR.The expression of downstream genes and proliferating genes Ki67,PCNA were detected by rescue experiment overexpressed NR2F2 gene and interfered with Smad 4 gene.The results showed that overexpression of NR2F2 gene significantly upregulated the expression of Ki67(P<0.01),and significantly upregulated the expression of PCNA(P<0.05),promoted cell cycle progression,inhibited cell apoptosis,and promoted cell proliferation.The CO-IP results proved that NR2F2 and Smad4 proteins were physically bound.After overexpression of NR2F2 gene,the expression of Smad 4 gene increased significantly(P<0.01),and the expression of downstream regulated genes by Smad 4 gene Cyclin B,CDK1,CDK 4 and Cyclin D 1 significantly increased(P<0.01).After interfering with NR2F2 gene,the expression of Smad 4 gene significantly decreased(P<0.01),and the expression of downstream genes regulated by Smad 4 gene was significantly decreased(P<0.01).The rescue experiments showed that after overexpression of NR2F2 gene,interfering with the expression of Smad 4 gene could significantly reduce the expression of Cyclin D1,Cyclin B,CDK1,Ki 67 and PCNA(P<0.01,P<0.05).In summary,overexpression of NR2F2 gene promoted cell proliferation and inhibited apoptosis,and NR2F2 protein could bind to Smad4 protein and affect the expression of Smad 4,which in turn affected the expression of downstream genes.