The Effects Of Circadian Gene PER2 Silencing On The Proliferation And Milk Fat Synthesis Of Bovine Mammary Epithelial Cell

Biological rhythms are ubiquitous in the body's cells and have a certain regulatory effect on cell cycle,proliferation,and apoptosis.In addition,studies have shown that there is a strong link between circadian clock genes and lipid synthesis,but its specific regulatory mechanism is not yet clear.This experiment was conducted to study the regulation of PER2 on the cell cycle,proliferation and apoptosis,as well as the molecular relationship between the circadian rhythm and the synthesis of milk fat.Experiment 1:The silence efficiency of PER2 and the effect of PER2 silence on the circadian genes of bovine mammary epithelial cellIn this study,dairy cows were selected as experimental animals and the primary bovine mammary epithelial cells were isolated from breast tissue by collaenase digestion method,which provided materials for subsequent experiments.In this study,three siRNA interference sequences(siRNA-PER2-a/b/c)were constructed to transfect dairy cow mammary epithelial cells and Realtime PCR was used to analyze PER2 mRNA expression of these interference sequences at five time points to screen for the best interference sequences and the best transfection time points.The mRNA expression of circadian clock genes were detected by Real-time PCR.The results show that the interference fragment siRNA-PER2-b had the maximum inhibitory effect(84.7%)at 36h after transfection.In addition,the PER2 silence could significantly down-regulate the mRNA expression of PER1,CLOCK,CRY2,BAML1,NR1D1 and Timeless(P<0.05),while no significance was found on CRY1 and RORA(P?0.05).Thus,to some extent,PER2 gene silencing disrupted the balance of the circadian rhythm in bovine mammary epithelial cell.Experiment 2:Effect of circadian gene PER2 silencing on proliferation and apoptosis of bovine mammary epithelial cellsIn this study,we down regulated PER2 in bovine mammary epithelial cells,and then detected the alterations of cell cycle,cell proliferation and apoptosis and all important genes involved in cell cycle and cell apoptosis.Cell vitality was determined using CCK8,cell cycle and apoptosis were tested by flow cytometry analysis.Genes involved in cell cycle and cell apoptosis signaling pathways were detected by Real-time PCR.The results analysis:The cck8 determination of cell proliferation activity showed no significant difference after PER2 silencing.The flow cytometry methods experiment showed that the PER2 silence geoup had a significantly increased number of cells in G0/G1 phase,decreased number of cells in S phase(P<0.05)and significantly decreaseed proliferation index(P<0.05)compared with the nontransfected group.PER2 silence group had a significantly decreased mRNA expression of CDK1,CDK2,CDK4,cyclinBl,cyclinDl,Cdc25B and C-cym(P<0.05),there was no significant difference in mRNA expression of CDK6(P>0.05).Furthermore,PER2 silence group had no significant difference on apoptosis index,but significantly up-regulated the mRNA expression of Caspase 8(P<0.05),but no significant difference was found in mRNA expression of Caspase 3 and P53(P>0.05).Experiment 3:Effect of circadian gene PER2 silencing on milk fat Synthesis in bovine mammary epithelial cellsThis experiment was aimed to investigate the effect of PER2 silencing on lipid droplet,triglyceride,and important genes in milk fat metabolism.Lipid drop synthesis was detected by oil red O staining,triglyceride enzymatic method was used to measure triglyceride content in mammary epithelial cells,and mRNA expression of genes in lipid synthesis were detected by RT-PCR.The results analysis:The OD of Oil Red O staining showed that the PER2 silence group significantly decreased accumulation of lipid droplet(P<0.05).Also,PER2 gene silencing significantly reduced cellular triglyceride levels(P<0.05).Compaired with the Nontransfected group,the PER2 silence group had significantly down-regulate the mRNA expression of ACC,ACACA,FAS,LPIN1 and SCD(P<0.05),while no significance was found on LPL(P>0.05).The results above can be concluded that the PER2 gene can participate in the regulation of lipid synthesis.In conclusion,1)PER2 silencing could down-regulate the mRNA expression of some circadian clock-related and cycle-regulatory genes,and block the transition from G0/G1 phase to S phase,mainly by inhibiting the expression of CyclinDl and CDK4.2)PER2 gene silencing had significantly decreased the cell proliferation index,while no significant difference was found apoptosis index,however,Caspase 8 mRNA expression was significantly up-regulated by PER2 silencing.3)PER2 gene silencing could directly or indirectly regulate fatty acid de novo synthesis,fatty acid desaturation,triglyceride production,and lipid droplet secretion by inhibiting the expression of the transcriptional regulators SREBF1 and PPARG.