Study on the Function of PCYOX1L Gene Regulating LPS-induced Bovine Endometrial Epithelial Cells
[Objective]This study was aimed to investigate the regulatory effects of prenylcysteine oxidase 1 like protein(PCYOX1L)on lipopolysaccharide(LPS)-induced inflammation,proliferation,and apoptosis in bovine endometrial epithelial cells(BEECs),so as to clarify the regulatory mechanism of PCYOX1L gene on the occurrence of endometritis in dairy cows.[Method]An in vitro inflammation model was constructed by stimulating BEECs with LPS,and small interfering RNA(si-PCYOX1L)and overexpression vector(pcDNA3.1-PCYOX1L)of PCYOX1L gene were designed and synthesized,Lipofectamine 3000 transfection reagent was used to transfect the BEECs with pcDNA3.1-PCYOX1L.The effects of interference and overexpression of PCYOX1L gene on the mRNA expression of cellular inflammation,proliferation and apoptosis marker genes were detected by Real-time quantitative PCR.Reactive oxygen species(ROS)level in cell was detected by kit,the protein expression of interleukin-1(IL-1)was detected by ELISA method,and the cell viability,proliferation and cycle were detected by EdU,CCK-8 and flow cytometry.Cellular mitochondrial damage was detected by a mitochondrial membrane potential kit,and the apoptosis was detected by flow cytometry.[Result]pcDNA3.1-PCYOX1L overexpression vector was successfully constructed in this experiment,and the interference effect of si-PCYOX1L-385 was screened out to be the best.Compared with control group,after interfering with PCYOX1L gene,the mRNA expression of inflammatory marker genes(IL-1β,IL-6 and IL-8)was extremely significantly down-regulated(P＜0.01),the IL-1 protein content was significantly increased(P＜0.05),and the intracellular ROS level was extremely significantly decreased(P＜0.01).The mRNA expressions of proliferative marker genes(CDK2,CDK4,PCNA and CCND2)were extremely significantly or significantly up-regulated(P＜0.01 or P＜0.05),and the cell viability was increased,promoting the transition of cells from S phase to G2 phase.The expression of pro-apoptotic gene Bax was significantly decreased(P＜0.05),and the expression of anti-apoptotic gene BCL2 was increased,reducing the apoptosis rate of BEECs(P＜0.01).However,the results were exactly the opposite after overexpression of PCYOX1L gene.[Conclusion]PCYOX1L gene promoted inflammation,inhibited cell proliferation,and promoted apoptosis in BEECs.This results provided basic data for further investigation of the molecular mechanism of PCYOX1L gene regulating the occurrence of endometritis in dairy cows.
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