Effect Of SiRNA Silencing ChOP Gene Expression On Apoptosis Of Type Ⅱ Alveolar Epithelial Cells Exposed To Hyperoxia

【Objective】Bronchopulmonary dysplasia(BPD)is a common lung disease that seriously threatens the health of premature infants,especially very early preterm infants.Long-term inhalation of high concentrations of oxygen in premature infants can cause BPD.Previous studies have found that type II alveolar epithelial cells(AECⅡs)increases the rate of apoptosis in hyperoxia-induced BPD,and the apoptotic pathway was activated by endoplasmic reticulum stress(ERS)which mediated by the CCAAT/enhancer-binding protein-homologous protein(CHOP).However,it is unclear whether the change in CHOP expression affects the apoptosis of AECⅡs exposed to hyperoxia.This study is mainly to detect the expression levels of CHOP,Bcl-2 and Bax and the rate of apoptosis in AECⅡs lines which exposed to high oxygen after transfected with CHOP-siRNA.【Methods】The siRNA based sequence targeting CHOP gene was designed and synthesized In vitro,and the pcDNA3.1(+)-CHOP-siRNA and pcDNA3.1(+)-empty plasmid were transiently transfected into AECII cells by Lipofectamine2000 liposome-mediated method.RT-PCR and Western blot were used to detect the effect of CHOP gene silencing after siRNA interference.The cells were exposed to hyperoxia after transfection for 24 h,and the cells were divided into air group,air + pcDNA3.1(+)-empty plasmid group,air + pcDNA3.1(+)-CHOP-siRNA group,hyperoxia group,hyperoxia+ pcDNA3.1(+)-empty plasmid group,hyperoxia + pcDNA3.1(+)-CHOP-siRNA group.Under inverted phase contrast microscope the morphology of the cells was observed.The cells of each group were harvested after 48 h of air or high oxygen exposure.Apoptosis was detected early by Annexin V/PI double staining flow cytometry.The expression levels of CHOP,Bcl-2 and Bax were detected by real-time quantitative PCR and Western blot.【Results】(1)AECⅡs in the air group protruded more pseudopods and adhered to the bottom of the bottle,showing an island-like growth pattern.The cell morphology in The air+pcDNA3.1(+)-empty plasmid group and air+pcDNA3.1(+)-CHOP-siRNA group had no significant difference compared to that in the air group;AECⅡs in the hyperoxia group stretched hypertrophy,cell adhesion was not tight,pseudopod decreased,nucleoli decreased or disappeared,the number of suspended cells in culture medium increased,and a few cells showed vacuolar changes.There was no significant difference between the hyperoxia + pcDNA3.1(+)-empty plasmid group and the hyperoxia group.The morphology of the cells in the hyperoxia + pcDNA3.1(+)-CHOP-siRNA group was slightly larger than that in the air group,but the degree of cell expansion in the hyperoxia group was smaller,the number of suspended cells was decreased,and the cells were free of vacuoles.(2)Compared to the air group,the rate of AECⅡs apoptosis was significantly increased in the hyperoxia group;the expression levels of CHOP mRNA and protein were increased in the hyperoxia group;the expression levels of Bcl-2 mRNA and protein were decreased in the hyperoxia group;and the expression levels of Bax mRNA and protein were increased in the hyperoxia group.(3)After the AECⅡs transfected with CHOP-siRNA,Compared with the hyperoxia group,the rate of AECⅡs apoptosis was decreased in the hyperoxia+pcDNA3.1(+)-CHOP-siRNA group,the expression levels of CHOP mRNA and protein were decreased,the expression levels of Bcl-2 mRNA and protein were increased,.the expression levels of Bax mRNA and protein were decreased.【Conclusions】(1)Hyperoxia could lead to increase the rate of AECⅡs apoptosis,increase the expression levels of CHOP,decreased the expression levels of anti-apoptotic Bcl-2,and increase the expression levels of proapoptotic gene Bax,suggesting that the high expression levels of CHOP could down-regulate the expression levels of anti-apoptotic gene Bcl-2 and promote the expression levels of proapoptotic gene Bax,and lead to increase the rate of AECⅡs apoptosis eventually.(2)CHOP-siRNA transfected to AECⅡs could block the expression levels of CHOP and induce the rate of AECⅡs apoptosis after high oxygen exposure.