Effects Of KGFR Deletion On A549Cells Exposed To Hyperoxia

Part Ⅰ Selection of the Effective KGFR-silence Sequence and thePreliminary Effect of KGFR Deletion on A549CellsExposed to Hyperoxia[Objective] The aim of this study was to select an effective small interference RNA（siRNA） tosuppress the expression of KGFR and to explore the preliminary effect of hyperoxia on A549cells under KGFR suppressed.[Method] A549cells were gained by serial sub cultivation in vitro. Afer transfectionwith KGFR sequence-specific si-RNA and NT si-RNA for48h, A549cells were harvestedto extract total RNA and protein. KGFR mRNA was detected by real-time polymerasechain reaction (real-time PCR). Western blot was used to detect the expression of KGFR.A549cells were randomly assigned to four groups: the normoxia group, the normoxiagroup with si-RNA, the hyperoxia group, and the hyperoxia group with si-RNA. The cellsof normal groups were exposed to5%CO2and95%air, while the hyperoxia groups wereexposed to5%CO2and95%oxygen. MTT method was utilized to test cell viability.[Results] Sequence-specific si-KGFR-RNA can significantly down-regulate theexpression of both mRNA and protein in A549cells, the most effective sequence wassi-KGFR-002. Compared with the normoxia group with si-RNA, cell viability wassignificantly decreased in the hyperoxia group with si-RNA(p<0.005).[Conclusion] The growth condition of KGFR-silent cells exposed to hyperoxoa wasnot well and suspension. Part Ⅱ Effects of KGFR Deletion on A549Cells Exposed to Hyperoxia[Objective] To investigate the effects of hyperoxia on A549cells under KGFR-silence, andexplore the protective roles of α-lipoic acid and rhKGF in KGFR-silence cells exposed tohyperoxia.[Method] A549cells were fed and subcultured in vitro and randomly divided into fourgroups: I. hyperoxia group; II hyperoxia with KGFR-silence group; III hyperoxia withKGFR-silence plus LA group; IV hyperoxia with KGFR-silence plus KGF group. All groups werecultured in95%O2and5%CO2in cell culture incubator. After exposed to oxygen for24h,48h,72h,cells were harvested and detected intracellular ROS levels and apoptosis cell by flow cytometry.WST method was utilized to test the superoxide dismutase activity. Western blot wereused to test the KGFR levels in all groups at the same time.[Results] Compared to hyperoxia control group,①KGFR siRNA significantlyincreased the generation of ROS in A549cells exposed to hyperoxia(p<0.005).②LA,KGF treatment can partly relieve ROS generation, but cannot reverse the effect ofKGFR-silence(p<0.05).③KGFR siRNA significantly increased apoptosis and necrosisin A549cells exposed to hyperoxia(p<0.005).④LA,KGF treatment can partly relievecell apoptosis, but cannot reverse the effect of KGFR-silence(p<0.005).⑤The activity ofsuperoxide dismutase was not significantly different in all groups.⑥LA, KGF treatmentincreased the expression of KGFR in A549cells exposed to hyperoxia(p<0.05).[Conclusion] KGFR siRNA significantly increased the generation of ROS andapoptosis in A549cells exposed to hyperoxia. LA, KGF treatment can partly relieve theeffect of KGFR-silence and hyperoxia.