Effects of OXSR1 Activity on Apoptosis of Madin-Darby Canine Kidney Cells Through p53-dependent and-independent Pathways
[Objective]This study was aimed to investigate the role of oxidative stress-responsive 1(OXSR1)in the apoptosis of Madin-Darby canine kidney cells(MDCK),so as to provide a reference for better understanding the apoptotic pathway in MDCK and study on interventional therapies targeting OXSR1 gene for MDCK apoptosis.[Method]OXSR1 gene knockout cell lines were produced using CRISPR/Cas9 technology on MDCK-Cas9 cells.OXSR1 gene competent and inactivae cell lines were generated by transfecting OXSR1 gene backtill and inactivation plasmids into OXSR1 gene knockout cells.MMC,JNJ-26854165 and Rotenone were used to stimulate wild-type,OXSR1 gene knockout,gene competent and inactive cells,and the apoptosis through p53-dependent and-independent pathways and ROS signaling pathway were detected using flow cytometry.The expression of relevant apoptotic genes were detected by Real-time quantitative PCR.[Result]There was apparent Indel features in the target sequences,it confirmed OXSR1 gene knockout cell lines were successfully constructed.Western blotting results indicated that OXSR1 gene competent and inactive cell lines were successfully constructed.Flow cytometry determined that MMC,JNJ-26854165 and Rotenone could induce varying degrees of cell apoptosis.When stimulated with MMC and Rotenone,the apoptosis rate of OXSR1 gene knockout cells was extremely significantly or significantly lower than that of wild-type and OXSR1 gene competent cells(P＜0.01 or P＜0.05),and there was no significant difference with OXSR1 gene inactive cells(P＞0.05).When stimulated with JNJ-26854165,the apoptosis rate of OXSR1 gene knockout cells was extremely significantly or significantly lower than that of wild-type,OXSR1 gene competent and inactive cells(P＜0.01 or P＜0.05).Real-time quantitative PCR results showed that when cells were stimulated with MMC and Rotenone,the expression of BCL-XL gene in OXSR1 gene knockout cells was extremely significantly or significantly higher than that of wild-type and OXSR1 gene competent cells(P＜0.01 or P＜0.05),and there was no significant difference with OXSR1 gene inactive cells(P＞0.05).When cells were stimulated with JNJ-26854165,the expression of BCL-XL gene in OXSR1 gene knockout cells was extremely significantly higher than that of wild-type,OXSR1 gene competent and inactive cells(P＜0.01).The expression of NOXA gene in OXSR1 gene knockout cells was extremely significantly lower than that of wild-type,OXSR1 gene competent and inactive cells(P＜0.01).[Conclusion]The kinase activity of OXSR1 gene regulated the transcription of BCL-XL gene in MDCK after adding MMC,JNJ-26854165 and Rotenone,and influenced the apoptosis through p53-dependent and-independent pathways and ROS pathway.
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