The Function Of OSR2 In Cell Cycle Arrest

Objective:Screen out the activated signal pathways and the differentially expressed genes in early renal ischemia-reperfusion injury(RIRI)by using bioinformatics technology.Observe expression pattern of pivotal transcription factor OSR2(odd-skip-related 2)in renal tubular epithelial cells both in vitro and in vivo.And preliminarily examine its regulation of cell cycle and apoptosis,and its role in the kidney injury.Methods:1.Searching the early renal ischemia injury datasets from GEO database.After uniformized process of these datasets,DEGs were obtained.Cisplatin and hydrogen peroxide were used to construct in vitro AKI model.Common differentially expressed genes(DEGs)were obtained from uniformized AKI model transcriptome sequencing results and GEO datasets.Besides,compare the conjunction DEGs numbers to screen suitable AKI model.Use David website to proceed GO pathway and KEGG pathway enrichment analysis by submitting the conjunction DEGs.STRING and Mogrify website are used to find hub-DEGs,R used to visualize those analysis results.2.The expression patterns of hub-DEGs in the in vitro hypoxia model were detected by qPCR and Western blot.In the cell cycle aspect,construct fluorescent ubiquitination-based cell cycle indicator(FUCCI)cell in vitro and then observe the fluorescence changes of FUCCI cells in response to hypoxia stimulation.The expression changes of cycle-associated proteins such as cyclin E1,cyclin D1,p21 and p27 were detected by Western blotting.Cells were stained with Propidium Iodide to analysis the cell cycle changes by using flow cytometry.In order to observe the apoptosis situation,the hypoxic-stimulated cells were stained with Hoechst 33342 and observe the number of hyperchromatic apoptosis bodies with fluorescence microscopy.The apoptotic associated proteins Bax,Bcl-2,Cleaved caspase-3,and Cleaved PARP were detected by Western Blotting.Annexin V and PI double staining detect changes of apoptotic rate of in vitro hypoxia model by flow cytometry.3.Establish ischemia-reperfusion mouse model,immunofluorescence staining was used to observe the expression and localization of hub-DEGs,HE staining was used to evaluate renal tubular injury.Analyzing the possible relation of hub-DEGs OSR2 expression with the renal injury.Results:1.DEGs were obtained by co-analyze eligible dataset GSE126805 screened from GEO website with the transcriptome sequencing results of in vitro AKI model.The analyzing result shows that up-regulated DEGs in GSE126805 data set,hypoxia group and cisplatin group were168,755 and 72 respectively.The value of common DEGs in the hydrogen peroxide stimulation group and GSE126805 was 74(including FOSB,OSR2 and Gadd family).The KEGG pathway enrichment analysis of these common DEGs shows the activation of apoptosis signaling pathway.In addition,these common DEGs were analyzed by STRING database and cytoscape software.The high degree scores gene concentrated on apoptosis-related proteins Gadd45g,TRIB1 and IER5.The OSR2 was screened based on STRING database and transcription factor score as hub-DEGs.2.The expression way of OSR2 in the hydrogen peroxide stimulation model will detected by qPCR and Western blot.Found that OSR2 reached its peak at 2h and gradually decreased to low level after 4h by comparing with control group.FUCCI detection cells show increased red fluorescence at the early stage and green fluorescence transformation after 4h than untreated group.Western blotting shows that compared with control group,the proteins of Cyclin D1 and Cyclin E1 which promote the transition from G₁ phase to S phase both increased at 4h.p27,the protein makes G₀ phase blocked,gradually decreased after 4h.Flow cytometry shows that the proportion of S phase cells in hydrogen peroxide stimulation group was increased than control group.Besides,Hoechst 33342 staining shows that the nuclear hyperchromatism increased significantly than control group after 4h.Simultaneously western blotting shows that compared with control group,the expression of Bcl-2 down-regulated significantly after 4h,But,the expression of apoptosis activation protein Bax cleaved caspase3 and cleaved PARP was up-regulated after 4h.Flow cytometry shows that the proportion of apoptotic cells increased significantly than control group after 4h.3.We also found that in the 26min renal arterial clipping with contralateral nephrectomy RIRI mouse,OSR2 was significantly expressed in the proximal tubule epithelium compared with sham group at 4h.In addition,HE staining renal tubule injury score showed significant proximal tubule injury in the 26min renal arterial clipping with contralateral nephrectomy group compared with the sham group.Conclusion:1.Bioinformatics analysis obtain common DEGS according to the GSE126805 dataset and transcriptome sequencing results of in vitro hypoxia model.Pathway enrichment revealed early increase in transcription levels and activation of apoptotic pathways.The key transcription factor OSR2 was screened out from the STRING and Mogrify datasets.2.In vitro model find that the G₁/S phase of renal tubular epithelial cells was blocked after OSR2 expression,and the apoptotic process of the cells began to activate.3.In vivo experiments in RIRI mice showed that OSR2 expression in renal tubular epithelial cells was associated with renal tubular epithelial cell injury.4.In the early stage of ischemia-reperfusion,OSR2 may be involved in the development of renal tubular injury by triggering cell cycle arrest and lead to the onset of apoptosis.