Bioinformatics Analysis Of CircRNA In Cells Exposed To Ionizing Radiation And Study On The Role And Mechanism Of Kremen2 In Non-small Cell Lung Cancer

Objective:Radiotherapy is one of the most common treatments for cancer.However,due to the various side effects after radiotherapy,the application of radiotherapy technology still has some limitation.Therefore,it is of great significance to comprehensively understand the response of cells to ionizing radiation(IR).Circular RNAs(circRNAs),as a structurally stable non-coding RNA,have recently been found to regulate a variety of cellular physiological processes.However,the changes of their expression and their role in cells after IR exposure are unclear.Therefore,in this study,we identified and analyzed the differentially expressed circRNA in irradiated cells and preliminarily predicted its role in ionizing radiation injury and its potential biological function,so as to provide a new idea for optimizing the effect of radiotherapy.Method:Human embryonic kidney(HEK)293T cells were divided into control group and irradiation group.The cells in the irradiation group were irradiated with 8Gy gamma rays.24 hours after irradiation,two groups of cells were collected at the same time and total RNAs were extracted.CircRNAs were identified by RNA-seq high-throughput sequencing and the differentially expressed circRNAs were screened.We performed qRT-PCR to verify the differentially expressed circRNAs.GO and KEGG analysis were used to enrich the source genes of differentially expressed circRNAs to predict the possible biological function and involved signal pathway of circRNAs.Finally,the network of circRNAmiRNA-mRNA was used to indirectly predict the function of differentially expressed circRNAs.Results:A total of 5592 circRNAs were identified and 1038 circRNAs were found new.The expression of 158 circRNAs was changed significantly after IR exposure withthe threshold of fold change?2 and P<0.05.Among them,61 circRNAs were up-regulated and 97 circRNAs were down-regulated in the irradiation group.Through the analysis of GO,KEGG and network map,we predicted that differentially expressed circRNAs may be involved in biological functions such as oxidative phosphorylation,epithelial growth factor receptor(EGFR)tyrosine kinase inhibitor resistance and mTOR signal pathway.Conclusion:In the process of cell injury induced by ionizing radiation,differentially expressed circRNAs may be involved in cell stress changes and cell damage response repair and other related processes.Objective:Lung cancer is one of the most common cancers in the world,which is characterized by low survival rate and poor prognosis.In recent years,some breakthroughs have been made in the diagnosis and treatment of lung cancer,but the five-year overall survival rate of lung cancer is still low.So finding new targets for the treatment of lung cancer is still a hot issue in cancer research.Previous studies have shown that Kremen2 is highly expressed in multiple myeloma and promotes its occurrence and development.However,there is no reports on related study in lung cancer.The purpose of this study is to investigate the expression of Kremen2 in non-small cell lung cancer(NSCLC)cells and its effects on proliferation,migration and apoptosis,and explore its molecular mechanism.It may provide theoretical basis and possible new therapeutic targets for the treatment and early prevention of NSCLC.Methods:NSCLC cells,A549 and H1703,were used to construct Kremen2 knockdown cell lines using lentivirus-mediated small interfering RNA.The cell survival and proliferation ability were observed by colony formation assay;cell migration ability was examined by Transwell assay and wound healing assay;apoptosis was measured by flow cytometry;tumor-bearing nude mice were used to test tumor formation and proliferation of NSCLC cells in vivo;the expression of proliferation-related protein was detected by immunohistochemistry;qRT-PCR was used to measure the mRNA levels of the genes involved in proliferation,apoptosis,and migration;the Kremen2-interacted proteins were examined by co-immunoprecipitation assay;the expression of proliferation-related pathway proteins was detected by Western blot.Results:1.Kremen2 is expressed at higher level in NSCLC cells than in normal cells.2.Knockdown of Kremen2 inhibits the proliferation and migration ability of NSCLC cells.3.Knockdown of Kremen2 promotes apoptosis in NSCLC cells.4.Knockdown of Kremen2 inhibits the tumorigenicity of NSCLC cells in nude mice.5.Knockdown of Kremen2 inhibits STAT3 phosphorylation and Kremen2 interacts with SOCS3,which is a negative regulator of STAT3.Conclusion:Kremen2 may promote the proliferation and migration of NSCLC cells through the phosphorylation and activation of STAT3,thereby driving the initiation and progression of lung cancer.