Significance Of Tumor Associated R-loop Binding Proteins In Tumorigenesis, Development And Treatment

Background and objective: R-loop is a special nucleic acid structure composed of DNA/RNA hybrid strand and single strand DNA,which is mainly formed in the process of transcription.In the normal physiological process,R-loop can participate in transcriptional regulation,immunoglobulin conversion and recombination,maintenance of telomere stability and other specific physiological activities,but when the imbalance of R-loop regulation leads to the persistence of this structure,it can cause replication pressure,DNA damage,genomic instability,and thus become a potential risk factor for the occurrence and development of cancer.Therefore,the precise regulation of R-loop is very important for maintaining normal physiological processes and genomic stability.There are a large number of proteins that interact directly with R-loop structure in the body,which are called R-loop binding proteins.The regulation of Rloop depends on this series of binding proteins,which play an important role in the regulation of R-loop structure.The purpose of this study is to reveal the expression correlation of R-loop-binding protein in pan-cancer by analyzing the expression difference of R-loop-binding protein in many kinds of cancer histology,and to analyze the drug sensitivity of R-loop expression.Through analysis and experimental verification,this study provides a prospect for the potential application of R-loop-binding protein as a prognostic marker or drug target in tumor therapy.Research methods: This study is mainly divided into three parts:histological data analysis,drug sensitivity analysis and cell level experimental verification.With reference to two independent experimental projects for the detection of R-loop-binding proteins,we identified 116Rloop-binding proteins as subjects.We analyzed the differential expression of these proteins at gene level and protein level in a variety of oncology databases,and found that 64 tumor-related R-loop-binding proteins with significant tumor consistency and high expression were found in four oncology databases.Through the immunohistochemical experiments on the protein MCM5 found above,the reliability of our above analysis results was verified.Then we clustered 64 R-loop-binding proteins into fourcluster protein interaction groups according to the expression distance of these proteins,and the correctness of typing was proved by protein coimmunoprecipitation experiments in the two groups.Finally,we analyzed the protein expression level of the four protein clusters and the drug sensitivity data in GDSC database.According to the results of drug screening,we selected U2AF2 gene to construct overexpression stable line.Through CCK8 cell proliferation experiment,drug treatment to detect cell damage and replication pressure level,immunofluorescence and other cytological experiments to verify the reliability of our analysis and prediction.Research results: After analyzing 116R-loop-binding proteins in a variety of oncology databases,we defined 64 tumor-related R-loopbinding proteins that were significantly overexpressed in tumors,and analyzed the expression of MCM5,which belongs to tumor-related R-loopbinding proteins,in cancer and paracancerous tissues by immunohistochemistry.It was found that MCM5 was highly expressed in tumors compared with adjacent tumors.According to the calculation of protein expression distance,we found that there was a close distance between many R-loop-binding proteins,which could be divided into four clusters,such as DDX18 and FBL,and their interaction was proved by immunoprecipitation.By analyzing the drug sensitivity of four clusters of R-loop-binding proteins,we found that the third cluster of genes was significantly resistant to MAPK inhibitors and sensitive to DNA replication pressure.Subsequently,we established a stable cell line of colorectal cancer cells of U2AF2.Through the drug sensitivity test of CCK8,it was found that U2AF2 was sensitive to DNA damage.Western blotting showed that the level of DNA damage and replication pressure increased after the cells overexpressing U2AF2 were administered,and the immunofluorescence experiment also proved that the repair of DNA homologous recombination slowed down.Conclusion: Our study reveals the relationship between newly discovered R-loop binding proteins and cancer,and provides some new ideas for the clinical application of these proteins as therapeutic strategies in the future.