The Function And Mechanism Of Targeting RBM19 In Radio-Sensitivity Of Glioblastoma

Gliomas are the most common malignant tumors of the central nervous system,and glioblastoma(GBM),as the most common type,has the characteristics of extremely high malignancy,difficult treatment,and easy recurrence.The current standard treatment for this type of disease in clinical practice is to ensure the largest range of surgical resections under safe conditions,followed by chemoradiation.However,because the intracranial anatomy is complex or the tumor is adjacent to important functional areas,when surgical resection is not possible,adjuvant therapy has become an important part of the treatment process.Among them,radiotherapy as an important adjuvant treatment method has played a key role in improving the quality of life and prolonging the survival time of patients.However,as patients further receive radiotherapy,most patients will gradually develop different degrees of resistance to radiotherapy,which will greatly reduce the treatment effect.This phenomenon reminds us that improving the sensitivity and reducing radiotherapy resistance of glioblastoma is an important entry point to improve the effect of radiotherapy,and it is a scientific problem urgently to be solved in related research on the treatment of glioblastoma.Our research group used Zhang Feng's CRISPR Cas9 whole-genome library in the early stage,and screened several gene related to radiosensitization of gliomas.In this study,RBM19 is selected for in-depth research to explore its value in radiosensitization of glioblastoma and its related molecular mechanisms.Previous studies have shown that the RNA motif-binding protein family(RBM)plays an important role in the development of a variety of tumors,including promoting tumor proliferation,apoptosis,and so on.Recent studies have shown that RBM19 is related to mouse early embryonic development,intestinal epithelial cell proliferation / differentiation,etc.However,there are currently no relevant literature reports on tumor radiotherapy and even tumors.DNA damage,which is an important cause of cell death caused by ionizing radiation,is an important part of the mechanism related to radiosensitization for the study of DNA damage repair.It is also the focus of research in this subject.In this project,we first used the gene expression data set of 4 glioblastoma patients downloaded from the GEO public database(containing 70 normal samples and 267 GBM)to perform differential expression analysis,respectively.We found that RBM19 was indeed There are differential expression in GBM and normal brain tissue,and the expression is up-regulated in GBM.Then we performed a functional enrichment analysis of RBM19 using GSEA and found that the RBM19 gene is closely related to DNA damage.Finally,we used the clinical data of patients in the downloaded TCGA database Data and follow-up data were screened and analyzed for survival.We found that in GBM patients receiving radiotherapy,the expression level of RBM19 was associated with the patient's overall survival(OS)and disease-free survival(DFS),but there is no obvious correlation with progression-free survival(PFS).In the next stage,in order to verify the results of the previous database analysis and further study the relevant mechanism of RBM19 in radiosensitization of glioblastoma,we constructed a knockdown plasmid of RBM19 and infected U251 and U87 cells with lentivirus to construct a stable knockdown.Low cell line to complete the next experiment.Using the constructed cell line,we performed a series of experiments in vitro and found that knockdown of RBM19 has a clear radiosensitization effect.CCK-8 experiments detected U251 and U87 cell lines after irradiation.We found that after RBM19 knockdown,cell proliferation and cell viability were significantly reduced.Then we used flow cytometry to detect the apoptosis of glioma cell lines after irradiation,and found that after knocking down RBM19,the apoptosis rates of U251 and U87 cell lines increased significantly.In the cell cycle test,no significant difference was found between RBM19 knockdown and control cells.The previous database analysis suggested that RBM19 is highly correlated with DNA damage.Therefore,we determined the location of RBM19 in cells with the help of immunofluorescence technology.As a result,we found that RBM19 was located in the nucleus.In order to verify that RBM19 participates in the process of DNA damage repair,we verified the expression levels of DNA damage repair related proteins by Western Blot.We found that compared with the NC control group,the DNA of UBM and U251 cell lines in the RBM19 knockdown group was compared with the negative control group The activation of damage repair pathway-related proteins was significantly reduced,demonstrating that in different glioma cell lines,the knockdown of RBM19 significantly inhibited the activation of the DDR pathway.Through Western Blot technology,we have demonstrated that knockdown of RBM19 plays a role in the increasing of radio-sensitivity of glioblastoma through the DNA damage repair pathway.In order to further find proteins that interact with RBM19,we found that CHEK1 can directly interact with RBM19 by immunoprecipitation technology,which may be an important medium for RBM19 to participate in the DNA damage repair pathway.Through the above in vitro experiments,we made a preliminary exploration of the molecular mechanism by which knockdown of RBM19 can enhance the sensitivity of glioblastoma to radiotherapy.Finally,we performed experiments in vivo to construct a model of glioblastoma tumor-bearing radiotherapy in nude mice.We inoculated negative control U87 cells and RBM19 knockdown U87 cells into the brain of nude mice and found that after irradiation The survival time of the tumor-bearing nude mice in the RBM19 knockdown group was significantly prolonged,and the tumor volume of the tumor-bearing nude mice in the RBM19 knock-down group was relatively smaller,and the sensitivity of radiotherapy was significantly improved under the same time period.In conclusion,this research firstly confirmed that knockdown of RBM19 has a significant radiosensitization effect on glioblastoma,and proved that RBM19 affects the radiosensitivity of glioblastoma by regulating DNA damage repair.At the same time,we also found the protein to interact with RBM19 was CHEK1.The above results show that rbm19 is a new molecular target for radiosensitization of glioblastoma.It provides a new research direction and potential treatment method for clinical radiotherapy and sensitization of glioblastoma,and has certain significance for enhancing the radiotherapy effect of glioblastoma.