Preliminary Study On BRAF Gene Mutation By ENU In Mouse Tumor Cells And Brain Metastasis

Objective:Breast cancer is the second most common malignant tumor that is prone to brain metastasis after lung cancer.With the improvement of diagnosis and treatment,the survival time of patients with recurrent and metastatic breast cancer is significantly prolonged,which significantly increases the chance of brain metastasis.Therefore,the breast The incidence of cancer brain metastases has increased year by year.The BRAF gene is a conserved serine/threonine protein kinase that is widely present in mammals and has the highest frequency of mutations in malignant tumors.In malignant tumor cells,if the BRAFV600E mutation occurs,the MEK-ERK pathway is continuously activated,which promotes the proliferation of cells and inhibits apoptosis,thereby causing tumor invasion and metastasis.Schindler et al.reported a high mutation rate in ganglion gliomas and astrocytomas in pleomorphic yellow astrocytoma by studying BRAF gene mutations in 1,320 cases of nervous system tumors.66%,18%and 33%.60%to 80%of star cell tumors show an increase in the activity of BRAF,which is caused by changes in the gene's replication process or point mutationsEthylnitrosourea(ENU)is a synthetic organic compound that causes random mutations,especially single-base mutations,in a variety of organisms.ENU can replace the ethane group into the group of some DNA bases by alkylation reaction,and alkylate the corresponding base,causing errors in base pairing,resulting in mutation of the biological gene,and the reaction does not depend on any Metabolic process.The mutagenic effect of ENU exists and functions in various tissues and organs of organisms.For nearly 50 years,ENU has been used as an effective mutagen in mouse developmental models.In this study,ENU was used to induce mutations in selected breast cancer cells,which could generate a large number of random mutations in mouse cells in a short period of time.To study the effect of BRAF gene on brain metastasis of breast cancer and explore the inheritance of organisms.Factors and animal models of human disease.These studies can be used to find treatment strategies to make drug-resistant tumors sensitive and to study the feasibility of being a therapeutic target for breast cancer brain metastasis.Methods:1.Using Dabrafenib to treat mouse breast cancer cells,CTG assay was used to find the appropriate drug treatment concentration and to detect the inhibitory effect of Dabrafenib on mouse tumor cells at different concentrations.2.4T1 cells after inhibition by Dabrafenib screening were treated with acetylnitrosourea(ENU).3.Pick monoclonal cell culture,extract DNA,and sequence to detect whether BRAF gene has mutation.4.Establish a mouse breast cancer model to determine whether BRAF mutations have an effect on brain metastasis,and the sensitivity of EMU-induced tumor sensitivity to Dabrafenib after mutation.To establish a brain metastasis model of mouse breast cancer tumors,compare them by group,and simultaneously pass Dabrafenib drug to verify whether they have inhibitory effect on brain metastasis of mouse breast cancer.Results:The results of previous experiments show that Dabrafenib drugs have a certain inhibitory effect on the growth of tumor cells,and ENU can still induce mutations in the BRAF gene in 4T1 cells screened and inhibited by Dabrafenib.In vivo experiments,Dabrafenib inhibited BRAF-M better than Origin cells.Conclusion:ENU can induce mutations in mouse breast cancer cells,which can make breast cancer cells sensitive to Dabrafenib,and has a clinical application prospect for the treatment of breast cancer brain metastasis.