Exploration Of Genes Conferring To BRAF^V600E Inhibitor Resistance By Bioinformatics Analysis Of Melanoma Database

BackgroundMelanoma is a malignant tumor of melanocytes that exists in the skin or mucous membranes.According to a survey conducted by the World Health Organization（WHO）in recent years,there are approximately 130,000 new cases in the world each year.The number of new melanoma patients in every year is gradually increasing.Melanoma is characterized by its deterioration,rapid metastasis,poor prognosis,and limited therapeutic effects from chemotherapy and radiotherapy.Therefore,melanoma is a huge threat to human health in the world.BRAF,as a proto-oncogene,belongs to the RAF gene family,which include ARAF,BRAF and CRAF.BRAF is frequently mutated in many kinds of tumors especially the melanoma and thyroid cancer.BRAF^V600E mutations in melanoma is about 80%among all the BRAF mutations.The kinase activity of BRAF active mutation will increase around five hundred times than that of wild-type BRAF,which will significantly promote the proliferation and immigration of tumor cells.The latest research showed that the role of BRAF mutation in melanoma is comprehensive.BRAF active mutation not only promotes melanoma cells itself proliferation and immigration,but also changes the status of various immune cells in the tumor microenvironment,which in turn causes immunosuppression and immune escape of tumor cells.In addition,BRAF active mutations can also induce some tumor-related chemokines,which indirectly facilitate tumor proliferation and metastasis.Given the high frequency and activity of BRAF^V600E mutation in tumors,the exploration of inhibitors targeting BRAF^V600E attracted increasing attentions in melanoma therapy.So far,Vemurafenib,Dabrafenib and Cornafenib have been approved by the U.S.Food and Drug Administration to treat BRAF^V600E mutation melanoma.Vemurafenib was the first to be approved for the treatment of melanoma patients harboring BRAF^V600E mutations.Vemurafenib is characterized by quick effects,and the condition of patients improved quickly after the medication,but most of the patients treated with Vemurafenib will develop resistance around nine months.Furthermore,the patients treated with dabrafenib will also develop resistance around seven months after treatment.Therefore,it is of importance to explore the underlying mechanisms for BRAF^V600E inhibitor resistance and to develop combined therapy strategies in melanoma therapy.Combined therapy,such as vemurafenib and MEK inhibitors,could dramatically delay the development of drug resistance and enhance the therapeutic effect and increase the survival rate of treated patients.However,the patients received combined therapy will also inevitably develop drug resistance.Therefore,it is urgent to investigate the underlying mechanisms of resistance to BRAF^V600E inhibitors in melanoma therapy.Gene chip technology,also known as c DNA microarray hybridization,is a technology that uses nucleotide hybridization to sequence genes.It is characterized by high-throughput,high-speed,and rapid parallel analysis of a large number of genes.The advent of gene chip technology makes it possible to search for differentially expressed genes in drug sensitive and drug resistant cells or patients in large database.By analyzing the different fluorescence density displayed on the gene chip,the relative expression profile of these genes between different melanoma cells and samples can be obtained.Further analysis of the differentially expressed genes,it is possible to screen the genes responsible for drug resistance in melanoma based on large database.ObjectiveThis study excavated melanoma gene chip data and screened the candidate genes related to melanoma drug resistance.Through the establishment of gene engineered cell lines,we further analyzed these candidate genes and ultimately confirmed the genes conferring drug resistance to BRAF^V600E inhibitors in melanoma.The established method will provide a certain experimental basis for the research of drug resistance in melanoma patients.MethodsIn this study,we analyzed the differentially expressed genes in the three data sets of GSE114443,GSE45648 and GSE148638(BRAF^V600E inhibitor resistant group versus response group).By taking the intersection of genes differentially expressed between different data sets,26 genes were primarily identified.Then,further functional enrichment analysis,including GO analysis and KEGG pathway analysis,was carried out.In addition,protein-protein network analysis was performed to reveal the potential relationships between genes,5 genes were primarily identified.By analyzing the expression of these 5 genes in primary melanoma and metastatic melanoma,we found that VCAM1 and PDGFD are highly expressed in metastatic melanoma.Finally,VCAM1 and PDGFD were screened out by comprehensive analysis,which were related to the drug resistance of melanoma to BRAF^V600E inhibitor.The credibility of the analysis results was confirmed by constructing gene knockdown cell lines.ConclusionsBy integrating information from different databases,screening drug resistance genes with bioinformatics methods,and further verifying through cell biology experiments,we initially found that the increase of VCAM1 and PDGFD would promote the resistance of A375 cells to the BRAF^V600E inhibitor PLX4032.