DDX39B Contributes To The Proliferation Of Colorectal Cancer Through Direct Binding To CDK6/CCND1

Background and PurposeColorectal cancer(CRC)has become the third most common cause of cancer and the leading cause of cancer death worldwide to threaten our lives and health.The dysregulation of cell proliferation and cell cycle is an important biological feature in the development of colorectal cancer.The cell cycle related genes are important to maintain the normal cell division and their abnormal expression will lead to tumorigenesis.Many critical checkpoints are responsible for replication errors detecting and arresting the cell cycle until repairs are completed between the different phases of the cell cycle.One of the most important and widely studied is the Gl/S checkpoint.Several kinds of cyclin-dependent kinases(CDKs)and their cyclin partners can form the complexes to play a fundamental role in the regulation of G1/S checkpoints,such as the cyclinDl(CCND1)/cyclin dependent kinase 4(CDK4)/cyclin dependent kinase 6(CDK6)or the cyclin E(CCNE)/cyclin dependent kinase 2(CDK2).DDX39B(also known as UAP56 or BAT1)is a DEAD box family helicase,which plays a key role in mRNA binding,splicing and export.It has been found that it is upregulated as an oncogene in a variety of tumors.It plays an important role in the export of mRNA from the nucleus to the cytoplasm,and is a basic splicing factor necessary for the association of U2 small nuclear ribonucleoprotein with pre-mRNA.Therefore,the abnormal expression of DDX39B will have many adverse effects on mRNA processing and export.The function of related proteins will also be affected.In a word,this study used used relevant experiments to verify the direct molecular relationship and post-transcriptional regulation mechanism of DDX39B in promoting abnormal proliferation of colorectal cancer.Our research provides new insights into the role of DDX39B in promoting tumor proliferation,and may be able to study more and more new drugs to prevent the progress of cell cycle.Method and Results1.The expression of DDX39B in colorectal cancer and its relationship with prognosis1)Using bioinformatics methods and public database,we found that DDX39B was highly expressed in colorectal cancer and was associated with poor prognosis.2.Overexpression of DDX39B can promote abnormal proliferation and cell cycle progression of colorectal cancer1)Firstly,we constructed colorectal cancer cell lines with stable overexpression and knockdown of DDX39B,qRT-PCR and Western blot were used to detect the transfection efficiency.2)Through CCK8 and colony forming assays and other functional experiments including cell cycle flow cytometry,we found that overexpression of DDX39B can promote abnormal proliferation of colorectal cancer cells in vitro.3)The nude mice xenograft models that were injected with the DDX39B knockdown cells and the control cells.and we used the immunohistochemistry staining to detect the protein expression levels of Ki67 and we discovered DDX39B can affect the proliferation of colorectal cancer in vivo.3.Mechanism exploration of DDX39B downstream targeted genes1)Through bioinformatics methods,enrichment analysis was used to find that DDX39B might affect the cell cycle changes of colorectal cancer cells,and correlation analysis was used to find the targeted genes CDK6 and CCND1.2)It was proved that DDX39B can regulate the CDK6/CCND1 expression by qRT-PCR and Western blotting experiments.3)RNA binding protein immunoprecipitation sequencing(RIP seq)confirmed that DDX39B directly binds to the first exon of CDK6/CCND1 pre mRNA and upregulates its expression.4)The splicing experiment in vitro determined by DNA gel electrophoresis confirmed that DDX39B can promote the splicing mature of CDK6/CCND1 precursor mRNA.5)The rescue experiment showed that CDK6/CCND1 was a downstream effector of the proliferation of colorectal cancer cells mediated by DDX39B.ConclusionsThis study verified that DDX39B can promote the abnormal proliferation of colorectal cancer using bioinformatics,cell experiments and the nude mice xenograft models,and the mechanism may be the direct binding to the first exon of the CCND1/CDK6 and upregulate their expression by DDX39B through assisting their splicing and export.