Target Gene Prediction Of MiR-196a-3p、preliminary Validation In Tumour Cells And Functional Analysis

[Objective]The aim of this research isto predict the target gene of miR-196a-3p by using microRNA target gene prediction website and functional analysis network to verify the target gene of miR-196a-3p in tumor cells.And provide guidance for further study of the regulatory mechanism of miR-196a-3p in tumor formation and progression.[Methods]Part Ⅰ:According to the scoring rules of different software,the predicted target genes were obtained from the microRNA target gene prediction software DIANA-MICROT,MIRDB,MICRORNA and TargetScan respectively,and the intersection of the four databases were selected as the reliable target gene.Part Ⅱ:1.The miR-196a-3p mimics was transfected into MDA-MB-231 and OCI-LY3 cells to achieve high expression of miR-196a-3p in cell lines.2.Real-time PCR was used to detect miR-196a-3p mimetic,the expression of the target gene in the cell changes.Part Ⅲ:1.NCBI Gene,KEGG,Genecards and other databases were used to annotate the functions of the validated target genes.String and Panther protein interaction search tools were used to analyze the protein interaction network of the target proteins and the proteins with protein interaction relationship were enriched in biological pathways and annotated:in biological processes.[Results]Part Ⅰ:The target gene of miR-196a-3p was predicted by the four bioinformatics websites combined with NCBI Gene,KEGG and Genecards databases.Proto-oncogene ARF4,CORO1C,ZNF280B,NRP2 were screen outas candidate target genes by the miR-Ontology Database、Tumor Suppressor Gene Database and Cancer Gene Census.Part Ⅱ:The expression of NRP2 in MDA-MB-231 cells transfected with miR-196a-3p mimic was significantly inhibited(P<0.05).The results of Western blot showed that the expression of NRP2 protein was positively correlated with TGF-β1 and negatively correlated with miR-196a-3p,suggesting that miR-196a-3p was involved in the development of tumor by down-regulating the downstream expression of NRP2.However,the expression of ARF4 and ZNF280B protein in OCI-LY3 cells decreased significantly after transfection of microRNAs-196a-3p mimic,suggesting that microRNAs-196a-3p may be involved in the development of diffuse large B lymphoma by down-regulating the expression of ARF4 and ZNF280B.PartⅢ:The interacting genes with ARF4 are ARF1,ASAP1,GBF1,ARFGAP3,COPG2,ARFGAP1,COPG1,PLD2,ARFGAP2,COPB2,and NRP2.The interacting genes with NRP2 are NRCAM,PLXNA4,PLXNA3,SEMA3A,PLXNA1,SEMA3F,FLT4,KDR,FLT1,and VEGFC,respectively.They are ZNF280A,PSIP1,POTEE,POTEI,POTEJ,POTEF,HSFY2,HSFY1,SETD4,RIPK4.The main biological processes involved are regulation of extracellular stimulation,cell growth,localization,metabolism and so on.[Conclusion]The target genes ARF4,CORO1C,ZNF280B and NRP2 were predicted by target gene prediction website and gene function analysis website.NRP2,ARF4,ZNF280B are the downstream reliable target genes of microRNAs-196a-3p.MicroRNAs-196a-3p may be involved in the tumorigenesis and development process through the up-regulation of the expression of downstream target genes NRP2,ARF4,ZNF280B.