Establishment And Functional Research Of "c-Myc/miRNA/target Genes" Interaction Molecular Network In NPC Cells

miRNA,a nova in gene regulation,negatively regulates gene expression.It plays an important role for tumorigenesis and tumor development by regulation of many oncogenes and tumor suppressor genes.A interaction molecular complex network is composed of transcription factors,miRNAs and target genes.There is abnormal expression or inactivation of some miRNAs in nasopharyngeal carcinoma.c-Myc,a highly conserved proto-oncogene.In recent years,many studies have discovered that abnormal activation and overexpression of c-myc oncogene closely correlate with the occurrence,development and prognosis of NPC.Western-blot results revealed that there was high-level expression of endogenous c-myc in NPC 5-8F cells.Previous studies showed that knock-down of c-Myc could inhibit cell growth and cell cycle progression,migration and invasion of NPC cells by down-regulating some molecules of Rb/E2F pathways.Several studies indicate that c-Myc also can regulats some miRNAs. To explore the miRNA regulation network in NPC cells with knockdown of endogenous c-Myc expression,miRNA microarray was applied to investigate the differentially expressed miRNAs in 5-8F/Si-c-Myc(with knockdown of endogenous c-Myc expression) contrasting with 5-8F/Si-control cells.Both stable transfection cell lines were established previously in our laboratory.To reveal the molecular mechanisms of microRNA-involved NPC pathogenesis,the following experiments were performed:1.MicroRNA microarray analysis and verification results revealed that hsa-mir-141 and hsa-mir-200c were significantly down -regulated by knockdown of c-Myc,and were highly expressed in nasopharyngeal carcinoma tissues than in normal nasopharyngal epithelial tissues.MicroRNA microarray results showed that there were 14 miRNAs up-regulated(fold change＞3),and 7 miRNAs down-regulated(fold change＞3) in 5-8F/Si-c-Myc(with knockdown of endogenous c-Myc expression) contrasting with 5-8F/Si-control NPC cells.Hsa-mir-141 and hsa-mir-200c down-regulated were accordant with the results from Northern-blot and qRT-PCR.qRT-PCR results revealed that expression levels of hsa-mir-141 and hsa-mir-200c were higher in nasopharyngeal carcinoma tissues than in normal nasopharyngal epithelial tissues purified by frozen section and laser capture microdissection(P＜0.025,P＜0.04).2.Hsa-mir-141 and hsa-mir-200c affected NPC cell cycle,migration and invasive ability by positive regulation of Rb/E2F and AKT pathwayMTT assays,flow cytometry analysis,wound healing assays and Matrigel invasion assays(Transwell) were performed in 5-8F, 5-8F/Si-c-Myc and 5-8F/Si-control cells which were transfected with mimics and inhibitors of hsa-mir-141 and hsa-mir-200c.Effectors of the relevant signaling pathways were examined in those transfected cells by Western-blot.The inhibition of hsa-mir-141 and hsa-mir-200c reduced the cell growth,G1-S phase cell cycle progression,migration and invasive ability in NPC cell line 5-8f,but the overexpression of hsa-mir-141 and hsa-mir-200c promoted the cell growth,G1-S phase cell cycle progression,migration and invasive ability in 5-8F/Si-c-Myc cells.The overexpression of hsa-mir-141 up-regulated some critical molecules of Rb/E2F pathway including cyclinD1,pRb,E2F3 and DP2 in 5-8F/Si-c-Myc cells;the overexpression of hsa-mir-200c up-regulated cyclin D1,pRb;the overexpression of hsa-mir-141 and hsa-mir-200c up-regulated pAKT,the critical molecule of AKT pathway.However,the inhibition of hsa-mir-141 down-regulated cyclinD1, pRb,E2F3 and DP2 in 5-8F/Si-control cells;the inhibition of hsa-mir-200c down-regulated cyclin D1,pRb;the inhibition of hsa-mir-141 and hsa-mir-200c down-regulated pAKT.The results showed that hsa-mir-141 and hsa-mir-200c affected NPC cell cycle,migration and invasive ability by positive regulation of Rb/E2F and AKT pathway3.Bioinformatics and experiments verification results confirmed BRD3 as a direct target of hsa-mir-141 and PTEN as a common direct target of hsa-mir-141 and hsa-mir-200c.Furthermore,the basic informations of hsa-mir-141 and hsa-mir-200c were collected from miRBase::Sequences database,and the possible target genes were predicted by online softwares Targetscan and Pictar.The potential target genes were classified by DAVID 2008 Functional Annotation Bioinformatics Microassay Analysis Tools and Tumor Suppressor Gene Database.The target genes were confirmed by Luciferase assays and Western-blot.The miRBase::Sequences database analysis revealed that hsa-mir-141 and hsa-mir-200c were located on the chromosome 12 p13.31 belonging to the same clustered miRNAs.The physical distance between hsa-mir-141 and hsa-mir-200c was less than 10kb,which implied there may be close relationship about their function.The online software analysis showed that many potential target genes of hsa-mir-141 and hsa-mir-200c,including PTEN,BRD3,UBAP1, CDC42,GRB2,MAP2K4,MAP3k3,etc.,some critical molecules of signaling pathways associated with NPC.Those potential target genes involved the pathways as Rb/E2F,AKT,Ras/MEK/ERK,and MAPK,etc. which were both important for tumorigenesis and tumor development.Luciferase assays and Western-blot results confirmed BRD3 as a direct target of hsa-mir-141 and PTEN as a common direct target of hsa-mir-141 and hsa-mir-200c.4.BRD3 negatively regulated Rb/E2F pathwayBRD3 recombinant vector(pCMV-Myc/BRD3) was constructed and transfected into NPC 5-8F cells.Over-expression of BRD3 in 5-8F cells down-regulated some critical molecules of Rb/E2F pathway including cyclinD1,pRb,E2F3 by Western-blot assays.The results showed that BRD3 negatively regulated Rb/E2F pathway.5.The Whole Human Genome Oligo Microarray analysed the effect of knockdown c-Myc in NPC 5-8F cells on Gene-Expression ProfileThe Whole Human Genome Oligo Microarray results revealed that there were 400 genes(EGR1,DMBT1,etc.) up-regulated(fold change＞2), and 535 genes(CDK2,HMGA2,etc.) down-regulated(fold change＞2) in 5-8F/Si-c-Myc contrasting with 5-8F/Si-control NPC cells..Those differential genes involved the pathways as MAPK,TGF-beta Receptor, JAK-STAT,Cell cycle,Cell proliferation,Adherens junction,Tight junction,Migration and Invasive,etc.,which were both important for tumorigenesis and tumor development.The differential genes,EGR1, DMBT1,CDK2,HMGA2 were accordant with the results from RT-PCR.CONCLUSION:1.Hsa-mir-141 and hsa-mir-200c are positively regulated by c-Myc, and are highly expressed in nasopharyngeal carcinoma tissues than in normal nasopharyngal epithelial tissues.So they may play a role as oncogene in NPC.2.Experiments confirm BRD3 as a direct target of hsa-mir-141 and PTEN as a common direct target of hsa-mir-141 and hsa-mir-200c.3.It is demonstrate that c-Myc positively regulate Rb/E2F,AKT signaling pathways and affect NPC cell cycle progression,migration and invasive ability by the targets(BRD3 and PTEN) of hsa-mir-141 and hsa-mir-200c.4.The Whole Human Genome Oligo Microarray and Bioinformatics analysis results verify that knock-down of c-Myc in NPC 5-8F cells up-regulate EGR1,DMBT1,etc.and down-regulate CDK2,HMGA2 etc.It imply that c-Myc affect the cell growth and proliferation in NPC cells by regulating those genes.5.Finally we establish the preliminary interaction molecular network of "c-Myc/miRNA/target genes" and achieve the network mapping.