MicroRNA 483 Modulates IGF2 Gene Imprinting Expression In Tumors And Mechanism

VBackground:Insulin-like growth factor II(IGF-II)is a fetal mitogen,with both growth promoting and metabolic effects.IGF-? expression mainly occurs in fetus and human liver,but is dysregulated in a variety of human malignancies.,IGF-?promotes tumor growth by binding to the type 1 IGF receptor(IGF1R)through autocrine,paracrine and endocrine pathways.By stimulating the MAPK and/ or PI3-K/AKT signaling cascades,IGF-? reduces tumor cell apoptosis,increases cell proliferation and drug resistance.As a result,the IGF2 and IGF1 R have been studied as a target for developing tumor-specific gene therapy.The gene encoding IGF-?,IGF2,is a maternally imprinted gene,located on chromosome 11p15.In postnatal life,four promoters regulate the expression of IGF2gene;promoter P1 directs biallelic expression,while promoters P2-4 stimulate monoallelic expression of IGF2 in most tissues except the brain.Monoallelic gene expression is regulated by an allele-specific epigenetic modificator in the imprinting control region(ICR),located between IGF2 and H19 on chromosome 11p15.5.Loss of IGF2 imprinting(LOI)with biallelic expression of IGF2 is a hallmark of many human tumors,especially of childhood tumors and cancer stem cells.LOI is associated with increased cellular proliferation,cancer therapy resistance,and sensitivity of the IGF1 R signaling pathway.Purpose:Little is known about the molecular mechanisms underlying the activation of the normally suppressed maternal IGF2 allele in tumors with IGF2 LOI.Reports regarding epigenetic modifications in the ICR are inconsistent,and epigenetic modulators in the IGF2 promoter regions have not been extensively studied.Therefore,we decided to identify molecular components in the major IGF2 promotercomplex that regulate IGF2 expression.Our study showed that miR-483-5p can induce the loss of IGF2 imprinting.Methods:1.We used a Cas9-guided chromatin immunoprecipitation assay(Cas IP)to pull down candidate molecules that interact with the IGF2 promoters.In this assay,target cancer cells were stably transfected with a lentiviral vector containing the mutated Cas9(d Cas9)and two Cas9 g RNAs.The d Cas9 is a catalytically dead CRISPR Cas9 mutant,which is defective in DNA cleavage function,but maintains the ability to bind with the g RNA-guided gene target.Thus,we are able to pull down the Cas9-IGF2 promoter complex by using anti-Cas9 antibody and beads,known as Ch IP assay.Micro RNA 483-5p(miR-483-5p)was identified as an IGF2 promoter-binding miRNA.The interaction was later confirmed by the biotinylated miR483 precipitation assay.Briefly,after transient transfection,the biotin-miR483-5p interacting chromatin complex was pulled down by streptavidin beads.The presence of the IGF2 promoter in the precipitated chromatin complex was measured by PCR.2.After confirming the interaction between miR-483 and IGF2,we produced miR-483,miR-483-5p,anti-miR-483,anti-miR-483-5p,and control vectors and lentiviruses to study the function of miR-483.Colon cancer cell line HCT116 and pancreatic cancer cell line ASPC-1 were infected with the viruses and stable cells were selected.We further detected the impact of miR-483 on the cell function and IGF2 signaling pathway by examine the difference between miR-483 overexpressing cells and normal cells on cell proliferation,invasion,migration,cloning formation,as well as downstream IGF2 m RNA,IGF-II protein,and phosphate AKP levels.3.Study the impact of miR-483 and miR-483-5p on IGF2 imprinting.IGF2 is an imprinted gene in HCT116 and ASPC-1 cell lines and the two alleles could be distinguished by the Apa1 polymorphic restriction enzyme.We tested the imprinting status after miR-483 and miR-483-5p overexpression.4.Using chromatin immunoprecipitation(Ch IP)assay,we examined histone 3lysine 27(H3K27)methylation in IGF2 promoter before and after miR-483-5p induced IGF2 LOI.A chromatin interacting complex containing CTCF(CCCTC binding factor)and SUZ12(multi protein complex binding factor)is required for specific monoallelic methylation of H3K27 in the IGF2 promoters.So we examinedwhether miR-483 affects the binding of CTCF and SUZ12 to the IGF2 promoter region with Ch IP assay.Results:1.We found that IGF2 promoter P2 can bind to IGF2 seventh intron miR-483-5p with Cas IP experiment.Using miR-483-5p IP,we precipitated miR-483-5p and the complex combined with it.q PCR of the complex also verified the interaction of the two.2.Colon cancer cell line HCT116 and pancreatic cancer cell line ASPC-1 were infected with lentivirus to overexpress miR-483 and miR-483-5p.Compared with control group,cells infected with miR-483 and miR-483-5p both showed increased in the proliferation,invasion,migration,and the colony forming ability,as well as the upregulation in the expression of IGF2 m RNA and IGF-II protein,and AKT phosphorylation level.was are all enhanced.However,cells transfected with anti-miR-483-5p showed opposite.3.Colon cancer cell line HCT116 and pancreatic cancer cell line ASPC-1 were all IGF2 imprinted tumor cell lines,expressing only the paternal allele.We used the polymorphic restriction enzyme Apa1 to distinguish the two parental alleles for imprinting analysis in these two cell lines.In the control cells,genomic DNA(g DNA)contained both the “A” and “C” alleles.In c DNAs,only the “A” allele product(171bp,can not be digested by Apa1 enzyme)was detected,exhibiting typical mono-allelic expression.In both the miR-483 and miR-483-5p clones,however,we found that the “C” allele products(cuted to 111 bp and 60 bp fragments by Apa1enzyme)were also detected.These data suggest that the imprinted maternal allele was reactivated by miR-483 and miR-483-5p.4.Compared with MOI control group,the H3K27 methylation of IGF2 imprinted promoters(P2-4)and the binding of CTCF and SUZ12 to the IGF2 imprinted promoters(P2-4)was significantly reduced in the cells transfected with miR-483 and miR-483-5p.But nonimprinted promoter P1 was not affected.Conclusions:1.IGF2 intronic miR 483-5p can combine with IGF2 promoter 2;2.Exogenic miR 483-5p can promote tumor genesis by stimulating IGF2 expression and activate IGF1 R pathway;3.IGF2 imprinting status of cell lines HCT116 and ASPC-1 can be altered LOI by miR 483-5p;4.The mechanism of miR483-5p induced IGF2 LOI is: maternal allelic expression is induced by decreasing of H3K27 methylation of IGF2 promoter by reducing CTCF and SUZ12 binding.The importance of this study is: we found miRNA regulating of imprinting status.Using Cas9 immunoprecipitation we identified the oncogenic miR-483 as a critical component in the regulatory complex of IGF2 imprinting.After binding to IGF2 promoter P2,miR-483 decreases the binding of CTCF and SUZ12 and consequently reduces H3K27 methylation.By altering the epigenotype in the promoter,miR-483 upregulates IGF2 production by relaxing IGF2 imprinting.The overexpressed IGF-? growth factor may then promote tumorigenesis through the IGF1R/AKT pathway,and anti-miR-483-5p can reverse this effect.These data demonstrate a new role of the oncogenic miR-483-5p in promoting tumor growth,also,a new way to regulate tumor IGF2.