Polymorphisms In The 3’UTR Of IGF1R Can Influence Prostate Cancer Susceptibility By Affecting MiRNA Binding

Objectives Prostate cancer(PCa)is a common malignant cancer in men worldwide.Numerous genetic variations have been associated with PCa,but their biological function remains unclear.Single nucleotide polymorphisms(SNPs)inside 3’untranslated region(UTR)affect gene expression,with one essential mechanism being regulation by miRNAs.Based on data from genome-wide association study of the Consortium for Chinese Consortium for Prostate Cancer Genetics(ChinaPCa),rs1815009 and rs2684788 inside 3’UTR of insulin-like growth factor 1 receptor(IGF1R)presented significant genotype distribution between PCa and control samples.In this study,we predicted targeting mi RNAs that bingding sites located in or near the rs1815009 of rs2684788 using prediction tools,calculated minimum free energy(MFE)of targeting miRNAs binding to IGF1 R 3’UTR with polymorphism for each stage of the binding process,identified effect on polymorphism sites in the 3’UTR binding to miRNAs,dectected the regulation relation between targetingmiRNAs and IGF1 R gene and genes that asscocited with tumor function.It aimed to explore influence on polymorphism sites in the 3’UTR binding to miRNAs,contributing to clarify the PCa susceptibility.Methods 1.Bioinformation analysis:(1)We predicted targeting miRNAs that binding sites located in or near the rs1815009 and rs2684788 using Target Scan database;(2)The predicted results with TargetScan were identified using miRanda database;(3)The MFE of targeting miRNAs binding to IGF1R3’UTR with polymorphism sites(rs1815009 or rs2684788)for each stage of the binding process was calculated with a parameter-free thermodynamic model;(4)PCa clinical data and miR-seq level 3 were downloaded from TCGA database;(5)Inclusive criteria was established,then GEO datasets that in agreement with our inclusive criteria were selected and downloaded from NCBI;(6)Based on inclusive tumor tissues and adjacent tissues from TCGA database and GEO databases,differential miRNAs analysis was performed using Student’s t test and GEO2 R,respectivly;2.Experiment validation for miRNA-target interaciton and regulation by targeting miRNAs :(1)Dual luciferase reporter assay was performed to detected effect on sites with polymorphism binding to targeting miRNAs;(2)PCa cell lines with overexpression of targeting miRNAs were established by lipofectamine transfection;(3)We detected regulation for IGF1 R gene or protein expression by targeting miRNAs in PCa cell lines using real-time PCR and western blot;(4)real-time PCR was performed to analysize influence by targeting miRNAs on genes associated with tumor behaviors.Results 1.Predicton by TargetScan 7.1 showed that among all the broadly conserved miRNAs,seed region of miR-133 a or mi R-133 b was located within the range of 30 bp surrounding the polymorphism site rs1815009,and that seed sequence of miR-455 was located within the range of 30 bpsurrounding the polymorphism site rs1815009,which was consistent with the results of prediction by miRanda database.2.Prediction by Thermodynamic model revealed that compared to 3’UTR containning the T allele of rs181009,3’UTR inside IGF1 R carrying C allele of rs1815009 have a higher binding affinity for miR-133 a or miR-133 b.In addition,compared to 3’UTR containning the C allele of rs2684788,3’UTR inside IGF1 R carrying C allele of rs2684788 was more accessible for miR-455 binding.Prediction by Thermodynamic model was validation by dual luciferase reporter assay.3.Analysis for mi RNA datasets from TCGA mi RNA-seq and two GEO datasets(GSE36803 and GSE8126)showed that with the exception of miR-133 b in the GSE8126 dataset showing no significance(P = 0.102),the expression levels of the three miRNAs(mi R-133 a,miR-133 b and mi R-455)in PCa tissues were lower than that of normal tissues in the three datasets(P < 0.05);4.With the exception of overexpression of miR-133 a in PC3 cells,PC3 cells or LNCa P cells with forced-expression of the targeting mi RNAs had significantly lower IGF1 R expression at the transcriptional level by real-time PCR(P < 0.05).In addition,the western blot analysis showed that IGF1 R expression at protein level significantly downregulated in LNCaP cells with overexpression of miR-133 b.A similar downregulated effect was observed in PC3 cells that overexpressed miR-455;5.The results of tumor relevant genes(MMP2,CDH1,VEGFA and BCL-2)that detected by real-time PCR showed MMP2 in transfected LNCaP cells with overexpression targeting miRNAs was found to be significantly downregulated,and was similarly suppressed in PC3 cells with forced-expression of miR-455.CDH1 was significantly upregulated in LNCaP cells that overexpressed miR-133 a,and in PC3 cells that overexpressed miR-455,but showed mild downregulation in LNCaP cells that overexpressed mi R-455.Forced-expressionof mi R-133 b and miR-455 in LNCaP cells resulted in mild suppression of VEGFA,but the overexpression of miR-133 a in LNCaP cells led to significant downregulation at the transcriptional level.BCL-2 showed significant upregulation in LNCaP cells with forced-expression of miR-133 a,but a suppression effect was observed in post-transfected LNCaP cells that overexpressed miR-133 b.Conclusions 1.Alleles polymorphism of rs1815009 and rs2684788 influence on the binding affinity between 3’UTR inside IGF1 R with interacting miRNAs(miR-133 a,miR-133 b or miR-455);2.Compared to adjective tissues,miR-133 a,miR-133 b and miR-455 in PCa tissues almostly showed lower expression.After overexpression targeting miRNAs in PCa cells,IGF1 R gene expression was downregulated and tumor relevant genes expression were affected.C allele of rs1815009 and T allele of rs2684788 may show a greater risk in the PCa development,and genetic mutation contributing to cancer susceptibility.