Analysis of gene expression regulation by miRNA using MiRaGE method
Analysis of gene expression regulation by miRNA using MiRaGE method Yh. Taguchi/Dept.Phys.,Chuo Univ.Jun Yasuda /School Med.,Tohoku Uinv.
Three Topics:Inference of transfection of miRNA to human lung cancer cellInference of gene regulation via miRNA in murine medulloblastoma Identification of critical miRNAs for ES stemness during differentiation to neuronal cells
Regulation of gene expression via miRNA Computer oriented prediction (uncertain) Target genes genome microRNA mRNA microRNA mRNA 3
miRNA target gene list Gene8 Gene7 Gene6 Gene5 Gene4 Gene3 Gene2 Gene1simple seed match(Virtual) miRNA1 ○×○○○○×× miRNA2 ○×○○××○○Prediction miRNA3 ×○○×○○×× miRNA4 ○○○×○○×× VS Human lung cancer Murine medulloblastoma gene1 Murine ES cell gene2 miRNA1 Real gene3 4
Control Treated Gather the information of miRNA targetsCompare the expressions of targets for each miRNAs (see Next Slides) Calculate False Discovery Rate Generate ranking 5
MiRaG miRNA Targets E Down Pvalue FDR miRa 54 3 0.5 0.4 miRb 120 54 0.0001 0.005 miRc 36 1 0.5 0.7 ... ... ... ... ... miRX 60 18 0.001 0.007Reject miRa & c because the FDR > 0.05Filtrate with miRNA expression profiles Ranking 6
Topics 1Inference of transfection of miRNA to human lung cancer cell
Gene expression byArray: AgilentatOne and three days after transfection ofMir107, mir185, and let7a.log(xg[miRNA]) vs log(xg[Control]) xg: gene expressionTarget gene list: simple seed match ICIC2011, LNBI in press. (2011/8/1114)
Transfected miRNA mir107 mir185 let7atime replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2day1 1[1st] 1[1st] 7[1st] 9[1st] 2[1st] 2[1st]day3 1[1st] 0 0[1st] 0 1[1st] 1[1st]Numbers of significant miRNAs. The ranks of transfected micriRNAs are shown in square brackets. Transfected miRNAs are correctly identified by MiRaGE method.
Topics 2Inference of gene regulation via miRNA in murine medulloblastoma
Materials(established at Tastuo Noda group)P6＝6 days after birth, normal but growingP6P30＝30 days after birth, normal and not P30growingMB＝a few month after birth, malignant MBneoplasm30% of the Ptc1 +/ mice suffers from MB. 11
mRNA/miRNA expression byArray: AgilentatP6, P30 and MB log(xg[mRNA/miRNA:MB or P6]) vs log(xg[mRNA/miRNA:P30]) xg: mRNA/miRNA expression 12Target gene list: simple seed match
t test for miRNA expressionlog(xg[miRNA:P6/MB]) vs log(miRNA:xg[P30]) of considered miRNA(*)(*) each miRNA is measured by multiple probe 13
t test for miRNA target genes (MiRaGE method)log(xg[mRNA:P6/MB]) – log(xg[mRNA:P30]) in target genes of considered miRNA V S log(xg[mRNA:P6/MB]) – log(xg[mRNA:P30]) in target genes of any of other miRNA 14
selected bymiRNA miRNA expression / MiRaGE miRNA P30<MB 1 mmu-miR-25 1 1 target gene P30>MB 2 m m u-m iR-466i-5p 1 1 3 mmu-miR-92a 0.75 1 4 mmu-miR-19a 1 0.69 miR17~92 cluster family 5 mmu-miR-19b 1 0.69 members are ranked in top 5 members 6 m m u-m iR-3082-5p 1 0.56 by combination of MiRaGE 7 m m u-m iR-130a 1 0.5 methods and miRNA 8 m m u-m iR-130b 1 0.5 expression profiling. 9 m m u-m iR-15b 1 0.5 10 m m u-m iR-2861 1 0.5 11 m m u-m iR-3096-5p 1 0.5 12 m m u-m iR-32 0.5 1 13 m m u-m iR-322 1 0.5 14 m m u-m iR-721 1 0.5 15 m m u-m iR-149* 0.5 0.88 16 m m u-m iR-3081* 1 0.38 17 m m u-m iR-574-5p 1 0.31 18 m m u-m iR-669n 0.5 0.81 suggested contribution to 15 19 m m u-m iR-1187 1 0.25 cancer formation
selected by miRNA P30>MBmiRNA miRNA expression / MiRaGEm m u-m iR-100 1 1 target gene P30<MBm m u-m iR-126-3p 1 1mmu-miR-29c 1 1 Some of the neuronmmu-miR-376a 1 1 specific miRNAs and specific miRNAm m u-m iR-451 1 1 tumorsuppressive m m u-m iR-99b 1 1 miRNAs seem to contribute miRNAsm m u-m iR-136* 1 0.9375 to the gene expression m m u-m iR-299* 0.75 1 profiles of P30.mmu-miR-26a 1 0.5mmu-miR-26b 1 0.5mmu-miR-29a 0.5 1mmu-miR-7a-1* 1 0.5m m u-m iR-3107 1 0.4375m m u-m iR-340-5p 1 0.3125m m u-m iR-369-5p 1 0.3125mmu-let-7a 1 0.25 tumorsuppressive miRNAs mmu-let-7e 1 0.25mmu-let-7g 1 0.25 neuronspecific miRNAs 16mmu-let-7i 1 0.25
selected bymiRNA miRNA expression / MiRaGE miRNA P30<P6 1 mmu-miR-106b 1.00 1.00 target gene P30>P6 2 m m u-m iR-130a 1.00 1.00 3 m m u-m iR-130b 1.00 1.00 4 m m u-m iR-15b 1.00 1.00 miR17~92, mir106b 5 mmu-miR-17 1.00 1.00 25,mir106a363 6 mmu-miR-20a 1.00 1.00 cluster family members are 7 mmu-miR-20b 1.00 1.00 ranked in top 5 by 8 m m u-m iR-301b 1.00 1.00 combination of MiRaGE 9 m m u-m iR-322 1.00 1.00 methods and miRNA 10 m m u-m iR-721 1.00 1.00 expression profiling. 11 mmu-miR-93 1.00 1.00 12 m m u-m iR-542-3p 1.00 0.94 13 m m u-m iR-3081* 1.00 0.88 14 m m u-m iR-335-3p 1.00 0.88 15 m m u-m iR-199a-5p 1.00 0.81 16 m m u-m iR-199b* 1.00 0.81 17 mmu-miR-19a 1.00 0.81 17 18 mmu-miR-1 9 b 1.00 0.81
selected bymiRNA miRNA expression / MiRaGE miRNA P30>P6m m u-m iR-29c 1.00 1.00 target gene P30<P6mmu-miR-376a 1.00 1.00m m u-m iR-451 1.00 1.00 Some of the neuronmmu-let-7b 1.00 0.94 specific miRNAs and specific miRNAmmu-let-7e 1.00 0.94 tumorsuppressive mmu-let-7g 1.00 0.94 miRNAs seem to contribute miRNAsmmu-let-7i 1.00 0.94m m u-m iR-98 1.00 0.94 to the gene expression profiles of P30.m m u-m iR-126-3p 0.75 1.00m m u-m iR-299* 0.75 1.00m m u-m iR-29a 0.75 1.00mmu-let-7a 0.75 0.94m m u-m iR-3070b-3p 1.00 0.69m m u-m iR-138 1.00 0.63m m u-m iR-3107 1.00 0.56m m u-m iR-181a-1* 0.50 1.00 tumorsuppressive miRNAs mmu-let-7d 0.50 0.94 neuronspecific miRNAs 18m m u-m iR-1937b 0.25 1.00
MiRaGE method + miRNA expression successfully pick up biologically important miRNAs. Further (wet) experiments which supress miRNA expression with tiny LNA is now planed.If it is successful, our method can find miRNAs which control tumor formation.Published in IPSJ Technical report, 2011SIGBIO25, No5, pp.16 19
Topics 3Identification of critical miRNAs for ES stemness during differentiation to neuronal cells
Materials:Gene expression data is downloaded from GEO by the Gene expressionaccession number GSE11523, “Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse ES Cells”. In this study, among those, data for the differentiation from ES cells to neuronal cells are used. In parallel, we analyzed the difference of miRNA expression profiles between 2 datasets from ES cells and 6 datasets from mature neuronal tissues(*) and listed up the ES cell specific miRNAs.*) http://www.mirz.unibas.ch/cloningprofiles/
m iRNAsm m u-m iR-200b ratio 1.00 miRNA ES> Neuronal Cell > m m u-m iR-200c (+) 1.00 target gene ES< Neuronal Cell < m m u-m iR-23a 1.00m m u-m iR-23b 1.00mmu-miR-291a-3p 1.00mmu-miR-429 1.00mmu-miR-294 0.98mmu-miR-295 0.98m m u-m iR-302a (+) 0.98m m u-m iR-302b (+) 0.97m m u-m iR-302d (+) 0.97m m u-m iR-199a-5p 0.94m m u-m iR-141 0.69m m u-m iR-200a 0.69m m u-m iR-409-3p 0.67m m u-m iR-369-3p (+) 0.36m m u-m iR-96 0.11m m u-m iR-674 0.08 Mallanna, S.K., and Rizzino., A.(2010)m m u-m iR-467b 0.06
Our method identified critical miRNAs for ES stemness including iPS inducing miRNAs (marked with “+”in the previous slide) recently reported by Miyoshi, et al.(2011, Cell Stem Cell). Published in IPSJ Technical report, 2011BIO25, No.38, pp.1 2
miRNA is short noncoding RNA which regulate many biological processes ranging from cancer formation to differentiation.We have proposed a new method, MiRaGE, MiRNA Ranking by Gene Expression, which assists to discriminate which miRNA really regulate target genes.Since there are some technical method which suppress miRNA machinery, it can also be a drug candidates related to several biological processes.
Acknowledgements: We thank Drs. Tetsuo Noda and Katsuyuki Yaginuma for providing reagents (Topics 2). These works were supported by KAKENHI (23300357) .