期刊论文详细信息
BMC Research Notes
Validation of miRNA-mRNA interactions by electrophoretic mobility shift assays
Carlos J Ciudad1  Véronique Noé1  Xenia Villalobos1  Núria Mencia1  Anna Solé1 
[1] Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Avenue Diagonal 643, Barcelona E-08028, Spain
关键词: EMSA;    Target validation;    miRNA;    Binding assay;    3′-UTR;   
Others  :  1140872
DOI  :  10.1186/1756-0500-6-454
 received in 2013-06-20, accepted in 2013-11-08,  发布年份 2013
PDF
【 摘 要 】

Background

MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3′-UTR of the mRNA of their target genes. This binding leads to a decrease in the protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3′-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA.

Findings

RNA molecules corresponding to miR-224 and to the 3′-UTR of SLC4A4 were incubated together and their interaction studied under different binding conditions using electrophoretic mobility shift assays. A direct and specific interaction between miR-224 and SLC4A4 mRNA was observed. This interaction was abolished in the presence of competitors.

Conclusions

In this study, we explored a new application for the electrophoretic mobility shift assay and we demonstrated that it is a useful alternative method to assess, in a direct and specific manner, whether a miRNA binds to a specific predicted target mRNA.

【 授权许可】

   
2013 Solé et al.; licensee BioMed Central Ltd.

【 预 览 】
附件列表
Files Size Format View
20150325133944529.pdf 1680KB PDF download
Figure 4. 83KB Image download
Figure 3. 43KB Image download
Figure 2. 75KB Image download
Figure 1. 65KB Image download
【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】
  • [1]Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116(2):281-297.
  • [2]Carrington JC, Ambros V: Role of microRNAs in plant and animal development. Science 2003, 301(5631):336-338.
  • [3]Pillai RS, Bhattacharyya SN, Filipowicz W: Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol 2007, 17(3):118-126.
  • [4]Huang Y, Shen XJ, Zou Q, Wang SP, Tang SM, Zhang GZ: Biological functions of microRNAs: a review. J Physiol Biochem 2010, 67(1):129-139.
  • [5]Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, Aldler H, Rattan S, Keating M, Rai K, et al.: Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2002, 99(24):15524-15529.
  • [6]Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M, Wojcik SE, Aqeilan RI, Zupo S, Dono M, et al.: miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A 2005, 102(39):13944-13949.
  • [7]Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tomida S, Yatabe Y, Kawahara K, Sekido Y, Takahashi T: A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res 2005, 65(21):9628-9632.
  • [8]Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, et al.: MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005, 65(16):7065-7070.
  • [9]Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A: High expression of precursor microRNA-155/BIC RNA in children with burkitt lymphoma. Genes Chromosomes Cancer 2004, 39(2):167-169.
  • [10]Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K: Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2006, 25(17):2537-2545.
  • [11]Voorhoeve PM, Le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, Van Duijse J, Drost J, Griekspoor A, et al.: A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 2006, 124(6):1169-1181.
  • [12]Garofalo M, Croce CM: microRNAs: Master regulators as potential therapeutics in cancer. Annu Rev Pharmacol Toxicol 2011, 51:25-43.
  • [13]Ma J, Dong C, Ji C: MicroRNA and drug resistance. Cancer Gene Ther 2010, 17(8):523-531.
  • [14]Iorio MV, Croce CM: MicroRNA involvement in human cancer. Carcinogenesis 2012, 33(6):1126-1133.
  • [15]Chi SW, Zang JB, Mele A, Darnell RB: Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 2009, 460(7254):479-486.
  • [16]Hsu PW, Huang HD, Hsu SD, Lin LZ, Tsou AP, Tseng CP, Stadler PF, Washietl S, Hofacker IL: MiRNAMap: genomic maps of microRNA genes and their target genes in mammalian genomes. Nucleic Acids Res 2006, 34(Database issue):D135-D139.
  • [17]Mencia N, Selga E, Noe V, Ciudad CJ: Underexpression of miR-224 in methotrexate resistant human colon cancer cells. Biochem Pharmacol 2011, 82(11):1572-1582.
  • [18]Coma S, Noe V, Eritja R, Ciudad CJ: Strand displacement of double-stranded DNA by triplex-forming antiparallel purine-hairpins. Oligonucleotides 2005, 15(4):269-283.
  • [19]Hellman LM, Fried MG: Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2007, 2(8):1849-1861.
  • [20]Morita T, Maki K, Aiba H: Detection of sRNA-mRNA interactions by electrophoretic mobility shift assay. Methods Mol Biol 2012, 905:235-244.
  • [21]Peyman A, Uhlmann E: Minimally modified oligonucleotides - combination of end-capping and pyrimidine-protection. Biol Chem Hoppe Seyler 1996, 377(1):67-70.
  • [22]Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003, 31(13):3406-3415.
  文献评价指标  
  下载次数:66次 浏览次数:38次