Variation of gene silencing involving endogenous microRNA in mammalian cells

Abstract

MicroRNAs (miRNAs) are small noncoding RNA and play a role in gene expression regulation by inhibiting translation of their target messenger RNAs (mRNAs). In this study, we investigated the effects of endogenous let-7 miRNA on the expression of target genes in various mammalian cells by means of two types of reporter plasmids possessing target sequences for let-7: one carries perfectly matched target sequence for let-7 in the 3′-untranslated region of the luciferase reporter gene to monitor RNA interference (RNAi) activity and the other has three bulged binding sites for let-7 to monitor translation-inhibition activity. The results indicate that different cells have different levels of gene silencing against the target reporter genes. The data presented here suggest that not only microRNA level but also target transcript level likely participate in the generation of a variety of gene silencing.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. 1.

    Lee Y, Ahn C, Han J et al (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419. doi:10.1038/nature01957

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Denli AM, Tops BB, Plasterk RH et al (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432:231–235. doi:10.1038/nature03049

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297. doi:10.1016/S0092-8674(04)00045-5

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Krichevsky AM, King KS, Donahue CP et al (2003) A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9:1274–1281. doi:10.1261/rna.5980303

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Lagos-Quintana M, Rauhut R, Yalcin A et al (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12:735–739. doi:10.1016/S0960-9822(02)00809-6

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Babak T, Zhang W, Morris Q et al (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10:1813–1819. doi:10.1261/rna.7119904

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Liu CG, Calin GA, Meloon B et al (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci USA 101:9740–9744. doi:10.1073/pnas.0403293101

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297:2056–2060. doi:10.1126/science.1073827

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Caudy AA, Myers M, Hannon GJ et al (2002) Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev 16:2491–2496. doi:10.1101/gad.1025202

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Schwarz DS, Hutvagner G, Du T et al (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208. doi:10.1016/S0092-8674(03)00759-1

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Zeng Y, Yi R, Cullen BR (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 100:9779–9784. doi:10.1073/pnas.1630797100

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Olsen PH, Ambros V (1999) The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol 216:671–680. doi:10.1006/dbio.1999.9523

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Liu J, Valencia-Sanchez MA, Hannon GJ et al (2005) MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol 7:719–723. doi:10.1038/ncb1274

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Pillai RS, Bhattacharyya SN, Artus CG et al (2005) Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309:1573–1576. doi:10.1126/science.1115079

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17:438–442. doi:10.1101/gad.1064703

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Yekta S, Shih IH, Bartel DP (2004) MicroRNA-directed cleavage of HOXB8 mRNA. Science 304:594–596. doi:10.1126/science.1097434

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Chen CZ, Li L, Lodish HF et al (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303:83–86. doi:10.1126/science.1091903

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Chen JF, Mandel EM, Thomson JM et al (2006) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 38:228–233. doi:10.1038/ng1725

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Schratt GM, Tuebing F, Nigh EA et al (2006) A brain-specific microRNA regulates dendritic spine development. Nature 439:283–289. doi:10.1038/nature04367

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Zhao Y, Samal E, Srivastava D (2005) Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436:214–220. doi:10.1038/nature03817

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Cheng AM, Byrom MW, Shelton J et al (2005) Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res 33:1290–1297. doi:10.1093/nar/gki200

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Hornstein E, Mansfield JH, Yekta S et al (2005) The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature 438:671–674. doi:10.1038/nature04138

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Calin GA, Dumitru CD, Shimizu M et al (2002) Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 99:15524–15529. doi:10.1073/pnas.242606799

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Eis PS, Tam W, Sun L et al (2005) Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA 102:3627–3632. doi:10.1073/pnas.0500613102

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    He L, Thomson JM, Hemann MT et al (2005) A microRNA polycistron as a potential human oncogene. Nature 435:828–833. doi:10.1038/nature03552

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Johnson SM, Grosshans H, Shingara J et al (2005) RAS is regulated by the let-7 microRNA family. Cell 120:635–647. doi:10.1016/j.cell.2005.01.014

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Hohjoh H, Fukushima T (2007) Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells. Biochem Biophys Res Commun 362:360–367. doi:10.1016/j.bbrc.2007.07.189

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Ohnishi Y, Tokunaga K, Kaneko K, Hohjoh H (2006) Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles. J RNAi Gene Silencing 2:154–160

    Google Scholar 

  29. 29.

    Hohjoh H, Fukushima T (2007) Expression profile analysis of microRNA (miRNA) in mouse central nervous system using a new miRNA detection system that examines hybridization signals at every step of washing. Gene 391:39–44. doi:10.1016/j.gene.2006.11.018

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Kiriakidou M, Nelson PT, Kouranov A et al (2004) A combined computational-experimental approach predicts human microRNA targets. Genes Dev 18:1165–1178. doi:10.1101/gad.1184704

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Lewis BP, Shih IH, Jones-Rhoades MW et al (2003) Prediction of mammalian microRNA targets. Cell 115:787–798. doi:10.1016/S0092-8674(03)01018-3

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    O’Donnell KA, Wentzel EA, Zeller KI et al (2005) c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435:839–843. doi:10.1038/nature03677

    PubMed  Article  Google Scholar 

  33. 33.

    Petersen CP, Bordeleau ME, Pelletier J et al (2006) Short RNAs repress translation after initiation in mammalian cells. Mol Cell 21:533–542. doi:10.1016/j.molcel.2006.01.031

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Schmitter D, Filkowski J, Sewer A et al (2006) Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res 34:4801–4815. doi:10.1093/nar/gkl646

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Hohjoh H (2002) RNA interference (RNAi) induction with various types of synthetic oligonucleotide duplexes in cultured human cells. FEBS Lett 521:195–199. doi:10.1016/S0014-5793(02)02860-0

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Sago N, Omi K, Tamura Y et al (2004) RNAi induction and activation in mammalian muscle cells where Dicer and eIF2C translation initiation factors are barely expressed. Biochem Biophys Res Commun 319:50–57. doi:10.1016/j.bbrc.2004.04.151

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20. doi:10.1016/j.cell.2004.12.035

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank T. Fukushima (Mitsubishi Rayon) for his helpful assistance. This work was supported in part by research grants from the Ministry of Health, Labour and Welfare of Japan and by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Hirohiko Hohjoh.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tamura, Y., Yoshida, M., Ohnishi, Y. et al. Variation of gene silencing involving endogenous microRNA in mammalian cells. Mol Biol Rep 36, 1413–1420 (2009). https://doi.org/10.1007/s11033-008-9330-4

Download citation

Keywords

  • MicroRNA
  • Let-7
  • Gene silencing
  • Variation
  • Target gene expression