Nature Biotechnology | Research | Letter

English version

miR-302およびmiR-372の複数の標的
はヒト線維芽細胞の人工多能性幹細胞
への初期化を促進する

Journal name:
Nature Biotechnology
Volume:
29,
Pages:
443–448
Year published:
DOI:
doi:10.1038/nbt.1862
Received
Accepted
Published online

胚性幹細胞に特異的な細胞周期を調節する（ESCC）マイクロRNA（miRNA）のファミリーは、マウス胚性線維芽細胞の人工多能性幹細胞への初期化を促進する。本論文では、ESCC miRNAのヒトのオーソログであるhsa-miR-302bおよびhsa-miR-372が、ヒト体細胞の初期化を促進することを明らかにした。さらに、この2つのmiRNAは複数の標的遺伝子を抑制したが、個々の標的の下方制御はmiRNAの全影響を部分的に再現するにとどまった。その標的は、細胞周期、上皮－間葉転換（EMT）、後生的調節、小胞輸送など、さまざまな細胞過程を調節している。ESCC miRNAは、独特の胚性幹細胞周期の調節で役割を担っていることが知られている。本論文では、ESCC miRNAが初期化時の間葉－上皮転換を加速し、ヒト上皮細胞のTGFβ誘導性EMTを遮断していることも示した。この結果は、ESCC miRNAが複数の下流経路に作用することによって脱分化を促進することを示している。個々のmiRNAは、一般に相乗作用によって細胞の運命決定を調節および強制する多数の経路を介して作用すると考えられる。

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  1. Hsa-miR-302b and/or hsa-miR-372 enhances reprogramming efficiency of human somatic cells.
    Figure 1: Hsa-miR-302b and/or hsa-miR-372 enhances reprogramming efficiency of human somatic cells.

    (a) Fold increase in number of human ESC-like colonies obtained per 15,000 cells compared to mock-transfected cells. Cells infected with 4Y ± miRNA were counted on day 21 after infection, whereas cells infected with 3Y ± miRNA were counted on day 31 after infection. *, a significant difference when compared to mock-transfected; P < 0.05; N = 6. Error bars represent mean ± s.e.m. (b) Expression of exogenous factors in iPSC lines that were picked and expanded after 3Y or 4Y infection and the indicated miRNA. Expression was determined by RT-qPCR using primers specific to only the exogenous factors. Data were normalized to BJ cells 3 d after retroviral infection with 4Y. (c) Expression of pluripotency markers in iPSC lines that were picked and expanded after 3Y or 4Y infection and the indicated miRNA. H9 hESCs shown as control. Expression was determined by RT-qPCR. Data were normalized to expression observed in BJ cells.

  2. Hsa-miR-302b and hsa-miR-372 regulate expression of a number of targets that influence reprogramming of human somatic cells.
    Figure 2: Hsa-miR-302b and hsa-miR-372 regulate expression of a number of targets that influence reprogramming of human somatic cells.

    (a) Heat map showing average expression from three independent experiments of 34 predicted targets of hsa-miR-302b and hsa-miR-372 on day 7 in the process of reprogramming. Expression was determined by qRT-PCR and was first normalized to GAPDH followed by normalization to mock-transfected cells. Statistically significant genes (P < 0.05; ANOVA) are labeled red. (b) Fold increase in reprogramming of human somatic cells infected with 4Y upon introduction of siRNAs against specific targets. Human ESC-like colonies were counted on day 21 after infection. N = 4. Error bars represent s.d. *, significant difference when compared to mock transfected; P < 0.05; measured by Kruskal-Wallis test. i, siRNA; ROCKi, ROCK inhibitor. (c) Fold increase in reprogramming of human somatic cells infected with 3Y upon introduction of siRNAs against specific targets. Human ESC-like colonies were counted on day 31 after infection. N = 3. Error bars represent s.d. *, significant difference when compared to mock-transfected; P < 0.05; measured by Kruskal-Wallis test. i, siRNA; ROCKi, ROCK inhibitor.

  3. Hsa-miR-302b and hsa-miR-372 enhance reprogramming by regulating mesenchymal-epithelial transition.
    Figure 3: Hsa-miR-302b and hsa-miR-372 enhance reprogramming by regulating mesenchymal-epithelial transition.

    (a) Western blot showing levels of TβRII from lysates prepared from BJ cells infected with either 4Y or 3Y, or uninfected cells plus the indicated miRNAs. N = 3. (b) Luciferase analysis of TGFBR2 and RHOC 3′UTRs. Seed matches for ESCC miRNAs in the 3′UTRs along with different mutant constructs are shown in the top panel. Luciferase results after co-transfection with ESCC miRNAs relative to mock transfection are shown in the lower panel after normalization to firefly luciferase values. All data are represented as mean ± s.d. *P < 0.05 by t-test. (c) RT-qPCR showing relative expression levels of mesenchymal (ZEB1 and SLUG) and epithelial (E-cadherin, CDH1, and occludin, OCLN) markers at day 7 after infection in the process of reprogramming normalized to GAPDH. N = 3. Error bars represent s.e.m. *, significant difference when compared to mock-transfected cells within each group (P < 0.05) by t-test. (d) Immunocytochemistry performed at different days during the course of reprogramming with 4Y or 3Y ± hsa-miR-372. Representative portions of the well are shown in each image. N = 2. Scale bars, 25 μm.

  4. Hsa-miR-302b, hsa-miR-372 and mmu-miR-294 inhibit TGF-[beta]-induced epithelial-mesenchymal transition in human cells.
    Figure 4: Hsa-miR-302b, hsa-miR-372 and mmu-miR-294 inhibit TGF-β–induced epithelial-mesenchymal transition in human cells.

    (a) Western blot showing levels of TGF-β receptors, phospho-SMAD2 and phospho-SMAD3 in HaCaT cells 0–60 min after TGF-β exposure in the presence of miRNA mimics. Cells were transfected with the indicated miRNAs 48 h before TGF-β treatment. miR-294m, mmu-miR-294 seed mutant mimic. Representative blot of N = 2. (b,c) HaCaT cells were transfected with the indicated miRNAs, then treated or not with TGF-β for 72 h and observed by phase contrast microscopy (b), or fixed and subjected to immunostaining for F-actin, E-cadherin and ZO-1 (c). N = 2. Scale bars, 100 μm (b), 20 μm (c). (d) HaCaT cells were transfected with the indicated miRNAs, then treated with TGF-β for 48 h before lysis and immunoblotting with the indicated antibodies. N = 2. (e) HaCaT cells were transfected with the indicated miRNAs treated or not with TGF-β for 24 h, before RNA was extracted and analyzed by RT-qPCR. Expression was normalized to RPL19. Representative graph of two independent experiments is shown. Error bars represent mean ± s.e.m.

References

  1. Judson, R.L., Babiarz, J.E., Venere, M. & Blelloch, R. Embryonic stem cell-specific microRNAs promote induced pluripotency. Nat. Biotechnol. 27, 459–461 (2009).
  2. Qi, J. et al. microRNAs regulate human embryonic stem cell division. Cell Cycle 8, 3729–3741 (2009).
  3. Wang, Y. et al. Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40, 1478–1483 (2008).
  4. Hochedlinger, K. & Plath, K. Epigenetic reprogramming and induced pluripotency. Development 136, 509–523 (2009).
  5. Gonzalez, F., Boue, S. & Belmonte, J.C. Methods for making induced pluripotent stem cells: reprogramming a la carte. Nat. Rev. Genet. 12, 231–242 (2011).
  6. Li, W. & Ding, S. Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming. Trends Pharmacol. Sci. 31, 36–45 (2010).
  7. Bartel, D.P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).
  8. Li, Z., Yang, C.S., Nakashima, K. & Rana, T.M. Small RNA-mediated regulation of iPS cell generation. EMBO J. 30, 823–834 (2011).
  9. Bar, M. et al. MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries. Stem Cells 26, 2496–2505 (2008).
  10. Card, D.A. et al. Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells. Mol. Cell. Biol. 28, 6426–6438 (2008).
  11. Morin, R.D. et al. Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res. 18, 610–621 (2008).
  12. Suh, M.R. et al. Human embryonic stem cells express a unique set of microRNAs. Dev. Biol. 270, 488–498 (2004).
  13. Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159 (2008).
  14. Chan, E.M. et al. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033–1037 (2009).
  15. Thomas, M., Lieberman, J. & Lal, A. Desperately seeking microRNA targets. Nat. Struct. Mol. Biol. 17, 1169–1174 (2010).
  16. Melton, C., Judson, R.L. & Blelloch, R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature 463, 621–626 (2010).
  17. Benetti, R. et al. A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases. Nat. Struct. Mol. Biol. 15, 998 (2008).
  18. Lin, S.L. et al. Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res. 39, 1054–1065 (2011).
  19. Sinkkonen, L. et al. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 15, 259–267 (2008).
  20. Ichida, J.K. et al. A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5, 491–503 (2009).
  21. Lin, T. et al. A chemical platform for improved induction of human iPSCs. Nat. Methods 6, 805–808 (2009).
  22. Maherali, N. & Hochedlinger, K. Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr. Biol. 19, 1718–1723 (2009).
  23. Riento, K. & Ridley, A.J. Rocks: multifunctional kinases in cell behaviour. Nat. Rev. Mol. Cell Biol. 4, 446–456 (2003).
  24. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
  25. Ohgushi, M. et al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7, 225–239 (2010).
  26. Chen, G., Hou, Z., Gulbranson, D.R. & Thomson, J.A. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7, 240–248 (2010).
  27. Rosa, A., Spagnoli, F.M. & Brivanlou, A.H. The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection. Dev. Cell 16, 517–527 (2009).
  28. Xu, J., Lamouille, S. & Derynck, R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 19, 156–172 (2009).
  29. Li, R. et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7, 51–63 (2010).
  30. Samavarchi-Tehrani, P. et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64–77 (2010).
  31. Thiery, J.P., Acloque, H., Huang, R.Y. & Nieto, M.A. Epithelial-mesenchymal transitions in development and disease. Cell 139, 871–890 (2009).
  32. Lin, S.L. et al. Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14, 2115–2124 (2008).
  33. Banito, A. et al. Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 23, 2134–2139 (2009).
  34. Hong, H. et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460, 1132–1135 (2009).
  35. Kawamura, T. et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460, 1140–1144 (2009).
  36. Li, H. et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460, 1136–1139 (2009).

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Author information

  1. These authors contributed equally to this work.

    • Samy Lamouille &
    • Robert L Judson

Affiliations

  1. Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, USA.

    • Deepa Subramanyam,
    • Samy Lamouille,
    • Robert L Judson,
    • Jason Y Liu,
    • Nathan Bucay,
    • Rik Derynck &
    • Robert Blelloch

  2. Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, California, USA.

    • Deepa Subramanyam,
    • Robert L Judson,
    • Jason Y Liu,
    • Nathan Bucay &
    • Robert Blelloch

  3. Department of Cell and Tissue Biology, Programs in Cell Biology and Developmental Biology, University of California San Francisco, San Francisco, California, USA.

    • Samy Lamouille &
    • Rik Derynck

Contributions

D.S. did the experiments described in Figures 1, 2 and 3. S.L. and R.L.J. did experiments described in Figure 4. J.Y.L. helped with experiments described in Figure 2a and performed experiments described in 3b. N.B. helped with experiments in Figure 1b,c. D.S. and R.B. wrote the manuscript with help from S.L., R.L.J. and R.D.

Competing financial interests

The authors declare no competing financial interests.

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