Inhibition and role of let-7d in idiopathic pulmonary fibrosis.
BACKGROUND
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.


OBJECTIVE
To determine changes in expression and role of microRNAs in IPF.


METHODS
RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.


RESULTS
Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells.


CONCLUSIONS
Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).
Pandit
Kusum V
KV
University of Pittsburgh Medical Center, NW 628 MUH, 3459 5th Avenue, Pittsburgh, PA 15261, USA. kaminskin@upmc.edu
Corcoran
David
D
Yousef
Hanadie
H
Yarlagadda
Manohar
M
Tzouvelekis
Argyris
A
Gibson
Kevin F
KF
Konishi
Kazuhisa
K
Yousem
Samuel A
SA
Singh
Mandal
M
Handley
Daniel
D
Richards
Thomas
T
Selman
Moises
M
Watkins
Simon C
SC
Pardo
Annie
A
Ben-Yehudah
Ahmi
A
Bouros
Demosthenes
D
Eickelberg
Oliver
O
Ray
Prabir
P
Benos
Panayiotis V
PV
Kaminski
Naftali
N
D000199:Actins / Q000378:metabolism; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism; D025743:HMGA2 Protein / Q000378:metabolism; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000378:metabolism* / Q000473:pathology; D017403:In Situ Hybridization; D008168:Lung / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011650:Pulmonary Alveoli / Q000378:metabolism; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000502:physiology; D014746:Vimentin / Q000378:metabolism
C541116:ACTA2 protein, human; D000199:Actins; D015820:Cadherins; D025743:HMGA2 Protein; D035683:MicroRNAs; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C470944:S100a4 protein, mouse; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D014746:Vimentin; C487931:mirnlet7 microRNA, human
Nat Genet. 2004 Oct;36(10):1090-8
15448693
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7
11983918
J Clin Invest. 2009 Jun;119(6):1420-8
19487818
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2999-3004
14973191
Am J Respir Crit Care Med. 2000 Feb;161(2 Pt 1):646-64
10673212
J Bone Miner Res. 2008 Oct;23(10):1619-28
18466073
EMBO Rep. 2008 Jun;9(6):582-9
18483486
Genes Dev. 2008 Apr 1;22(7):894-907
18381893
Am J Pathol. 2003 May;162(5):1495-502
12707032
Am J Pathol. 2005 May;166(5):1321-32
15855634
Genes Dev. 2006 Aug 15;20(16):2202-7
16882971
PLoS One. 2008 May 14;3(5):e2142
18478089
Science. 2002 Oct 25;298(5594):799-804
12399584
Cancer Res. 2007 Feb 15;67(4):1419-23
17308078
Nature. 2000 Feb 24;403(6772):901-6
10706289
Science. 2007 Mar 16;315(5818):1576-9
17322030
Am J Physiol Lung Cell Mol Physiol. 2007 Sep;293(3):L525-34
17631612
Nature. 2005 Dec 1;438(7068):685-9
16258535
PLoS Med. 2008 Mar 4;5(3):e62
18318599
Genome Res. 2005 Jun;15(6):840-7
15930494
Am J Clin Pathol. 1950 Jul;20(7):661-4
15432364
Am J Respir Crit Care Med. 2007 Dec 1;176(11):1108-19
17761615
Cells Tissues Organs. 2005;179(1-2):11-23
15942189
Nature. 1995 Aug 31;376(6543):771-4
7651535
Nucleic Acids Res. 1991 May 11;19(9):2499
2041787
Circulation. 2007 Jul 17;116(3):258-67
17606841
J Immunol. 2009 Apr 15;182(8):4994-5002
19342679
Cancer Res. 2007 Aug 15;67(16):7713-22
17699775
Science. 2001 Oct 26;294(5543):858-62
11679671
Cell. 2002 Jul 12;110(1):19-32
12150994
Cell Signal. 2008 Jan;20(1):50-9
17881189
Am J Physiol Lung Cell Mol Physiol. 2000 Sep;279(3):L562-74
10956632
PLoS One. 2009;4(4):e5279
19390574
Nat Cell Biol. 2008 May;10(5):593-601
18376396
J Cell Biol. 2006 Jul 17;174(2):175-83
16831886
Proc Am Thorac Soc. 2006 Jun;3(4):364-72
16738202
J Biol Chem. 2003 Mar 21;278(12):10304-13
12525489
Nat Rev Genet. 2009 Oct;10(10):704-14
19763153
Am J Respir Crit Care Med. 2009 Jul 15;180(2):167-75
19363140
Am J Respir Cell Mol Biol. 2009 Nov;41(5):583-9
19251946
J Biol Chem. 2008 May 30;283(22):14910-4
18411277
J Clin Invest. 2009 Apr;119(4):772-87
19287097
Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13180-5
16924102
Cell. 2004 Jan 23;116(2):281-97
14744438
PLoS Med. 2008 Apr 29;5(4):e93
18447576
Cell. 2008 Aug 8;134(3):521-33
18692474
PLoS Med. 2005 Sep;2(9):e251
16128620
Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18704-9
19843690
PLoS One. 2008;3(11):e3694
19002258
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11400-5
17600087
Ann Intern Med. 2001 Jan 16;134(2):136-51
11177318
Am J Respir Crit Care Med. 1998 Apr;157(4 Pt 1):1301-15
9563754