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Abstract

MicroRNAs (miRNAs) regulate myriad biological processes; however, their role in cell fate choice is relatively unexplored. Pluripotent NT2/D1 embryonal carcinoma cells differentiate into an epithelial/smooth muscle phenotype when treated with bone morphogenetic protein-2 (BMP-2). To identify miRNAs involved in epithelial cell development, we performed miRNA profiling of NT2/D1 cells treated with BMP-2 at 6, 12, and 24 h, and on days 6 and 10. Integration of the miRNA profiling data with previously obtained gene expression profiling (GEP) data of NT2/D1 cells treated with BMP-2 at the same time points identified miR-18b and miR-518b as the top two miRNAs with the highest number of up-regulated predicted targets with known functions in epithelial lineage development. Silencing of miR-18b and miR-518b in NT2/D1 cells revealed several up-regulated TFs with functions in epithelial lineage development; among these, target prediction programs identified FOXN1 as the only direct target of both miRNAs. FOXN1 has previously been shown to play an important role in keratinocyte differentiation and epithelial cell proliferation. NT2/D1 and H9 human embryonic stem cells with silenced miR-18b and miR-518b showed up-regulation of FOXN1 and the epithelial markers CDH1, EPCAM, KRT19, and KRT7. A 3′UTR luciferase assay confirmed FOXN1 to be a target of the two miRNAs, and up-regulation of FOXN1 in NT2/D1 cells led to the expression of epithelial markers. Overexpression of the two miRNAs in BMP-2-treated NT2/D1 cells led to down-regulation of FOXN1 and epithelial lineage markers. These results show that miR-18b and miR-518b are upstream controllers of FOXN1-directed epithelial lineage development.

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References

Bartel

. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116:281–297.

Baek

, Villén

, Shin

, Camargo

, Gygi

and Bartel

. (2008). The impact of microRNAs on protein output. Nature, 455:64–71.

Marson

, Levine

, Cole

, Frampton

, Brambrink

, Johnstone

, Guenther

, Johnston

, Wernig

, et al. (2008). Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell, 134:521–533.

Anokye-Danso

, Trivedi

, Juhr

, Gupta

, Cui

, Tian

, Zhang

, Yang

, Gruber

, Epstein

and Morrisey

. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, 8:376–388.

Chen

, Mandel

, Thomson

, Wu

, Callis

, Hammond

, Conlon

and Wang

. (2006). The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet, 38:228–233.

Ivey

, Muth

, Arnold

, King

, Yeh

, Fish

, Hsiao

, Schwartz

, Conklin

, Bernstein

and Srivastava

. (2008). MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell, 2:212–229.

Conaco

, Otto

, Han

and Mandel

. (2006). Reciprocal actions of REST and microRNA promote neuronal identity. Proc Natl Acad Sci U S A, 103:2422–2427.

Nissan

, Denis

, Saidani

, Lemaitre

, Peschanski

and Baldeschi

. (2011). miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification. Dev Biol, 356:506–515.

, Poy

, Stoffel

and Fuchs.

(2008). Skin microRNA promotes differentiation by repressing “stemness”. Nature, 452:225–229.

10.

Chaganti

and Houldsworth

. (2000). Genetics and biology of adult human male germ cell tumors. Cancer Res, 60:1475–1482.

11.

Pal

and Ravindran

. (2006). Assessment of pluripotency and multilineage differentiation potential of NTERA-2 cells as a model for studying human embryonic stem cells. Cell Prolif, 39:585–598.

12.

Houldsworth

, Heath

, Bosl

, Studer

and Chaganti

. (2002). Expression profiling of lineage differentiation in pluripotential human embryonal carcinoma cells. Cell Growth Differ, 13:257–264.

13.

Chadalavada

, Korkola

, Houldsworth

, Olshen

, Bosl

, Studer

and Chaganti

. (2007). Constitutive gene expression predisposes morphogen-mediated cell fate responses of NT2/D1 and 27X-1 human embryonal carcinoma cells. Stem Cells, 25:771–778.

14.

Chadalavada

, Houldsworth

, Olshen

, Bosl

, Studer

and Chaganti

. (2005). Transcriptional program of bone morphogenetic protein-2-induced epithelial and smooth muscle differentiation of pluripotent human embryonal carcinoma cells. Funct Integr Genomics, 5:59–69.

15.

Caricasole

, Ward-van Oostwaard

, Zeinstra

, van den Eijnden-van Raaij

and Mummery

. (2000). Bone morphogenetic proteins (BMPs) induce epithelial differentiation of NT2D1 human embryonal carcinoma cells. Int J Dev Biol, 44:443–450.

16.

Hildesheim

, Kühn

, Yee

, Foster

, Yancey

and Vogel

. (2001). The hSkn-1, a POU transcription factor enhances epidermal stratification by promoting keratinocyte proliferation. J Cell Sci, 114:1913–1923.

17.

, O'Carroll

, Pasolli

, Zhang

, Dietrich

, Tarakhovsky

and Fuchs

. (2006). Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat Genet, 38:356–362.

18.

Leask

, Byrne

and Fuchs.

(1991). Transcription factor AP2 and its role in epidermal-specific gene expression. Proc Natl Acad Sci U S A, 88:7948–7952.

19.

Page

, Zhang

, Steward

, Blumenthal

and Priess

. (1997). ELT-1, a GATA-like transcription factor is required for epidermal cell fates in Caenorhabditis elegans embryos. Genes Dev, 11:1651–1661.

20.

Creyghton

, Markoulaki

, Levine

, Hanna

, Lodato

, Sha

, Young

, Jaenisch

and Boyer

. (2008). H2AZ is enriched at polycomb complex target genes in ES cells and is necessary for lineage commitment. Cell, 135:649–661.

21.

Frank

, Pignata

, Panteleyev

, Prowse

, Baden

, Weiner

, Gaetaniello

, Ahmad

, Pozzi

et al. (1999). Exposing the human nude phenotype. Nature, 398:473–474.

22.

Cunliffe

, Furley

and Keenan

. (2002). Complete rescue of the nude mutant phenotype by a wild-type Foxn1 transgene. Mamm Genome, 13:245–252.

23.

Pignata

, Fusco

and Amorosi

. (2009). Human clinical phenotype associated with FOXN1 mutations. Adv Exp Med Biol, 665:195–206.

24.

Manley

and Condie

. (2010). Transcriptional regulation of thymus organogenesis and thymic epithelial cell differentiation. Prog Mol Biol Transl Sci, 92:103–120.

25.

Potter

, Pruett

, Kern

, Baybo

, Godwin

, Potter

, Peterson

, Sundberg

and Awgulewitsch

. (2011). The nude mutant gene Foxn1 is a HOXC13 regulatory target during hair follicle and nail differentiation. J Invest Dermatol, 131:828–834.

26.

Prowse

, Lee

, Weiner

, Jiang

, Magro

, Baden

and Brissette

. (1999). Ectopic expression of the nude gene induces hyperproliferation and defects in differentiation: implications for the self-renewal of cutaneous epithelia. Dev Biol, 212:54–67.

27.

Baxter

and Brissette

. (2002). Role of the nude gene in epithelial terminal differentiation. J Invest Dermatol, 118:303–309.

28.

Zhao

, Samal

and Srivastava

. (2005). Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature, 436:214–220.

29.

Zhao

, Ransom

, Li

, Vedantham

, von Drehle

, Muth

, Tsuchihashi

, McManus

, Schwartz

and Srivastava

. (2007). Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1–2. Cell, 129:303–317.

30.

Bracken

, Gregory

, Kolesnikoff

, Bert

, Wang

, Shannon

and Goodall

. (2008). A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res, 68:7846–7854.

31.

Przyborski

, Christie

, Hayman

, Stewart

and Horrocks

. (2004). Human embryonal carcinoma stem cells: models of embryonic development in humans. Stem Cells Dev, 13:400–408.

32.

Andrews

, Damjanov

, Berends

, Krumpf

, Zappavigna

, Mavilio

and Sampath

. (1994). Inhibition of proliferation and induction of differentiation of pluripotent human embryonal carcinoma cells by osteogenic protein-1 (or bone morphogenic protein-7). Lab Investig, 71:243–251.

33.

Sperger

, Chen

, Draper

, Antosiewicz

, Chon

, Jones

, Brooks

, Andrews

, Brown

and Thomson

. (2003). Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci U S A, 100:13350–13355.

34.

Dar

, Majid

, Rittsteuer

, de Semir

, Bezrookove

, Tong

, Nosrati

, Sagebiel

, Miller

3rd and Kashani-Sabet

. (2013). The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression. J Natl Cancer Inst, 105:433–442.

35.

Zhang

, Zhou

, Zhang

, Cai

, Zhu

, Huang

and Wang

. (2012). miR-518b is down-regulated, and involved in cell proliferation and invasion by targeting Rap1b in esophageal squamous cell carcinoma. FEBS Lett, 58:3508–3521.

36.

Katoh

and Katoh

. (2004). Human FOX gene family (Review). Int J Oncol, 25:1495–1500.

37.

Romano

, Palamaro

, Fusco

, Iannace

, Maio

, Vigliano

, Giardino

and Pignata

. (2012). From murine to human nude/SCID: the thymus, T-cell development and the missing link. Clin Dev Immunol, 2012:467101

38.

Lee

, Prowse

and Brissette

. (1999). Association between mouse nude gene expression and the initiation of epithelial terminal differentiation. Dev Biol, 208:362–374.

39.

Brissette

, Li

, Kamimura

, Lee

and Dotto.

(1996). The product of the mouse nude locus, Whn, regulates the balance between epithelial cell growth and differentiation. Genes Dev, 10:2212–2221.

40.

Janes

, Ofstad

, Campbell

, Watt

and Prowse

. (2004). Transient activation of FOXN1 in keratinocytes induces a transcriptional programme that promotes terminal differentiation: contrasting roles of FOXN1 and Akt. J Cell Sci, 117:4157–4168.

41.

Schlake

, Schorpp

, Maul-Pavicic

, Malashenko

and Boehm

. (2000). Forkhead/Winged-Helix transcription factor Whn regulates hair keratin gene expression: molecular analysis of the nude skin phenotype. Dev Dyn, 217:368–376.

42.

, Baxter

, Weiner

, Goetinck

, Calauttin

and Brissette

. (2007). Foxn1 promotes keratinocyte differentiation by regulating the activity of protein kinase C. Differentiation, 75:694–701.

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miR-18b and miR-518b Target FOXN1 During Epithelial Lineage Differentiation in Pluripotent Cells

Abstract

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Supplementary Material