Generation and Validation of miR-142 Knock Out Mice
September
Generation and Validation of miR-142 Knock Out Mice
Amit Shrestha 0 1 2
Gianni Carraro 0 1 2
Elie El Agha 0 1 2
Regina Mukhametshina 0 1 2
Cho-Ming Chao 0 1 2
Albert Rizvanov 0 1 2
Guillermo Barreto 0 1 2
Saverio Bellusci 0 1 2
0 1 German Center for Lung Research, Excellence Cluster Cardio-Pulmonary System, Universities of Giessen and Marburg Lung Center , Giessen, Hessen, Germany , 2 Lung and Regenerative Medicine Institutes, Department of Medicine, Cedars-Sinai Medical Center , Los Angeles , California, 3 Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University , Kazan, Russian Federation , 4 LOEWE Research Group Lung Cancer Epigenetic Max Plank Institute , Bad Nauheim, Germany , 5 Member of the German Center for Lung Research , Giessen, Germany , 6 Developmental Biology Program, Division of Surgery, Saban Research Institute of Children's Hospital Los Angeles, University of Southern California Keck School of Medicine , Los Angeles, California
1 Funding: SB was supported by grants from the Deutsche Forschungsgemeinschaft (BE4443/4-1 and BE4443/6-1), Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz, the Universtat Klinikum Giessen Marburg, the University of Giessen Marburg Lung Center , as well as the
2 Editor: Vladimir V. Kalinichenko, Cincinnati Children's Hospital Medical Center , UNITED STATES
microRNA-142 (miR-142) is an important regulator of many biological processes and associated signaling pathways during embryonic development, homeostasis and disease. The miR-142 hairpin gives rise to the “guide strand” miR-142-3p and the sister "passenger" strand miR-142-5p. miR-142-3p has been shown to play critical, non-redundant functions in the development of the hematopoietic lineage. We have recently reported that miR-1423p is critical for the control of Wnt signaling in the mesenchyme of the developing lung. miR142-5p has been proposed to control adaptive growth in cardiomyocytes postnatally and its increase is associated with extensive apoptosis and cardiac dysfunction in a murine heart failure model. Using homologous recombination, we now report the generation and validation of miR-142-null mice. miR-142-null mice show a significant decrease in th expression levels of both the 3p and 5p isoforms. The expression of Bzrap1, a gene immediately flanking miR-142 is not altered while the expression of a long non-coding RNA embedded within the miR-142 gene is decreased. miR-142-null newborn pups appear normal and are normally represented indicating absence of embryonic lethality. At embryonic day 18.5, miR142-null lungs display increased Wnt signaling associated with the up-regulation of Apc and p300, two previously reported targets of miR-142-3p and -5p, respectively. Adult miR-142null animals display impaired hematopoietic lineage formation identical to previously reported miR-142 gene trap knockdown mice. We report, for the first time, the homologous recombination-based miR-142-null mice that will be useful for the scientific community working on the diverse biological functions of miR-142.
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program of competitive growth of Kazan Federal
University. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
microRNAs (miRNAs) are 22 to 25 nucleotide-long, single stranded RNAs that are processed
from hairpin transcripts. The maturation of the hairpin transcript gives rise to the 3p guide
strand and 5p sister passenger strand, both of which allow the silencing of specific sets of genes
through base pairing to a minimal recognition sequence [1,2]. We previously described that
miR-142-3p is among a few miRs expressed at high level in the lung mesenchyme during early
embryonic development [3]. Such restricted expression pattern suggested that miR-142-3p
could play critical functions in controlling cell lineage formation in the mesenchyme. Using
invitro approaches with embryonic lungs cultured in presence of morpholinos for miR-142-3p,
we showed that knockdown of miR-142-3p leads to arrested proliferation and premature
differentiation of smooth muscle progenitor cells. We established that miR-142-3p positively
regulates Ctnnb1 (ß-catenin) signaling during lung development by targeting Adenomatous
polyposis coli (Apc) mRNA for degradation. Apc negatively regulates Ctnnb1 via direct binding
to Ctnnb1 thereby antagonizing the interaction of Ctnnb1 with the transcription factor Tcf. In
combination with Axin and Gsk3b, Apc induces ubiquitination and degradation of Ctnnb1 [4].
Using genetic tools, we showed that up-regulation of Ctnnb1 signaling specifically in the
mesenchyme via the induced expression of a stable form of Ctnnb1 or the deletion of a copy of Apc
is sufficient to rescue miR-142-3p morpholino-mediated loss-of-function and that Apc is a
critical target of this miRNA.
Beyond its proposed fu (...truncated)