Abstract
Background
Micro-RNA 21 (miR-21) has been shown to contribute to cardiac fibrosis in many diseases.
In this study we investigated the role of miR-21 in excessive production of collagen
in diabetic cardiomyopathy.
Methods
The proliferation rate of cardiac fibroblasts was analyzed by Western blot, Cell Counting
Kit-8 kit (Dojindo Molecular Technologies, Kumamoto, Japan), and Cell-Light EdU Apollo
488 In Vitro Imaging Kit (RiboBio, Guangzhou, China). Real-time polymerase chain reaction
and Western blotting were conducted to determine gene expression levels. A luciferase
reporter assay was used to verify the interaction between miR-21 and the 3′ untranslated
region (3′UTR) of dual specific phosphatase 8 (DUSP8).
Results
Our results show that high glucose promoted the proliferation and collagen synthesis
of rat cardiac fibroblasts, which was accompanied by an increase of miR-21. Gain-of-function
and loss-of-function assays confirmed that miR-21 mediated this effect, suggesting
the crucial role of miR-21 in diabetic cardiomyopathy. Our study also identified a
direct target of miR-21, DUSP8, which regulates cell proliferation and collagen synthesis
in cardiac fibroblasts through p38 and c-Jun N-terminal kinase (JNK)/stress-activated
kinase (SAPK) signalling. Our results show that miR-21 bound to the 3′UTR of DUSP8
post-transcriptionally repressed its expression. In addition, enforced expression
of miR-21 activated the JNK/SAPK and p38 signalling pathways.
Conclusions
Our study shows that miR-21 promotes high glucose–induced cardiac fibrosis through
the JNK/SAPK and p38 signalling pathways by suppressing DUSP8 expression.
Résumé
Introduction
Il a été démontré que la microARN 21 (miARN-21) contribue à la fibrose cardiaque dans
plusieurs maladies. Dans cette étude, nous avons examiné le rôle de la miARN-21 dans
la production excessive de collagène lors de cardiomyopathie diabétique.
Méthodes
Le taux de prolifération des fibroblastes cardiaques a été analysé par le buvardage
de Western, le Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japon),
et Cell-Light EdU Apollo 488 In Vitro Imaging Kit (RiboBio, Guangzhou, Chine). La
réaction en chaîne par polymérase en temps réel et le buvardage de Western ont été
effectués pour déterminer les taux d’expression génique. Un dosage du gène rapporteur
de la luciférase a été utilisé pour vérifier l’interaction entre la miARN-21 et la
séquence non traduite en 3′ (3′UTR : untranslated region) et le DUSP8 (dual specific phosphatase 8 : phosphatase 8 à double spécificité).
Résultats
Nos résultats montrent qu’un glucose élevé stimule la prolifération des fibroblastes
cardiaques et favorise la synthèse du collagène chez le rat, et qu’il est accompagné
d’une augmentation de la miARN-1. Les dosages « gain de fonction » et « perte de fonction
» ont confirmé que la miARN-21 médiait cet effet, ce qui suggère le rôle crucial de
la miARN-21 dans la cardiomyopathie diabétique. Notre étude a de plus déterminé une
cible directe de la miARN-21, le DUSP8, qui régule la prolifération des cellules et
la synthèse du collagène dans les fibroblastes cardiaques par la signalisation de
p38 et de JNK/SAPK (c-Jun N-terminal kinase : protéine JNK/ stress-activated kinase : protéine SAPK). Nos résultats montrent que la miARN-21 liée à la 3'UTR du DUSP8 réprime son expression
de manière post-transcriptionnelle. De plus, l’expression renforcée de la miARN-21
a activé les voies de signalisation de JNK/SAPK et de p38.
Conclusions
Notre étude montre que la miARN-21 favorise la fibrose cardiaque induite par le glucose
élevé par les voies de signalisation de JNK/SAPK et de p38 en réprimant l’expression
du DUSP8.
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Article info
Publication history
Published online: August 19, 2014
Accepted:
July 30,
2014
Received:
July 31,
2013
Footnotes
See page 1698 for disclosure information.
Identification
Copyright
© 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.
