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MiR?449a?5p suppresses CDK6 expression to inhibit cardiomyocyte proliferation.

ABSTRACT: Induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRs) have been reported to regulate CM proliferation. In particular, miR?449a?5p has been identified to be associated with CM proliferation in previous high throughput functional screening data. However, whether miR?449a?5p regulates CM proliferation has not been thoroughly investigated. This study aimed to explore whether miR?449a?5p modulates CM proliferation and to identify the molecular mechanism via which miR?449a?5p regulates CM proliferation. The current study demonstrated that miR?449a?5p expression levels were significantly increased during heart development. Furthermore, the results suggested that miR?449a?5p mimic inhibited CM proliferation in vitro as determined via immunofluorescence for ki67 and histone H3 phosphorylated at serine 10 (pH3), as well as the numbers of CMs. However, miR?449a?5p knockdown promoted CM proliferation. CDK6 was identified as a direct target gene of miR?449a?5p, and CDK6 mRNA and protein expression was suppressed by miR?449a?5p. Moreover, CDK6 gain?of?function increased CM proliferation. Overexpression of CDK6 also blocked the inhibitory effect of miR?449a?5p on CM proliferation, indicating that CDK6 was a functional target of miR?449a?5p in CM proliferation. In conclusion, miR?449a?5p inhibited CM proliferation by targeting CDK6, which provides a potential molecular target for preventing myocardial injury.

SUBMITTER: Li B

PROVIDER: S-EPMC7673318 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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miR‑449a‑5p suppresses CDK6 expression to inhibit cardiomyocyte proliferation.

Li Bing B Wang Zhi Z Yang Fan F Huang Jing J Hu Xingwei X Deng Shiyan S Tian Maobo M Si Xiaoyun X

Molecular medicine reports 20201112 1

Induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRs) have been reported to regulate CM proliferation. In particular, miR‑449a‑5p has been identified to be associated with CM proliferation in previous high throughput functional screening data. However, whether miR‑449a‑5p regulates CM proliferation has not been thoroughly investigated. This study aimed to explore whether miR‑449a‑5p modulates CM proliferation ...[more]

PMID: 33179102

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Project description:BackgroundLong non-coding ribonucleic acids (lncRNAs) are believed to play crucial roles in cardiovascular diseases; however, details of the underlying mechanisms by which this occurs remain unclear.MethodsA mouse heart failure (HF) model was established using isoproterenol (ISO), and confirmed by immunostaining and echocardiography. RNA-sequencing was performed to screen the differential lncRNA expression profiles and heart failure relative lncRNA (HFRL) was selected as the target which was validated by quantitative real-time polymerase chain reaction (qRT-PCR). In HL-1 cells, the cardiac function, inflammatory, and fibrosis-related genes expression changes were examined by qRT-PCR after silencing of HFRL by lentivirus. Meanwhile, Cell Counting Kit-8 (CCK-8) assays were used to detect the effects of HFRL on the cell proliferation and viability. Reactive oxygen species (ROS) assays were also used to explore the role of HFRL in oxidative damage. Next, bioinformatics analysis was conducted to predict the potential binding microRNAs (mmu-miR-149-5p) to HFRL, which was confirmed by RNA-pulldown assays. The target gene of miR-149-5p was also predicted and further validated by Dual-luciferase reporter assays, qRT-PCR, and western blot. To investigate the synergistic regulatory effect of HFRL and miR-149-5p, HL-1 cells were infected with the lentivirus of HFRL with or without simultaneous knockdown of miR-149-5p. Then, qRT-PCR and western blot were used to examine cardiac function, inflammatory, and fibrosis-related gene expression changes, respectively. In HL-1 cells, CCK-8 assays were performed to detect the proliferation and viability. ROS assays were used to explore the oxidative damage.ResultsThe administration of ISO induced mice fibrosis, inflammation, and HF. The in-vitro results showed that knockdown of HFRL suppressed cardiomyocyte proliferation and viability, attenuated inflammatory, cardiac function, and fibrosis-related gene expression, and promoted oxidative damage. HFRL was found to bind to mmu-miR-149-5p and inversely target the 3'-untranscripted region of the collagen 22A1 gene. Thus, HFRL affected cardiomyocyte inflammation, proliferation, viability, oxidative damage, and pro-fibrotic function via sequestration to miR-149-5p.ConclusionsThe HFRL/miR-149-5p axis plays an important role in regulating cardiac inflammation, proliferation, and fibrosis via a synergistic effect, which suggests that HFRL might be a novel target for HF.

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MiR?449a?5p suppresses CDK6 expression to inhibit cardiomyocyte proliferation.

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miR‑449a‑5p suppresses CDK6 expression to inhibit cardiomyocyte proliferation.

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