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MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2
We tested the hypothesis that Let-7d-3p contributes to cardiac cell protection during hypoxic challenge. Myoblast H9c2 cells and primary neonatal rat ventricular cardiomyocytes (NRVM) were transfected with five selected miRNA mimics. Both cell lines were subjected to 0.2% oxygen hypoxia. The protect...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480063/ https://www.ncbi.nlm.nih.gov/pubmed/30934671 http://dx.doi.org/10.3390/ijms20071522 |
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author | Wong, Lee Lee Saw, Eng Leng Lim, Jia Yuen Zhou, Yue Richards, Arthur Mark Wang, Peipei |
author_facet | Wong, Lee Lee Saw, Eng Leng Lim, Jia Yuen Zhou, Yue Richards, Arthur Mark Wang, Peipei |
author_sort | Wong, Lee Lee |
collection | PubMed |
description | We tested the hypothesis that Let-7d-3p contributes to cardiac cell protection during hypoxic challenge. Myoblast H9c2 cells and primary neonatal rat ventricular cardiomyocytes (NRVM) were transfected with five selected miRNA mimics. Both cell lines were subjected to 0.2% oxygen hypoxia. The protective effects of these miRNAs were determined by assessment of cell metabolic activity by CCK8 assay and measurement of lactate dehydrogenase (LDH) release as a marker of cell injury. Apoptosis and autophagy flux were assessed by Annexin V/7-AAD double staining and the ratio of LC3 II/I with Baf-A1 treatment, an autophagy flux inhibitor, respectively. Luciferase-reporter assay, RT-qPCR and Western blots were performed to identify the changes of relevant gene targets. Among five miRNA mimic transfections, Let-7d-3p increased CCK8 activity, and decreased LDH release in both H9c2 and NRVM during hypoxia. Apoptosis was significantly reduced in H9c2 cells transfected with Let-7d-3p mimic. Autophagy and autophagy flux were not affected. In silico, mRNAs of HMGA2, YY1, KLF9, KLF12, and MEX3C are predicted targets for Let-7d-3p. Luciferase-reporter assay confirmed that Let-7d-3p bound directly to the 3’-UTR region of HMGA2, MEX3C, and YY1, the down-regulations of these mRNAs were verified in both H9c2 and NRVM. The protein expression of HMGA2, but not others, was downregulated in H9c2 and NRVM. It is known that HMGA2 is a strong apoptosis trigger through the blocking of DNA repair. Thus, we speculate that the anti-apoptotic effects of Let-7d-3p mimic during hypoxia challenge are due to direct targeting of HMGA2. |
format | Online Article Text |
id | pubmed-6480063 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-64800632019-04-29 MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2 Wong, Lee Lee Saw, Eng Leng Lim, Jia Yuen Zhou, Yue Richards, Arthur Mark Wang, Peipei Int J Mol Sci Article We tested the hypothesis that Let-7d-3p contributes to cardiac cell protection during hypoxic challenge. Myoblast H9c2 cells and primary neonatal rat ventricular cardiomyocytes (NRVM) were transfected with five selected miRNA mimics. Both cell lines were subjected to 0.2% oxygen hypoxia. The protective effects of these miRNAs were determined by assessment of cell metabolic activity by CCK8 assay and measurement of lactate dehydrogenase (LDH) release as a marker of cell injury. Apoptosis and autophagy flux were assessed by Annexin V/7-AAD double staining and the ratio of LC3 II/I with Baf-A1 treatment, an autophagy flux inhibitor, respectively. Luciferase-reporter assay, RT-qPCR and Western blots were performed to identify the changes of relevant gene targets. Among five miRNA mimic transfections, Let-7d-3p increased CCK8 activity, and decreased LDH release in both H9c2 and NRVM during hypoxia. Apoptosis was significantly reduced in H9c2 cells transfected with Let-7d-3p mimic. Autophagy and autophagy flux were not affected. In silico, mRNAs of HMGA2, YY1, KLF9, KLF12, and MEX3C are predicted targets for Let-7d-3p. Luciferase-reporter assay confirmed that Let-7d-3p bound directly to the 3’-UTR region of HMGA2, MEX3C, and YY1, the down-regulations of these mRNAs were verified in both H9c2 and NRVM. The protein expression of HMGA2, but not others, was downregulated in H9c2 and NRVM. It is known that HMGA2 is a strong apoptosis trigger through the blocking of DNA repair. Thus, we speculate that the anti-apoptotic effects of Let-7d-3p mimic during hypoxia challenge are due to direct targeting of HMGA2. MDPI 2019-03-27 /pmc/articles/PMC6480063/ /pubmed/30934671 http://dx.doi.org/10.3390/ijms20071522 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Wong, Lee Lee Saw, Eng Leng Lim, Jia Yuen Zhou, Yue Richards, Arthur Mark Wang, Peipei MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2 |
title | MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2 |
title_full | MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2 |
title_fullStr | MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2 |
title_full_unstemmed | MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2 |
title_short | MicroRNA Let-7d-3p Contributes to Cardiac Protection via Targeting HMGA2 |
title_sort | microrna let-7d-3p contributes to cardiac protection via targeting hmga2 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480063/ https://www.ncbi.nlm.nih.gov/pubmed/30934671 http://dx.doi.org/10.3390/ijms20071522 |
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