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. 2020 Nov 17:8:e10371.
doi: 10.7717/peerj.10371. eCollection 2020.

MiR-26a-5p inhibits GSK3β expression and promotes cardiac hypertrophy in vitro

Liqun Tang  1 , Jianhong Xie  1 , Xiaoqin Yu  2 , Yangyang Zheng  1
Affiliations

MiR-26a-5p inhibits GSK3β expression and promotes cardiac hypertrophy in vitro

Liqun Tang et al. PeerJ. .

Abstract

Background: The role of miR-26a-5p expression in cardiac hypertrophy remains unclear. Herein, the effect of miR-26a-5p on cardiac hypertrophy was investigated using phenylephrine (PE)-induced cardiac hypertrophy in vitro and in a rat model of hypertension-induced hypertrophy in vivo.

Methods: The PE-induced cardiac hypertrophy models in vitro and vivo were established. To investigate the effect of miR-26a-5p activation on autophagy, the protein expression of autophagosome marker (LC3) and p62 was detected by western blot analysis. To explore the effect of miR-26a-5p activation on cardiac hypertrophy, the relative mRNA expression of cardiac hypertrophy related mark GSK3β was detected by qRT-PCR in vitro and vivo. In addition, immunofluorescence staining was used to detect cardiac hypertrophy related mark α-actinin. The cell surface area was measured by immunofluorescence staining. The direct target relationship between miR-26a-5p and GSK3β was confirmed by dual luciferase report.

Results: MiR-26a-5p was highly expressed in PE-induced cardiac hypertrophy. MiR-26a-5p promoted LC3II and decreased p62 expression in PE-induced cardiac hypertrophy in the presence or absence of lysosomal inhibitor. Furthermore, miR-26a-5p significantly inhibited GSK3β expression in vitro and in vivo. Dual luciferase report results confirmed that miR-26a-5p could directly target GSK3β. GSK3β overexpression significantly reversed the expression of cardiac hypertrophy-related markers including ANP, ACTA1 and MYH7. Immunofluorescence staining results demonstrated that miR-26a-5p promoted cardiac hypertrophy related protein α-actinin expression, and increased cell surface area in vitro and in vivo.

Conclusion: Our study revealed that miR-26a-5p promotes myocardial cell autophagy activation and cardiac hypertrophy by regulating GSK3β, which needs further research.

Keywords: Autophagy; Cardiac hypertrophy; GSK3β; LC3; MiR-26a-5p; α-actinin.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. MiR-26a promotes myocardial cell autophagy activation in PE-induced cardiac hypertrophy.
(A) The mRNA expression levels of miR-26a-5p in H9C2 cells treated by different concentrations of PE. (B–E) Western blot analysis results showing the expression of LC3II, Beclin-1 and p62. (F–H) The expression of LC3II and p62 in H9C2 cells in the presence or absence of Baf-A1. Data were presented as mean ±  SD (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001.
Figure 2
Figure 2. Immunofluorescence results showing the effect of miR-26a-5p on LC3 protein expression in PE-induced cardiac hypertrophy.
(A–R) Representative images of immunofluorescence results (×200). (S) The expression of LC3 protein was measured in H9C2 cells treated with 200 µM PE. Data were presented as mean ±  SD (n = 3). ∗p < 0.05; ∗∗∗∗p < 0.0001.
Figure 3
Figure 3. MiR-26a-5p inhibits GSK3β expression and promotes cardiac hypertrophy in vitro.
(A, B) qRT-PCR results showing the expression levels of miR-26a-5p and GSK3β in H9C2 cells treated with PE. (C, D) Western blot showing the effect of miR-26a-5p on the expression of GSK3 β and α-actinin proteins. (E, F) Dual luciferase report results confirmed that miR-26a-5p could bind to GSK3β 3′ UTR. (G) The effect of α-actinin on the mRNA expression of α-actinin according to qRT-PCR results. (H–Z) Immunofluorescence results showing the effect of miR-26a-5p on α-actinin protein in PE-induced cardiac hypertrophy (×200). The marker of α-actinin was stained by green color. Data were presented as mean ±  SD (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 4
Figure 4. Effects of miR-26a-5p on the surface areas in PE-induced cardiac hypertrophy.
(A–R) Representative images of TRITC-phalloidin fluorescence staining. (S) Relative cardiomyocyte surface areas. Data were presented as mean ±  SD (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001.
Figure 5
Figure 5. miR-26a-5p promotes cardiac hypertrophy by regulating GSK3 β.
(A–C) qRT-PCR results showing the effects of miR-26a-5p on PE-induced H9C2 cells. (D, E) GSK3β was successfully overexpressed and silenced in H9C2 cells. (F–H) According to qRT-PCR results, miR-26a-5p could promote the expression levels of cardiac hypertrophy-related markers by regulating GSK3β. Data were presented as mean ±  SD (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 6
Figure 6. MiR-26a-5p inhibits GSK3 β expression and promotes cardiac hypertrophy in vivo.
(A, B) qRT-PCR showing the mRNA expression levels of miR-26a-5p and GSK3β in SHR injected with miR-26a agomir. (C–G) The protein expression levels of GSK3β, LC3II, Beclin-1 and p62 were detected in myocardial tissues by western blot. (H–P) Hematoxylin and eosin (H&E) staining of myocardial tissues in the three groups (×200). (Q–S) qRT-PCR results showing the mRNA expression levels of cardiac hypertrophy-related markers including ANP, ACTA1 and MYH7 in myocardial tissues. Data were presented as mean ±  SD n = 3). ∗∗p-value < 0.01; ∗∗∗∗p-value <0.0001; ns, no statistical significance.
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Grants and funding

This work was funded by the Zhejiang Analytical Testing Fund (2017C37068) and the Zhejiang Medical and Health Research Fund (2018KY259). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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