MicroRNA-208a-3p contributes to connexin40 remolding in human chronic atrial fibrillation - PubMed (original) (raw)

MicroRNA-208a-3p contributes to connexin40 remolding in human chronic atrial fibrillation

Shanshan Li et al. Exp Ther Med. 2017 Dec.

Abstract

Previous studies have demonstrated that connexin40 (Cx40) remolding is involved in atrial fibrillation (AF). GJA5 encoding Cx40 is a potential target mRNA of microRNA-208a-3p (miR-208a-3p), as indicated by preliminary bioinformatics analyses. However, the exact effect of miR-208a-3p on Cx40 in human chronic AF has remained elusive. The present study demonstrated the role of miR-208a-3p in human chronic AF and further investigated the effect of miR-208a-3p on Cx40 expression. A total of 19 patients with AF and 18 patients with sinus rhythm (SR) were enrolled. The AC16 cell line was treated with miR-208a-3p inhibitor or mimics. The miR-208a-3p in right atrial appendage (RAA) tissues of patients was measured by in situ hybridization and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Furthermore, the expression of Cx40 in the RAA of patients and in AC16 cells treated with miR-208a-3p inhibitor or mimics were detected by RT-qPCR and western blot analysis. A luciferase assay was performed to confirm whether Cx40 was directly targeted by miR-208a-3p. The miR-208a-3p levels in patients with AF were significantly increased compared with those in patients with SR. Conversely, the Cx40 protein levels were significantly decreased and lateralization of Cx40 was observed in patients with AF. miR-208a-3p inhibitor led to a significant upregulation of the protein expression of Cx40 in AC16 cells, while miR-208a-3p mimics led to a significant downregulation. However, the luciferase assay demonstrated that GJA5 was not a direct target gene of miR-208a-3p. The findings still suggested that miR-208a-3p may be involved in human chronic AF by mediating atrial Cx40 remolding, and may represent a potential therapeutic target for AF.

Keywords: AC16 cell line; atrial fibrillation; connexin40; human; microRNA-208a-3p.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Predicted site of miR-208a-3p binding to GJA5 mRNA. miR, microRNA; UTR, untranslated region; hsa, Homo sapiens.

Figure 2.

Figure 2.

Expression of miR-208a-3p in human RAA. (A) Representative histological sections with in situ hybridization staining for miR-208a-3p (violet staining; magnification, ×100). (B) Relative expression of miR-208a-3p in human RAA determined by reverse-transcription quantitative polymerase chain reaction (n=18 in SR group, n=19 in AF group). Logarithmic transformation was performed for the data that was not normally distributed. **P<0.01 vs. SR group. miR, microRNA; RAA, right atrial appendage; SR, sinus rhythm; AF, atrial fibrillation.

Figure 3.

Figure 3.

Expression of Cx40 in human RAA. (A) Relative expression of Cx40 mRNA in human RAA determined by reverse-transcription quantitative polymerase chain reaction. Logarithmic transformation was performed for the data that was not normally distributed. (B) Relative expression of Cx40 in human RAA by western blot analysis (n=18 in SR group, n=19 in AF group). *P<0.05 vs. SR group. (C) Representative histological sections with immunohistochemical staining for Cx40 (brown; the white arrow represent Cx40 at the end-to-end connection between the cardiomyocytes; the black arrow represent Cx40 at the side-to-side connection between the cardiomyocytes. Magnification, ×200). Cx40, connexin 40; RAA, right atrial appendage; SR, sinus rhythm; AF, atrial fibrillation.

Figure 4.

Figure 4.

miR-208a-3p negatively regulates Cx40 expression in AC 16 cells. (A) miR-208a-3p inhibitor was not significantly affected by Cx40 mRNA in AC16 cells as determined by RT-qPCR. (B) miR-208a-3p inhibitor significantly elevated Cx40 protein levels in AC16 cells as indicated by western blot analysis. (C) miR-208a-3p mimics did not significantly affect Cx40 mRNA levels in AC16 cells as determined by RT-qPCR. (D) miR-208a-3p mimics significantly decreased Cx40 protein levels in AC16 cells as indicated by western blot analysis. **P<0.01 vs. miR mimics/inhibitor negative control group or blank control group. miR, microRNA; Cx40, connexin 40; RAA, right atrial appendage; SR, sinus rhythm; AF, atrial fibrillation; RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Figure 5.

Figure 5.

GJA5 is not a direct target gene of miR-208a-3p. (A) Predicted site of miR-208a-3p binding to GJA5 with the wild-type 3′-UTR and design of the MUT sequence. (B) Luciferase assay; overexpression of miR-208a-3p did not significantly inhibit the luciferase activity of the reporter plasmid carrying the wild-type 3′-UTR sequence of GJA5 in 293T cells. miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant-type; hsa, Homo sapiens.

References

    1. Rahman F, Kwan GF, Benjamin EJ. Global epidemiology of atrial fibrillation. Nat Rev Cardiol. 2014;11:639–654. doi: 10.1038/nrcardio.2014.118. - DOI - PubMed
    1. Nattel S, Dobrev D. Electrophysiological and molecular mechanisms of paroxysmal atrial fibrillation. Nat Rev Cardiol. 2016;13:575–590. doi: 10.1038/nrcardio.2016.118. - DOI - PubMed
    1. Wakili R, Voigt N, Kääb S, Dobrev D, Nattel S. Recent advances in the molecular pathophysiology of atrial fibrillation. J Clin Invest. 2011;121:2955–2968. doi: 10.1172/JCI46315. - DOI - PMC - PubMed
    1. Bikou O, Thomas D, Trappe K, Lugenbiel P, Kelemen K, Koch M, Soucek R, Voss F, Becker R, Katus HA, Bauer A. Connexin 43 gene therapy prevents persistent atrial fibrillation in a porcine model. Cardiovasc Res. 2011;92:218–225. doi: 10.1093/cvr/cvr209. - DOI - PubMed
    1. Segretain D, Falk MM. Regulation of connexin biosynthesis, assembly, gap junction formation, and removal. Biochim Biophys Acta. 2004;1662:3–21. doi: 10.1016/j.bbamem.2004.01.007. - DOI - PubMed

LinkOut - more resources