MiR-199 Aggravates Doxorubicin-Induced Cardiotoxicity by Targeting TAF9b

Abstract

The clinical application of doxorubicin (DOX) is limited because of its cardiotoxicity. However, the pathogenic mechanism of DOX and the role of miRNA in DOX-induced cardiotoxicity remain to be further studied. This study aimed to investigate the role of miR-199 in DOX-mediated cardiotoxicity. A mouse model of myocardial cell injury induced by DOX was established. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression changes of miR-199 and TATA-binding protein associated factor 9B (TAF9b) in DOX-induced cardiac injury. Cell apoptosis was detected by TUNEL staining and flow cytometry. The expression levels of apoptosis-related proteins, namely, Bax and Bcl-2, were detected by qPCR. The expression of Beclin-1 and LC3b was detected by western blotting. The binding effect of miR-199 with TAF9b was verified by dual-luciferase reporter gene assay. In this study, overexpression of miR-199 could promote cardiotoxicity. Inhibition of miR-199 could alleviate DOX-mediated myocardial injury. Further studies showed that miR-199 targeted TAF9b. Moreover, miR-199 promoted apoptosis of myocardial cells and aggravated autophagy. Furthermore, we demonstrated that TAF9B knockdown reversed the myocardial protective effect of miR-199 inhibitors. Therefore, miR-199 promoted DOX-mediated cardiotoxicity by targeting TAF9b, thereby aggravating apoptosis and regulating autophagy.

Figure 1

Cardiomyocytes (AC16 cells) was injured by DOX. (a) The cell proliferation rate in the DOX induction group was distinctly lower than that in the control group. (b) The levels of TNF-α, IL-1β, and IL-6 in the DOX induction group were obviously higher. (c) The LDH and MDA in the DOX induction group was higher than that in the control group, while SOD in the DOX induction group decreased relatively to the control group. (d) Cell apoptosis detection by flow cytometry. (e) Detection of apoptosis cells by TUNEL staining. (f-g) Apoptosis-related protein expression detection by qPCR. (h) Beclin-1 and LC3B expression detection by western blotting. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 2

DOX promotes the expression of miR-199. (a) Detection of miR-199 in DOX-mediated cell model. (b) Detection of miR-199 in DOX-mediated mice model. (c) The myocardial tissue injury was observed by HE staining. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 3

Overexpression of miR-199 promotes the DOX-mediated cardiotoxicity. (a) Detection of miR-199 expression level in AC16 cells model after different treatment. (b) Detection of TNF-α, IL-1β, and IL-6 after different treatment. (c) Detection of LDH, SOD, and MDA by ELISA method. (d) Cell apoptosis test by TUNEL staining. (e) Cell apoptosis test by flow cytometry. (f) & (g) Detection of mRNA levels of Bax and Bcl-2 by RT-qPCR. (h) Beclin-1 and LC3B detection by western blotting. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Magnification: 200x.

Figure 4

TAF9b binds to miR-199 as a target gene. (a) The schematic diagram of the combination of miR-199 and TAF9b. (b) Detection of TAF9b in DOX-mediated AC16 cell model. (c) Detection of TAF9b expression level in DOX-mediated mice model. (d) Detection of TAF9b expression level after different treatment by RT-qPCR. (e) Detection of TAF9b expression level after different treatment by RT-qPCR. ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

Figure 5

Knockdown of TAF9b reverses the cardioprotective influence on miR-199 inhibitor. (a) Detection of TAF9b expression level. (b) Detection of TAF9b expression level in AC16 cells model after different treatment. (c) Cell viability test by CCK-8 method. (d) Detection of TNF-α, IL-1β, and IL-6 after different treatment. (e) Detection of the content of LDH, SOD, and MDA in DOX-mediated cell model by ELISA method. (f) Bax and Bcl-2 test by RT-qPCR. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 6

Overexpression of TAF9b reverses injury mediated by miR-199. (a) Transfection efficiency of TAF9b overexpressing plasmid. (b) Detection of AC16 cell apoptosis by flow cytometry. (c) AC16 cell apoptosis test by TUNEL staining. (d, e) Detection of mRNA level of Bax and Bcl-2 by RT-qPCR. (f) Beclin-1 and LC3B expression detection by western blotting. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.