MicroRNA-194-5p Attenuates Doxorubicin-Induced Cardiomyocyte Apoptosis and Endoplasmic Reticulum Stress by Targeting P21-Activated Kinase 2

Abstract

Objective: Many studies have reported that microRNAs (miRs) are involved in the regulation of doxorubicin (DOX)-induced cardiotoxicity. MiR-194-5p has been reported significantly upregulated in patients with myocardial infarction; however, its role in myocardial diseases is still unclear. Various stimuluses can trigger the endoplasmic reticulum (ER) stress and it may activate the apoptosis signals eventually. This study aims to explore the regulatory role of miR-194-5p in DOX-induced ER stress and cardiomyocyte apoptosis.

Methods: H9c2 was treated with 2 μM DOX to induce apoptosis, which is to stimulate the DOX-induced cardiotoxicity model. The expression of miR-194-5p was detected by quantitative real-time PCR (qRT-PCR); the interaction between miR-194-5p and P21-activated kinase 2 (PAK2) was tested by dual luciferase reporter assay; terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and caspase-3/7 activity were used to assess apoptosis; trypan blue staining was applied to measure cell death; Western blotting was performed to detect protein expressions; and ER-related factors splicing X-box binding protein 1 (XBP1s) was detected by polyacrylamide gel electrophoresis and immunofluorescence to verify the activation of ER stress.

Results: MiR-194-5p was upregulated in cardiomyocytes and mouse heart tissue with DOX treatment, while the protein level of PAK2 was downregulated. PAK2 was predicted as the target of miR-194-5p; hence, dual luciferase reporter assay indicated that miR-194-5p directly interacted with PAK2 and inhibited its expression. TUNEL assay, caspase-3/7 activity test, and trypan blue stain results showed that either inhibition of miR-194-5p or overexpression of PAK2 reduced DOX-induced cardiomyocyte apoptosis. Silencing of miR-194-5p also improved DOX-induced cardiac dysfunction. In addition, DOX could induce ER stress in H9c2, which led to XBP1 and caspase-12 activation. The expression level of XBP1s with DOX treatment increased first then decreased. Overexpression of XBP1s suppressed DOX-induced caspase-3/7 activity elevation as well as the expression of cleaved caspase-12, which protected cardiomyocyte from apoptosis. Additionally, the activation of XBP1s was regulated by miR-194-5p and PAK2.

Conclusion: Our findings revealed that silencing miR-194-5p could alleviate DOX-induced cardiotoxicity via PAK2 and XBP1s in vitro and in vivo. Thus, the novel miR-194-5p/PAK2/XBP1s axis might be the potential prevention/treatment targets for cancer patients receiving DOX treatment.

Keywords: ER stress; apoptosis; cardiotoxicity; doxorubicin; miR-194-5p.

FIGURE 1

MiR-194-5p participated in doxorubicin (DOX)-induced cardiomyocyte apoptosis. (A) H9c2 cells were treated with 2 μM DOX for the indicated times. The expression levels of miR-194-5p were measured by qRT-PCR. *p < 0.01 vs. control. (B) MiR-194-5p expression after transfection with miR-194-5p inhibitor for 24 h was measured by real-time quantitative PCR (qRT-PCR). (C) MiR-194-5p expression after transfection with miR-194-5p mimic for 24 h was measured by qRT-PCR. (D–F) Suppressed miR-194-5p expression with miR-194-5p inhibitor for 24 h and exposed cells to 2 μM DOX for 24 h. Apoptosis was detected by terminal deoxinucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (D). Green, TUNEL-positive nuclei; blue, 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei; scale bar, 200 μm. Statistical analysis of TUNEL-positive cells (E) and caspase 3/7 activity (F) are shown. (G,H) Enhanced miR-194-5p expression with miR-194-5p mimic for 24 h and exposed cells to 0.2 μM DOX for 24 h. Caspase-3/7 activity (G) and cell death rate (H) are shown. All the experiments have been performed independently in triplicate, and the data were expressed as mean ± SD. *p < 0.01 as indicated.

FIGURE 2

MiR-194-5p directly targeted PAK2. (A) Prediction of the PAK2 3′UTR potential binding site of miR-194-5p via bioinformatics program TargetScan. Potential complementary bases are shown in red. (B) H9c2 were treated with 2 μM DOX for the indicated times. The expression levels of PAK2 were detected by Western blots and the quantitative histogram was shown. ∧p < 0.05 vs. control. *p < 0.01 vs. control. (C) Schematic diagram of the reporter containing the putative miR-194-5p binding site in the PAK2 3′UTR region. WT, wild-type; MT, mutant. (D) Luciferase activity detected in HEK-293 cells transfected with miR-194-5p mimic or negative control along with luciferase reporter constructs, as indicated. (E) H9c2 was transfected with miR-194-5p inhibitor for 24 h. The expression levels of PAK2 were detected by Western blot and the quantitative histogram was shown. (F) H9c2 was transfected with miR-194-5p mimic for 24 h. The expression levels of PAK2 were detected by Western blot and the quantitative histogram was shown. All the experiments have been performed independently in triplicate, and the data were expressed as mean ± SD. *p < 0.01 as indicated.

FIGURE 3

PAK2 attenuated DOX-induced cardiomyocyte apoptosis. (A) H9c2 was transfected with PAK2-overexpressing plasmid for 24 h. The expression levels of PAK2 were detected by Western blot and the quantitative histogram was shown. (B) H9c2 was transfected with PAK2 small-interfering RNA (siRNA) for 24 h. The expression levels of PAK2 were detected by Western blot and the quantitative histogram was shown. (C–E) Enhanced PAK2 expression with PAK2-overexpressing plasmid for 24 h and exposed to 2 μM DOX for 24 h. Apoptosis was detected by TUNEL assay (C). Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei; scale bar, 200 μm. Statistical analysis of TUNEL-positive cells (D) and caspase-3/7 activity (E) are shown. (F) H9c2 was cotransfected with miR-194-5p mimic and PAK2-overexpressing plasmid for 24 h and then exposed to 0.2 μM DOX for 24 h. Cell death rate was analyzed. (G) H9c2 was transfected with PAK2 siRNA for 24 h and exposed to 0.2 μM DOX for 24 h. Cell death rate was analyzed. All the experiments have been performed independently in triplicate, and the data were expressed as mean ± SD. *p < 0.01 as indicated.

FIGURE 4

X-box binding protein 1 (XBP1) participated in DOX-induced cardiotoxicity. (A–E) H9c2 was treated with 2 μM DOX for the indicated times. The XBP1s levels were detected using Western blots (A) and the quantitative histogram was shown (B,C), and also detected using qRT-PCR (D). ∧p < 0.05 vs. control. *p < 0.01 vs. control. Spliced bands of XBP1 were detected by polyacrylamide gel electrophoresis (E). (F) H9c2 was treated with 2 μM DOX for 3 and 24 h. The localization of XBP1s in cells was detected by immunofluorescence experiment. Green, XBP1s; blue, DAPI-stained nuclei; scale bar, 50 μm. (G,H) H9c2 was treated with 2 μM DOX or 50 nM TG for the indicated times. The XBP1s levels were detected by Western blots and the quantitative histogram was shown (G), and the spliced bands were detected by polyacrylamide gel electrophoresis (H). TG, thapsigargin. (I) H9c2 was treated with 2 μM DOX for 3 h, which was pre-treated with 5 mM 4-PBA for 3 h. The XBP1s levels were detected by Western blots and the quantitative histogram was shown. All the experiments have been performed independently in triplicate, and the data were expressed as mean ± SD. ∧p < 0.05 vs. control. *p < 0.01 vs. control.

FIGURE 5

XBP1s attenuated DOX-induced cardiomyocyte endoplasmic reticulum (ER) stress and apoptosis. (A) H9c2 was transfected with XBP1s-overexpressing plasmid for 24 h. The expression levels of XBP1s were determined using Western blot and the quantitative histogram was shown. (B–D) Enhanced XBP1s expression in H9c2 cells with XBP1s-overexpressing plasmid for 24 h and exposed to 2 μM DOX for 24 h. The expression levels of cleaved caspase-12 were determined using Western blot and the quantitative histogram was shown (B). Caspase-3/7 activity (C) and cell death rate (D) were analyzed. All the experiments have been performed independently in triplicate, and the data were expressed as mean ± SD. ∧p < 0.05 vs. control. *p < 0.01 vs. control.

FIGURE 6

MiR-194-5p participated in DOX-induced ER stress and cardiomyocyte apoptosis through PAK2 and XBP1s. (A) Suppressed miR-194-5p expression with miR-194-5p inhibitor for 24 h and exposed to 2 μM DOX for 24 h. The expression levels of PAK2, XBP1s, and cleaved caspase-12 were detected by Western blot and the quantitative histogram was shown. (B) Enhanced PAK2 expression with PAK2-overexpressing plasmid for 24 h and exposed to 2 μM DOX for 24 h. The expression levels of PAK2, XBP1s, and cleaved caspase-12 were detected by Western blot and the quantitative histogram was shown. (C,D) H9c2 was cotransfected with miR-194-5p mimic and XBP1s-overexpressing plasmid for 24 h, then exposed to 0.2 μM DOX for 24 h. The expression levels of cleaved caspase-12 were detected by Western blot and the quantitative histogram was shown (C) and cell death rate was analyzed (D). (E,F) H9c2 was co-transfected with PAK2 siRNA and XBP1s-overexpressing plasmid for 24 h, then exposed to 0.2 μM DOX for 24 h. The expression levels of cleaved caspase-12 were detected by Western blot and the quantitative histogram was shown (E) and cell death rate was analyzed (F). All the experiments have been performed independently in triplicate, and the data were expressed as mean ± SD. ∧p < 0.05 vs. control. *p < 0.01 vs. control.

FIGURE 7

MiR-194-5p was involved in DOX-induced cardiotoxicity in vivo. (A) The expression levels of miR-194-5p in mice heart tissue were detected after DOX treatment by qRT-PCR. (B) Adenovirus-harbored anta-miR-194-5p was injected into the mice 1 week before DOX treatment. The expression levels of PAK2, XBP1s and cleaved caspase-12 were detected by Western blot. Echocardiographic analysis of left ventricular cardiac function in mice, EF (C) and FS (D) results are shown. EF, ejection fraction; FS, fractional shortening. Apoptosis was measured by TUNEL assay (F) and apoptotic rates were analyzed (E). Green, TUNEL-positive nuclei; blue, DAPI (4,6-diamidino-2-phenylindole)-stained nuclei; scale bar, 500 μm. (G) Masson trichrome staining for collagen performed. scale bar, 200 μm. Anta-194, adenovirus-harbored miR-194-5p antagomir. N = 4, and the data were expressed as mean ± SD. ∧p < 0.05 vs. control. *p < 0.01 vs. control.