MicroRNA‑133b alleviates doxorubicin‑induced cardiomyocyte apoptosis and cardiac fibrosis by targeting PTBP1 and TAGLN2

Figure 1. miR-133b expression is decreased by doxorubicin treatment in HL-1 cardiomyocytes and cardiac tissues. (A) Apoptosis of HL-1 cardiomyocytes induced by different concentrations of doxorubicin was determined by flow cytometry. *P<0.05 and **P<0.01 vs. 0 µm. (B) RT-qPCR assays were used to measure miR-133b expression in the HL-1 cardiomyocytes treated with or without doxorubicin. **P<0.01 vs. CON. Echocardiography analysis was adopted to examine (C) left ventricular end-diastolic diameter, (D) left ventricular end-systolic diameter, (E) left ventricular ejection fraction and (F) left ventricular ejection shortening in mice (n=8/group). (G) RT-qPCR assays were used to measure miR-133b expression in the sham and DOX groups (n=8/group). *P<0.05 vs. sham. miR, microRNA; DOX, doxorubicin; CON, control; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2. Overexpression of miR-133b inhibits the apoptosis and collagen accumulation in the doxorubicin-treated HL-1 cardiomyocytes. (A) Overexpression efficiency of miR-133b mimics was evaluated by reverse transcription-quantitative PCR in the doxorubicin-treated HL-1 cardiomyocytes. (B) Effect of miR-133b overexpression on apoptosis of the doxorubicin-treated HL-1 cardiomyocytes was assessed by flow cytometry. Western blot assays of the changes in the protein expression levels of (C and D) Bcl-2, Bax and cleaved caspase-3, and (E and F) collagen I, III and IV, and fibronectin in the doxorubicin-treated HL-1 cardiomyocytes transfected with miR-NC or miR-133b mimics. *P<0.05, **P<0.01 and ***P<0.001 vs. miR-NC. miR, microRNA; DOX, doxorubicin; NC, negative control.

Figure 3. Overexpression of miR-133b alleviates apoptosis and cardiac fibrosis in vivo. (A) Overexpression efficacy of AAV-miR-133b was confirmed by reverse transcription-quantitative PCR (n=8/group). (B) Protein expression levels of Bcl-2, Bax and cleaved caspase-3 in cardiac tissues were determined by western blot assays (n=8/group). (C and D) Masson's trichrome staining was used to evaluate interstitial fibrosis in the mouse heart (n=8/group). Scale bar, 20 µm. (E) Collagen content in cardiac tissues (n=8/group). (F) Masson's staining of myocardial peripheral vessels and the percentage of perivascular fibrosis (n=8/group). Scale bar, 50 µm. (G) Protein expression levels of collagen I, III and IV, and fibronectin in cardiac tissues were determined by western blot assays (n=8/group). *P<0.05, **P<0.01 and ***P<0.001 vs. sham; #P<0.05, ##P<0.01 and ###P<0.001 vs. DOX+AAV-miR-NC. miR, microRNA; DOX, doxorubicin; NC, negative control; AAV, adeno-associated virus.

Figure 4. PTBP1 and TAGLN2 serve as targets of miR-133b. (A) mRNA expression levels of predicted mRNAs in HL-1 cardiomyocytes treated with or without doxorubicin. *P<0.05 and **P<0.01 vs. CON. (B) Binding sites between miR-133b and PTBP1 or TAGLN2. (C) Luciferase reporter assay was adopted to confirm the interaction between miR-133b and PTBP1 or TAGLN2. *P<0.05 vs. miR-NC. (D) mRNA expression levels of PTBP1 and TAGLN2 in the sham and DOX groups. *P<0.05. (E) Western blot assays of the changes in the PTBP1 and TAGLN2 protein expression levels in the doxorubicin-treated HL-1 cardiomyocytes transfected with miR-NC or miR-133b mimics. **P<0.01 vs. miR-NC. (F) RF classifier score and SVM classifier score of the combination of PTBP1 and the TAGLN2 3′-UTR or TAGLN2. (G) RNA pulldown and (H) RNA immunoprecipitation assays were utilized to evaluate the interaction between PTBP1 and TAGLN2. miR, microRNA; DOX, doxorubicin; CON, control; NC, negative control; UTR, untranslated region; PTBP1, polypyrimidine tract binding protein 1; TAGLN2, transgelin 2; Wt, wild-type; Mut, mutant; Bio, biotin; RF, random forest; SVM, support vector machine.

Figure 5. Overexpression of PTBP1 or TAGLN2 reverses the effects of miR-133b on apoptosis and collagen accumulation. Overexpression of (A) PTBP1 or (B) TAGLN2 in the doxorubicin-treated HL-1 cardiomyocytes following miR-133b overexpression was assessed by reverse transcription-quantitative PCR assays. (C) Flow cytometry was used to examine the apoptosis in the indicated groups. Western blot assays were performed to explore the protein expression levels of (D) Bcl-2, Bax and cleaved caspase-3, and (E) collagen I, III and IV, and fibronectin in the indicated groups. *P<0.05 and **P<0.01 vs. miR-NC; #P<0.05 and ##P<0.01 vs. miR-133b. miR, microRNA; DOX, doxorubicin; NC, negative control; PTBP1, polypyrimidine tract binding protein 1; TAGLN2, transgelin 2.