Downregulation of miR‑16 protects H9c2(2‑1) cells against hypoxia/reoxygenation damage by targeting CIAPIN1 and regulating the NF‑κB pathway

Abstract

The aim of the present study was to determine the function of microRNA‑16 (miR‑16) in myocardial hypoxia/reoxygenation (H/R)‑induced cardiomyocyte injury and the possible mechanism underlying its involvement. An H/R model was constructed using H9c2(2‑1) cells in vitro. The results of reverse transcription‑quantitative PCR demonstrated that the expression levels of miR‑16 were significantly upregulated in H9c2(2‑1) cells in the H/R group compared with the sham group (1.53±0.09 vs. 1.0±0.08; P=0.0019). Cell Counting Kit‑8 assays revealed that the relative proliferative ability of H9c2(2‑1) cells was significantly decreased in the H/R + negative control (NC) group compared with the sham group (0.53±0.05 vs. 1.0±0.08; P=0.00005). Upregulation of miR‑16 using miR‑16 mimics further decreased the proliferative ability of cells (0.31±0.03 vs. 0.53±0.05; P=0.0097), whereas downregulation of miR‑16 using an miR‑16 inhibitor increased the proliferative ability of cells compared with the H/R+NC group (0.89±0.08 vs. 0.53±0.05; P=0.000385). Flow cytometric analysis found that the apoptotic rate of H9c2(2‑1) cells was increased significantly following H/R compared with the sham group (25.86±2.62% vs. 9.29±0.82%, P=0.000014). Upregulation of miR‑16 further increased the apoptotic rate (38.62±2.04% vs. 25.86±2.62%; P=0.000099), whereas downregulation of miR‑16 decreased the apoptotic rate compared with the H/R+NC group (15.14±0.92% vs. 25.86±2.62%; P=0.000343). miR‑16 directly bound to the 3'‑untranslated region of cytokine‑induced apoptosis inhibitor 1 (CIAPIN1) and negatively modulated CIAPIN1 expression. Overexpression of CIAPIN1 reversed the changes in the expression of apoptosis‑associated proteins caused by H/R. Western blot analysis revealed that the levels of phospho‑(p‑)nuclear factor‑κB (NF‑κB) and p‑NF‑κB inhibitor α (IκBα) were upregulated following H/R (1.82±0.11 vs. 1.0±0.08; P=0.000152; and 1.77±0.07 vs. 1.0±0.00; P=0.000024, respectively), and these changes were further enhanced when miR‑16 expression levels were increased (3.10±0.14 vs. 1.82±0.11; P=0.000006; and 2.19±0.10 vs. 1.77±0.07; P=0.0017, respectively). Downregulation of miR‑16 exhibited the opposite effect on p‑NF‑κB and p‑IκBα expression levels. The present study illustrates that downregulation of miR‑16 may protect against H/R‑induced injury partially by targeting CIAPIN1 and the NF‑κB signaling pathway.

Figure 1.

mRNA expression levels of miR-16 in H9c2 cells following H/R. (A) mRNA expression levels of miR-16 were significantly upregulated in H9c2 cells following H/R compared to the sham treatment. **P<0.01. (B) mRNA expression levels of miR-16 were increased or decreased in H9c2 cells following transfection with miR-16 mimics or miR-16 inhibitor, respectively. **P<0.01 vs. miR-16 mimics NC, ^##P<0.01 vs. miR-16 inhibitor NC. (C) mRNA expression levels of miR-16 were increased or decreased by miR-16 mimics or the miR-16 inhibitor, respectively, in the H/R model. **P<0.01 vs. sham; ^##P<0.01 vs. miR-16 mimics NC, ^$$P<0.01 vs. miR-16 inhibitor NC. miR, microRNA; NC, negative control; H/R, hypoxia/reoxygenation.

Figure 2.

Effect of miR-16 expression on the viability of H9c2 cells following H/R, determined using a cell counting kit-8 assay. **P<0.01 vs. sham; ^##P<0.01 vs. H/R+NC. miR, microRNA; NC, negative control; H/R, hypoxia/reoxygenation.

Figure 3.

miR-16 regulates the apoptosis of H9c2 cells following H/R. Effect of miR-16 expression on apoptosis of H9c2 cells following H/R was determined using flow cytometry. **P<0.01 vs. sham; ^##P<0.01 vs. H/R+NC. miR, microRNA; NC, negative control; H/R, hypoxia/reoxygenation.

Figure 4.

Effect of miR-16 on the expression of apoptosis-associated proteins in H9c2 cells following H/R. (A) Protein expression levels of Bcl-2, Bax and cleaved-caspase-3 were determined by western blotting. Relative expression levels of (B) Bcl-2, (C) Bax and (D) cleaved caspase-3. **P<0.01 vs. sham; ^##P<0.01 vs. H/R+NC. miR, microRNA; NC, negative control; H/R, hypoxia/reoxygenation.

Figure 5.

miR-16 directly binds to the 3′-untranslated region of CIAPIN1. (A) Predicted binding site of miR-16 with CIAPIN1 and the mutated binding site used in the present study. (B) Luciferase activity in cells transfected with miR-16 mimics + WT or miR-16 mimics + Mut compared with cells transfected with miR-16 mimics NC + WT and miR-16 mimics NC + Mut. **P<0.01 vs. H/R+miR-16 mimic NC. CIAPIN1, cytokine-induced apoptosis inhibitor 1; MUT, mutated; WT, wildtype; miR, microRNA; NC, negative control; H/R, hypoxia/reoxygenation.

Figure 6.

Transfection with miR-16 mimics decreases the expression levels of CIAPIN1 following H/R. (A) mRNA expression levels of CIAPIN1 in H9c2 cells were detected by qPCR. **P<0.01 vs. sham; ^##P<0.01 vs. H/R+NC. (B) Protein expression levels of CIAPIN1 in H9c2 cells were determined by western blotting. **P<0.01 vs. sham; ^#P<0.05, ^##P<0.01 vs. H/R+NC. CIAPIN1, cytokine induced apoptosis inhibitor 1; miR, microRNA; NC, negative control; H/R, hypoxia/reoxygenation.

Figure 7.

Effect of CIAPIN1 overexpression on the expression of apoptosis-associated proteins in H9c2 cells following H/R. (A) mRNA expression levels of CIAPIN1 in H9c2 cells transfected with pcDNA3.1-CIAPIN1 compared with cells transfected with the empty vector. **P<0.01. (B) Protein expression levels of CIAPIN1 in H9c2 cells was detected by western blotting. (C) Relative protein expression levels of CIAPIN1 in H9c2 cells transfected with pcDNA3.1-CIAPIN1 compared with cells transfected with the empty vector. **P<0.01. (D) Expression levels of Bcl-2 and Bax were determined by western blotting. (E) Relative expression levels of Bcl-2 and Bax. *P<0.05. CIAPIN1, cytokine induced apoptosis inhibitor 1; H/R, hypoxia/reoxygenation; OE, overexpression; vector, empty vector.

Figure 8.

Effect of miR-16 on the expression of NF-κB, p-NF-κB, IκBα and p-IκBα. (A) Protein expression levels of NF-κB, p-NF-κB, IκBα and p-IκBα were determined by western blot. (B) Relative protein expression levels of p-NF-κB in cells following H/R and/or transfection of the miR-mimics or inhibitors. **P<0.01 vs. sham; ^##P<0.01 vs. H/R+NC. (C) Relative protein expression levels of p-IκBα in cells following H/R and/or transfection of the miR-mimics or inhibitors. **P<0.01 vs. sham; ^##P<0.01 vs. H/R+NC. miR, microRNA; H/R, hypoxia/reoxygenation; p-, phospho; NC, negative control; NF-κB, nuclear factor-κB; IκBα, NF-κB inhibitor α.