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

Zhang,Hai‑Jin; Zhang,Yi‑Na; Teng,Zong‑Yan

doi:10.3892/mmr.2019.10568

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

Authors:
- Hai‑Jin Zhang
- Yi‑Na Zhang
- Zong‑Yan Teng
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Affiliations: Department of Geriatrics, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
Published online on: August 7, 2019 https://doi.org/10.3892/mmr.2019.10568
Pages: 3113-3122
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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.

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