Identification of miR‑145 as a regulator of the cardiomyocyte inflammatory response and oxidative stress under hyperglycemia

Figures

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  Figure 1. miR-145 and ARF6 expressions are dysregulated in HG-treated H9c2 cells. Cells were treated with 5.5 or 33 mmol/l glucose, or co-treated with 24.5 mmol/l mannitol and 5.5 mmol/l glucose for 2 h. (A) RT-qPCR was performed to detect miR-145 expression. (B) RT-qPCR and (C) western blotting were also performed to measure the mRNA and protein expression of ARF6. **P<0.01 vs. the NG group. miR, microRNA; ARF6, ADP ribosylation factor 6; HG, high glucose; RT-qPCR, reverse transcription-quantitative PCR; NG, normal glucose.

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  Figure 2. miR-145 ameliorates the inflammatory response and oxidative stress injury in HG-treated H9c2 cells. Following treatment with 5.5 or 33 mmol/l glucose for 2 h, H9c2 cells were transfected with miR-145 mimics or mimic NCs. (A) miR-145 expression was detected by reverse transcription-quantitative PCR. The expression of (B) IL-6, (C) TNF-α and (D) MCP-1 was determined via ELISA. (E) LDH content was detected using an LDH assay. (F) ROS content was examined using an ROS detection kit. (G) ELISA was performed to determine MDA content using a lipid peroxidation (MDA) assay kit. (H) The activities of antioxidant enzymes were evaluated by ELISA. (I) Flow cytometry detected the apoptotic rate of H9c2 cells. *P<0.05, **P<0.01 and ***P<0.001 vs. indicated groups. miR, microRNA; HG, high glucose; NC, negative controls; MCP-1, monocyte chemoattractant protein 1; LDH, lactate dehydrogenase; ROS, reactive oxygen species; MDA, malondialdehyde; NG, normal glucose; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase.

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  Figure 3. Inhibitory effect of ARF6 knockdown on the inflammatory response and oxidative stress injury in HG-treated H9c2 cells. Following 2 h of 33 mmol/l glucose treatment, H9c2 cells were transfected with pcDNA3.1-ARF6, sh-ARF6 or corresponding negative controls, and the transfection efficiency was examined by (A) reverse transcription-quantitative PCR and (B) western blotting. Levels of (C) IL-6, (D) TNF-α and (E) MCP-1 were assessed by ELISA. (F) LDH content was determined via LDH assays. (G) ROS content was measured by ROS kits. (H) MDA content and (I) activities of antioxidant enzymes were evaluated by ELISA. (J) Detection of H9c2 cell apoptotic rate by flow cytometry. *P<0.05, **P<0.01 and ***P<0.001 vs. indicated groups. ARF6, ADP ribosylation factor 6; HG, high glucose; sh, short hairpin; MCP-1, monocyte chemoattractant protein 1; LDH, lactate dehydrogenase; ROS, reactive oxygen species; MDA, malondialdehyde; NC, negative control.

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  Figure 4. miR-145 negatively regulates ARF6. Following transfection of miR-145 mimics or mimic NCs into H9c2 cells, mRNA and protein expression levels were detected through (A) reverse transcription-quantitative PCR and (B) western blotting. (C) The potential binding sites between ARF6 and miR-145 were predicted by StarBase and WT and MT sites were designed. (D) Detection of the miR-145-ARF6 interaction by luciferase reporter assay following co-transfection of WT-ARF6 or MT-ARF6 with miR-145 mimics or mimic NCs into 293T cells. **P<0.01 vs. indicated groups. miR, microRNA; ARF6, ADP ribosylation factor 6; NCs, negative controls; WT, wild-type; MT, mutant; ns, not significant.

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  Figure 5. miR-145 protects H9c2 cells from the HG-induced inflammatory response and oxidative stress injury. Following 2 h of 33 mmol/l glucose treatment, H9c2 cells were transfected with miR-145 mimics, pcDNA3.1-ARF6, miR-145 + pcDNA3.1-ARF6 or corresponding negative controls, and the expression levels of (A) miR-145 and ARF6 were determined by (B) reverse transcription-quantitative PCR and (C) western blotting. Levels of (D) IL-6, (E) TNF-α and (F) MCP-1 were detected by ELISA. (G) LDH content was detected by a LDH kit. (H) ROS content was determined using an ROS kit. (I) MDA content and (J) activities of antioxidant enzymes were evaluated by ELISA. (K) Detection of H9c2 cell apoptotic rate by flow cytometry. *P<0.05, **P<0.01 and ***P<0.001 vs. indicated groups. miR, microRNA; HG, high glucose; MCP-1, monocyte chemoattractant protein 1; LDH, lactate dehydrogenase; ROS, reactive oxygen species; MDA, malondialdehyde; ns, not significant.