The lncRNA HOTAIR attenuates pyroptosis of diabetic cardiomyocytes by recruiting FUS to regulate SIRT3 expression

Figures

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  FIGURE 1. HG induced pyroptosis and the inflammatory response in cardiomyocytes. H9C2 cells were treated with NG (5.5 mM) or HG (30 mM) for 24 h. (A) RT–qPCR was used to evaluate HOTAIR and SIRT3 expression. (B) RT–qPCR was performed to analyze IL‐1β and IL‐18 levels. (C) ELISA was utilized to assess the secretion of IL‐1β and IL‐18. (D) Flow cytometry was conducted to detect pyroptosis. (E) Western blot analysis of pyroptosis‐ and inflammation‐related proteins. The results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 2. Overexpressed HOTAIR inhibited the HG‐induced pyroptosis and inflammatory response in cardiomyocytes. H9C2 cells were transfected with a HOTAIR overexpression plasmid and treated with HG. (A) The efficiency of HOTAIR overexpression was detected by RT–qPCR. (B) RT–qPCR was used to analyze IL‐1β and IL‐18 levels. (C) ELISA was performed to assess the secretion of IL‐1β and IL‐18. (D) Flow cytometry was utilized to detect pyroptosis. (E) Western blot analysis of pyroptosis‐ and inflammation‐related proteins. The results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 3. HOTAIR upregulated the level of SIRT3 through FUS. (A, B) RNA pulldown and RIP experiments were conducted to verify the binding relationship between FUS and HOTAIR in H9C2 cells. (C) The efficiency of HOTAIR knockdown was detected by RT–qPCR. (D, E) RT–qPCR and Western blot analysis of FUS expression after knockdown of HOTAIR. (F) RT–qPCR detection of FUS overexpression efficiency. (G) The level of FUS was analyzed by Western blotting. (H) RIP analysis of the binding relationship between SIRT3 and FUS. The results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 4. SIRT3 overexpression suppressed HG‐triggered cardiomyocyte pyroptosis. H9C2 cells were transfected with a SIRT3‐overexpressing plasmid and treated with HG. (A, B) The transfection efficiency of SIRT3 was detected by RT–qPCR and Western blotting. (C) RT–qPCR was performed to analyze IL‐1β and IL‐18 levels. (D) ELISA was used to assess the secretion of IL‐1β and IL‐18. (E) Flow cytometry detection of pyroptosis. (F) Western blot analysis of pyroptosis‐ and inflammation‐related proteins. The results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 5. SIRT3 knockdown reversed the inhibitory effect of HOTAIR on HG‐induced cardiomyocyte pyroptosis. HOTAIR overexpression and SIRT3 knockdown plasmids were transfected into HG‐exposed H9C2 cells. (A, B) The transfection efficiency of sh‐SIRT3 was detected by RT–qPCR and Western blotting. (C) RT–qPCR was utilized to analyze IL‐1β and IL‐18 levels. (D) ELISA was used to assess the secretion of IL‐1β and IL‐18. (E) Flow cytometry detection of pyroptosis. (F) Western blot analysis of pyroptosis‐ and inflammation‐related proteins. The results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.