CircularRNA circ_0071269 knockdown protects against from diabetic cardiomyopathy injury by microRNA-145/gasdermin A axis

Figure 1.. The expression of circ_0071269 and circRNA characterization in H9c2 cells. a: qRT-PCR was used to detect circ_0071269 expression in H9c2 cells treated with high glucose. b: After RNase R treatment, the expression levels of linear-circ_0071269 and circular-circ_0071269 in H9c2 cells were measured by qRT-PCR. c: After treatment with actinomycin D in H9c2 cells at the specified time point, the relative RNA levels were determined by qRT-PCR. All experimental results were obtained by repeating three times. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.. Circ_0071269 knockdown suppresses the pyroptosis of H9c2 cells. a: Two different siRNAs targeting circ_0071269 were detected by qRT-PCR to evaluate the efficiency of circ_0071269 knockdown in H9c2 cells. b: The viability of H9c2 cells treated with high glucose after circ_0071269 knockdown was detected by CCK-8. c: The LDH content of H9c2 cells treated with high glucose after circ_0071269 knockdown was detected using an LDH kit. d-e: The levels of IL-1β and IL-18 in H9c2 cells treated with high glucose after circ_0071269 knockdown were determined using an ELISA kit. f: The apoptosis rate of H9c2 cells treated with high glucose after circ_0071269 knockdown was detected by flow cytometry. G: The protein expression levels of NLRP3 and caspase-1 in H9c2 cells treated with high glucose after circ_0071269 knockdown were detected by Western blotting. All experimental results were obtained by repeating three times. *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.

Figure 3.. Circ_0071269 acts as a sponge for miR-145. a: Bioinformatics predictions show the targeting relationship between circ_0071269 and miR-145. b: Luciferase activity experiments were performed to verify the targeting relationship between circ_0071269 and miR-145. c: Relative mRNA enrichment of circ_0071269 was captured using biotin-miR-145. d: miR-145 expression reduced in H9c2 cells treated with high glucose. E: miR-145 was elevated in H9c2 cells induced by high glucose that knocked down circ_0071269. All experimental results were obtained by repeating three times. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.. miR-145 restores the inhibitory effect of circ_0071269 on the progression of H9c2. a: The level of miR-145 in H9c2 cells treated with miR-145 inhibitor and miR-145 mimic was measured by qRT-PCR. b: Cell viability detected by CCK-8 assay was suppressed in glucose-stimulated H9c2 cells co-transfected with miR-145 inhibitor and si-circ_0071269. c: The LDH content of H9c2 cells induced by high glucose, which inhibited miR-145 and knocked down circ_0071269, was detected using an LDH kit. d-e: Detection of IL-1β and IL-18 content in H9c2 cells induced by high glucose that inhibited miR-145 and knocked down circ_0071269 were determined using an ELISA kit. F: Flow cytometry detection inhibited miR-145 and knocked down circ_0071269’s high glucose-induced apoptosis rate in H9c2 cells. g: The protein expression levels of NLRP3 and caspase-1 in H9c2 cells induced by high glucose, which inhibited miR-145, while knocking down circ_0071269, were detected by Western blotting. All experimental results were obtained by repeating three times. *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.

Figure 5.. GSDMA is the direct target of miR-145 in H9c2 cells. a: Online prediction software was used to identify the targeting relationship between GSDMA and miR-145. b: Luciferase activity experiments was performed to verify the targeting relationship between GSDMA and miR-145. c: Relative mRNA enrichment of GSDMA was captured using biotin-miR-145. d: GSDMA levels elevated in H9c2 cells treated with high glucose. E: GSDMA increased and decreased in H9c2 cells treated with miR-145 inhibitor and si-circ_0071269, respectively. All experimental results were obtained by repeating three times. *P< 0.05, **P< 0.01, ***P< 0.001.

Figure 6.. Overexpression of GSDMA reversed the effect of miR-145 on H9c2. a: The level of GSDMA in H9c2 cells overexpressing GSDMA was measured by qRT-PCR. b: Cell viability of miR-145 mimic transfected simultaneously with overexpressing GSDMA was detected by CCK-8. c: The LDH content of cells overexpressing GSDMA upon transfection of the miR-145 mimic was determined using the LDH kit. D-E: The levels of IL-1β and IL-18 in cells overexpressing GSDMA upon transfection of the miR-145 mimic were determined using an ELISA kit. F: Flow cytometry was used to detect the high apoptotic rate associated with GSDMA overexpression in cells transfected with the miR-145 mimic. g: Western blotting was used to detect the protein expression levels of NLRP3 and caspase-1 in cells transfected with miR-145 mimic and overexpressing GSDMA. All experimental results were obtained by repeating three times. *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.

Figure 7.. Knockdown of circ_0071269 attenuated cardiac dysfunction of DM mice. a-b: mRNA expression levels of IL-1β and IL-18 in cardiac tissue of the DM mice determined by qRT-PCR. c-d: The echocardiographic data including EF and FS were determined by Echocardiography. e: Images of cardiac tissue stained by HE staining assay. f: The expression of GSDMA obtained from IHC method. All experimental results were obtained by repeating three times. **P < 0.01; ##P < 0.01.