MIR22HG Aggravates Oxygen-Glucose Deprivation and Reoxygenation-Induced Cardiomyocyte Injury through the miR-9-3p/SH2B3 Axis

Figure 1. The treatment with OGD/R promotes cell apoptosis and ECM accumulation in HL-1 cells. (a) The viability of HL-1 cells treated with or without OGD/R was measured by CCK-8 assay (n = 3). (b) The apoptosis rate of HL-1 cells treated with or without OGD/R was determined by flow cytometry analysis (n = 3). (c and d) The protein levels of Bax, Bcl-2, collagen I, fibronectin, and collagen III protein levels in HL-1 cells were measured by western blotting analysis (n = 3). (e) The expression level of MIR22HG in OGD/R-treated HL-1 cells was measured by RT-qPCR analysis (n = 3). ∗∗p < 0.01,∗∗∗p < 0.001.

Figure 2. MIR22HG knockdown attenuated OGD/R-treated cardiomyocyte injury. (a) The knockdown efficiency of sh-MIR22HG was determined by RT-qPCR (n = 3). (b) The effect of sh-MIR22HG on the viability of OGD/R-treated HL-1 cells was assessed by CCK-8 assay (n = 3). (c) The effect of sh-MIR22HG on the apoptosis of OGD/R-treated HL-1 cells was evaluated by flow cytometry analysis (n = 3). (d and e) The effect of sh-MR22HG on the protein levels of Bax, Bcl-2, collagen I, fibronectin, and collagen III in OGD/R-treated HL-1 cells was measured by western blotting analysis (n = 3). ∗p < 0.05, ∗∗p < 0.01.

Figure 3. MIR22HG interacts with miR-9-3p to regulate SH2B3 expression. (a) The possible miRNAs that possibly bind to MIR22HG predicted in starBase. (b) The expression level of miR-9-3p and miR-298-5p in OGD/R-treated HL-1 cells was determined by RT-qPCR (n = 3). (c) The overexpression efficiency of miR-9-3p was evaluated by RT-qPCR (n = 3). (d and e) The binding sequence between miR-9-3p and MIR22HG is shown. Luciferase reporter assay was used to confirm the binding capacity between miR-9-3p and MIR22HG (n = 3). (f) The expression level of candidate target mRNAs in OGD/R-treated HL-1 cells was determined by RT-qPCR (n = 3). (g) The binding sequence between miR-9-3p and SH2B3 3′ UTR. (h) Luciferase reporter assay was used to validate the binding between miR-9-3p and SH2B3 3′ UTR (n = 3). (i and j) The mRNA and protein levels of SH2B3 in the context of miR-9-3p overexpression or MIR22HG knockdown were measured by RT-qPCR and western blotting analyses (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4. Overexpression of SH2B3 reverses the effect of MIR22HG knockdown in OGD/R-treated HL-1 cells. (a) The overexpression efficiency of pcDNA3.1/SH2B3 was tested by RT-qPCR (n = 3). (b) The protein level of SH2B3 in each group was measured by western blotting (n = 3). (c) The cell viability in different groups was detected by CCK-8 assay (n = 3). (d) Cell apoptosis in different groups was assessed by flow cytometry (n = 3). (e and f) The protein levels of Bax, Bcl-2, collagen I, fibronectin, and collagen III in OGD/R-treated HL-1 cells transfected with different vectors were measured by western blotting (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5. MIR22HG overexpression reverses the effect of SH2B3 knockdown in OGD/R-treated HL-1 cells. (a) The knockdown efficiency of sh-SH2B3 was examined by RT-qPCR (n = 3). (b) The overexpression efficiency of pcDNA3.1/MIR22HG was tested by RT-qPCR (n = 3). (c) The protein level of SH2B3 in each group was measured by western blotting (n = 3). (d) The cell viability in each group was assessed by CCK-8 assay (n = 3). (e) The cell apoptosis in each group was determined by flow cytometry analysis (n = 3). (f–i) The protein levels of Bax, Bcl-2, collagen I, fibronectin, and collagen III in OGD/R-treated HL-1 cells transfected with different vectors were measured by western blotting analysis (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6. MIR22HG knockdown alleviates cardiomyocyte apoptosis in MIRI mice. (a) RT-qPCR was used to determine the expression of MIR22HG in MIRI mouse models (n = 6). (b) HE staining of myocardium tissues (n = 6). (c) The analysis of infarct size of MIRI mice (n = 6). (d) TUNEL staining was used to assess the apoptosis of cardiomyocytes in each group (n = 6). (e) The protein levels of Bax, Bcl-2, collagen I, fibronectin, and collagen III in each group (n = 6). N = 6 mice for each group. ∗∗∗p < 0.001 vs. the control group or LV-sh-MIR22HG group; &p < 0.05, &&p < 0.01, &&&p < 0.001 vs. the LV-sh-NC group.