To investigate the effects of circSERPINE2 on hypoxia/reoxygenation (H/R) -induced cardiomyocyte damage and the mechanism.

Rat cardiomyocytes H9c2 were cultured in vitro and divided into control group (cells were cultured in a conventional incubator for 7 hours) H/R group (cells were cultured in serum-free low-glucose DMEM under hypoxia [no oxygen] for 3 hours, and then cultured in DMEM containing 10﹪ FBS under reoxygenation [19.95﹪ oxygen] for 4 hours) , H/R+pcDNA group (H9c2 cells transfected with pcDNA then treated by H/ R) , H/R+pcDNA-circSERPINE2 group (H9c2 cells transfected with pcDNA-circSERPINE2 then treated by H/R) , H/R+anti-miR-NC group (H9c2 cells transfected with anti-miR-NC then treated by H/R) , H/R+anti-miR-106b- 5p group (H9c2 cells transfected with anti-miR-106b-5p then treated by H/R) , H/R+pcDNA- circSERPINE2+miR- NC group (H9c2 cells co-transfected with pcDNA-circSERPINE2+miR-NC then treated by H/ R) and H/R+pcDNA- circSERPINE2+miR-106b-5p group (H9c2 cells transfected with pcDNA- circSERPINE2+miR-106b-5p then treated by H/R) . RT- qPCR was used to detect the expression of circSERPINE2 and miR-106b-5p. Flow cytometry was used to detect cell apoptosis. Western blotting was used to detect the protein expression of cleaved caspase-3 and cleaved caspase-9 in cells. The kits were used to detect the content of MDA and the activity of SOD and GSH-Px. The regulatory relationship between circSERPINE2 and miR- 106b- 5p was detected by dual luciferase reporter assay. The homogeneity of variance was compared between the two groups by t-test. The variance was uneven, and t' test was used.

Compared with the control group, circSERPINE2 expression in H9c2 cells of H/R group was decreased (1.00±0.06 vs 0.43±0.04) , SOD[ (77.84±6.49) U/ mL vs (23.19±2.21) U/mL] and GSH-Px activity [ (59.21±5.31) U/mL vs (17.62±1.53) U/ mL] decreased, while miR-106b- 5p expression (1.00±0.07 vs 2.66±0.23) , apoptosis rate [ (6.53±0.52) ﹪ vs (24.21±2.18) ﹪], cleaved caspase-3 (0.24±0.02 vs 0.72± 0.05) and cleaved caspase-9 protein expression (0.13±0.02 vs 0.65±0.04) were increased. The content of MDA (5.23±0.47) nmol/L was higher than (45.56±4.33) nmol/L, and the difference was statistically significant (P < 0.05) . Compared with H/R + pcDNA group, the apoptosis rate of H9c2 cells [ (26.34±2.23) ﹪ vs (10.08±0.81) ﹪], cleaved caspase-3 (0.75±0.05 vs 0.32±0.04) , cleaved caspase-9 protein expression (0.67±0.05 vs 0.25±0.03) and MDA content [ (49.18±4.89) nmol/L vs (6.79±0.59) nmol/ L] in H/R + pcDNA-circSERPINE2 group were lower, However, SOD [ (21.42 ± 2.03) U/mL vs (63.57± 6.44) U/ mL] and GSH-Px activity [ (16.06 ± 1.42) U/ mL vs (47.94±5.52) U/mL] were increased, and the difference was statistically significant (P < 0.05) . Compared with H/R + anti-miR-NC group, the apoptosis rate of H9c2 cells[ (26.23± 2.15) ﹪ vs (13.87±1.26) ﹪], cleaved caspase- 3 (0.78±0.06 vs 0.38±0.03) , cleaved caspase-9 protein expression (0.68±0.05 vs 0.31±0.03) and MDA content [ (48.38±4.21) nmol/L vs (11.39±1.42) nmol/ L] in H/R + anti-miR-NC group were lower. However, SOD [ (22.19±2.21) U/ mL vs (54.33 ± 5.25) U/mL] and GSH- Px activity [ (15.84±1.68) U/mL vs (40.42±3.51) U/mL]were increased, and the difference was statistically significant (P < 0.05) . circSERPINE2 targeted and negatively regulated the expression of miR-106b-5p. Compared with the H/R+pcDNA-circSERPINE2+miR- NC group, the apoptosis rate [ (9.86±1.04) ﹪vs (18.47±1.24) ﹪], the protein expression of cleaved caspase-3 (0.31±0.03 vs 0.66±0.04) and cleaved caspase-9 (0.23±0.02 vs 0.56±0.06) , and the content of MDA [ (6.56±0.61) nmol/ L vs (36.97±3.14) nmol/L] of H9c2 cells in the H/ R+pcDNA-circSERPINE2+miR- 106b- 5p group were increased, but the SOD[ (64.14±6.32) U/ mL vs (32.26±3.58) U/mL] and GSH- Px activity [ (48.40±5.06) U/mL vs (25.58±3.11) U/mL] were decreased (P < 0.05) .

Up-regulation of circSERPINE2 can inhibit H/R-induced cardiomyocyte apoptosis and oxidative stress in rats, and its mechanism may be related to the targeted down-regulation of miR- 106b-5p expression.