Abstract:

Objective

To explore the targeted regulatory mechanism of circ-SESN2 silencing on miR-23a-5p and ULK1 pathway in a neuronal oxidative stress model.

Methods

（1） B35 cells were treated with different concentrations （0，100，200，400，600，800 μmol/L）of H₂O₂ for 24 h.The cell status was detected using CCK-8 method and flow cytometry to determine the optimal conditions for H₂O₂ induced oxidative stress damage in B35 cells. （2） B35 cells were transfected with wild-type （WT） and mutant（MT）plasmids containing circ-SESN2 and divided into four groups：circ-SESN2-3’UTR WT+miRNC group，circ-SESN2-3’UTR WT+miR-23a-5p group，circ-SESN2-3’UTR MT+miR-NC group，and circ-SESN2-3’UTR MT+miR-23a-5p group. Dual luciferase activity was used to detect the expression of circ-SESN2 gene and verify the targeted binding relationship between circ-SESN2 and miRNA-23a-5p. （3）The transfection effects of siRNA at different concentrations （0.1，0.01 μmol/L）and transfection durations（24，48 h），as well as the silencing effects of 3 different targets（siRNA-ccirc-SESN2-1156，1227，1692）on circ-SESN2 were compared， in order to determine the optimal transfection conditions and most effective circ-SESN2 siRNA targets for siRNA. （4） B35 cells were divided into blank group， model group， H/R model+siRNA negative control group， and H/R model+circ-SESN2-siRNA group. Except for the blank group， all three groups were treated with 400 μmol/L f H₂O₂ for 24 h. After intervention， the cell status was detected using CCK-8 and flow cytometry， and oxidative stress indicators such as manganese superoxide dismutase （Mn-SOD）， total superoxide dismutase （T-SOD）， NO， and 3-nitrotyrosine （3-NT）were detected using a biochemical kit. miRNA-23a-5p and ULK1 gene expression were detected using qRT-PCR， and ULK1 protein content was detected using Western blot.

Results

（1） The CCK-8 detection and SOD activity detection results showed that the survival rate and SOD activity of B35 cells gradually decreased with the increase of H₂O₂ concentration. Among them， the survival rate of the 400 μmol/L group decreased significantly compared to the 0， 100， and 200 μmol/L groups， and the SOD activity of the 400 μmol/L group decreased significantly compared to the 0 and 100 μmol/L groups， with statistical significance （P＜0.05）. Therefore， 400 μmol/L H₂O₂ was used as the oxidative stress damage condition for subsequent experiments.（2）The luciferase activity of the circ-SESN2-3’UTR WT+miR-23a-5p group was significantly lower than that of the circ-SESN2-3’UTR WT+miR-NC group，circ-SESN2-3’UTR MT+miR-NC group， and circ-SESN2-3’UTR MT+miR-23a-5p group， with statistical significance （P＜0.05）. （3） The transfection efficiency of 0.1 μmol/L siRNA transfection for 48 h was significantly better than other transfection conditions （0.01 μmol/L siRNA transfection for 24 h， 0.01 μmol/L siRNA transfection for 48 h， 0.1 μmol/L siRNA transfection for 24 h）. In target screening， the gene levels of the siRNA-ccirc-SESN2-1156 group，siRNA-ccirc-SESN2-1227 group，and siRNA-ccirc-SESN2-1692 group were significantly lower than those of the blank group and siRNA-NC group， with siRNA-ccirc-SESN2-1692 showing the best silencing effect. （4） The cell survival rate of the H/R model+circ-SESN2-siRNA group was lower than that of the blank group， but higher than that of the H/R model+siRNA negative control group and the model group； The apoptosis rate of cells in the H/R model+circ-SESN2-siRNA group was higher than that in the blank group， but lower than that in the H/R model+siRNA negative control group and model group， and the differences were statistically significant （P＜0.05）. The Mn-SOD and T-SOD contents in the H/R model+circ-SESN2-siRNA group were lower than those in the blank group， but higher than those in the model group and the H/R model+siRNA negative control group； The levels of NO and 3-NT increased compared to the blank group， and decreased compared to the model group and the H/R model+siRNA negative control group， with statistically significant differences （P＜0.05）. Fluorescence quantitative detection showed that miRNA-23a-5p in the H/R model+circ-SESN2-siRNA group was higher than that in the model group and H/R model+siRNA negative control group，while ULK1 was lower than that in the model group and H/R model+siRNA negative control group， with statistical significance （P＜0.05）.

Conclusion

Silencing circRNA-SESN2 may reduce the expression of ULK1 through the SESN2/miRNA-23a-5p/ULK1 pathway and play a protective role in B35 neurons under oxidative stress.

Key words: circ-SESN2, miRNA-23a-5p, Cerebral ischemia-reperfusion injury, Oxidative stress injury, ULK1, Gene expression regulation