Circular RNA COL1A2 Mediates High Glucose-Induced Oxidative Stress and Pyroptosis by Regulating MiR-424-5p/SGK1 in Diabetic Nephropathy

Abstract

Diabetic nephropathy (DN) represents a major diabetes-related complication, which could undermine renal function. CircCOL1A2 has been previously reported to show abnormal expression during DN. However, its functional role in the progression of DN, as well as the potential molecular mechanisms, remains unclear. The present work examined the expression of circCOL1A2 in the plasma of DN patients, and employed high glucose (HG)-challenged HK-2 cells as the in vitro cell model of hyperglycemia (HG)-induced DN. CircCOL1A2 was silenced using siRNA in HK-2 cells to clarify the functional engagement of circCOL1A2 in HG-induced DN. We examined the roles of circCOL1A2 in regulating oxidative stress by measuring reactive oxygen species (ROS), lipid peroxidation, and superoxide dismutase (SOD) levels. Besides, the effects of circCOL1A2 silencing on pyroptosis were investigated by RT-qPCR, western blot (WB), and ELISA assays. StarBase (version 2.0) was used to identify the downstream effector of circCOL1A2, and their interactions were further verified through dual-luciferase reporter analysis, RNA pull-down assays, and RNA immunoprecipitation (RIP) assay. CircCOL1A2 was highly expressed in DN patients and HG-induced HK-2 cells. Knocking down circCOL1A2 alleviated oxidative stress and pyroptosis upon HG treatment. In addition, we demonstrated that circCOL1A2 knockdown could promote miR-424-5p expression while inhibiting Serum/Glucocorticoid Regulated Kinase 1 (SGK1) level. Furthermore, miR-424-5p inhibitor or SGK1 overexpression impaired the effects of circCOL1A2 knockdown on HG-induced oxidative stress and pyroptosis. Hence, our results demonstrated that the circCOL1A2 mediates HG-exposed pyroptosis and oxidative stress through modulating miR-424-5p/SGK1 axis in diabetic nephropathy, indicating that silencing circCOL1A2 is a potential intervention strategy for DN management.

Keywords: CircCOL1A2; DN; MiR-424-5p; SGK1.

Fig. 1

CircCOL1A2 is up-regulated in diabetic nephropathy and HG-induced HK-2 cells. A CircCOL1A2 expression was measured through RT-qPCR in healthy controls and DN plasma samples. Three asterisks (***) P < 0.001 vs. NC group. B CircCOL1A2 expression was measured through RT-qPCR in control and HG-induced HK-2 cells. Three asterisks (***) P < 0.001 vs. control

Fig. 2

CircCOL1A2 silencing alleviates the oxidative stress in HG-treated HK-2 cells. A CircCOL1A2 expression was assessed by RT-qPCR upon the transfection of control siRNA and siRNA targeting circCOL1A2. Three asterisks (***) P < 0.001, one asterisk (*) P < 0.05 vs. the si-NC group. B Level of ROS was measured in control, HG-treated HK-2 cells, and HG-treated HK-2 cells with circCOL1A2 silencing. Three asterisks (***) P < 0.001 vs. si-NC group; two number signs (^##) P < 0.01 vs. HG + si-NC group. C Level of MDA in control, HG-treated HK-2 cells, and HG-treated HK-2 cells with circCOL1A2 silencing. Three asterisks (***) P < 0.001 vs. si-NC group; two number sign (^##) P < 0.01 vs. HG + si-NC group. D Level of SOD activity in control, HG-treated HK-2 cells, and HG-treated HK-2 cells with circCOL1A2 silencing. Two asterisks (**) P < 0.01 vs. si-NC group; number sign (^#) P < 0.05 vs. HG + si-NC group

Fig. 3

CircCOL1A2 silencing suppresses HG-induced HK-2 cell pyroptosis. A RT-qPCR was carried out for measuring Caspase-1, NLRP3, IL-1β and GSDMD-N mRNA levels in control, HG-treated HK-2 cells, and HG-treated HK-2 cells with circCOL1A2 silencing. Two number signs (^##) P < 0.01, one number sign (^#) P < 0.05 vs. HG + si-NC group; three asterisks (***) P < 0.001, two asterisks (**) P < 0.01 vs. control. B Caspase-1, NLRP3, GSDMD-N and IL-1β protein levels were assessed through WB assay in control, HG-treated HK-2 cells, and HG-treated HK-2 cells with circCOL1A2 silencing. Three number signs (^###) P < 0.001, two number signs (^##) P < 0.01, one number sign (^#) P < 0.05 vs. HG + si-NC group; three asterisks (***) P < 0.001 vs. control. C IL-18 and IL-1β expression was assessed through ELISA in control, HG-treated HK-2 cells, and HG-treated HK-2 cells with circCOL1A2 silencing. Two number signs (^##) P < 0.01, one number sign (^#) P < 0.05 vs. HG + si-NC group; three asterisks (***) P < 0.001 vs. control

Fig. 4

CircCOL1A2 silencing promotes miR-424-5p expression. A StarBase was adopted for predicting binding sites between miR-424-5p for circCOL1A2. B Dual-luciferase reporter assay was conducted for detecting luciferase activities of circCOL1A2-WT or circCOL1A2-MUT reporter in the presence of miR-424-5p mimic or miR-NC co-transfection. Two asterisks (**) P < 0.01 vs. miR-NC group. C Bio-NC or Bio-circCOL1A2 probe was utilized to precipitate miR-424-5p in RNA pull-down analysis, followed by RT-qPCR quantification. Two asterisks (**) P < 0.01 vs. Bio-NC group. D Association of circCOL1A2 and miR-424-5p in Ago-2 protein complex was verified through RIP assay. Two asterisks (**) P < 0.01 vs. anti-IgG group. E miR-424-5p expression was measured through RT-qPCR in the plasma samples of control and DN patients. Three asterisks (***) P < 0.001 vs. NC group. F Spearman correlation coefficient analysis of circCOL1A2 and miR424-5p in DN patient samples. G miR-424-5p expression was measured through RT-qPCR in control and HG-treated HK-2 cells. Three asterisks (***) P < 0.001 vs. control group. H miR-424-5p expression was determined through RT-qPCR in HK-2 cells transfected with control or cicCOL1A2 siRNA. Three asterisks (***) P < 0.001 vs. si-NC group

Fig. 5

MiR-424-5p targets SGK1 in HK-2 cells. A StarBase was utilized for predicting bindings sites between SKG1 mRNA 3’ UTR and miR-424-5p. B Dual-luciferase reporter assay was carried out using SGK1-WT/SGK1-MUT reporter in the presence of miR-424-5p mimic or miR-NC co-transfection. Two asterisks (**) P < 0.01 vs. miR-NC group. C SGK1 protein level was evaluated through WB assay in HK-2 cells transfected with miR-NC or miR-424-5p mimic. One asterisk (*) P < 0.05 vs. miR-NC group. D SGK1 protein level was evaluated through WB assay in HK-2 cells transfected with circCOL1A2 siRNA with or without miR-424-5p inhibitor. Number sign (^#) P < 0.05 vs. si-circCOL1A2 group; three asterisks (***) P < 0.001 vs. miR-NC group. E SGK1 level was evaluated through RT-qPCR in DN patient and healthy control plasma samples. Three asterisks (***) P < 0.001 vs. NC. F Spearman correlation coefficient analysis of miR-424-5p and SGK1 mRNA level. G Spearman correlation coefficient analysis of circCOL1A2 level and SGK1 mRNA level. H SGK1 protein level was assessed through WB assay in control and HG-treated cells. Two asterisks (**) P < 0.01 vs. control

Fig. 6

CircCOL1A2 regulates HG-triggered HK-2 cells injury through miR-424-5p/SGK1 axis. The following experimental groups were established in HK-2 cells: control, HG treatment, HG + circCOL1A2 silencing, HG + circCOL1A2 silencing plus miR-424-5p inhibitor, and HG + circCOL1A2 silencing plus SGK1 expression vector. A SGK1 protein level was assessed through WB assay. Circumflex accent (^) P < 0.05 vs. HG + circCOL1A2 group; two number signs (^##) P < 0.01 vs. HG + si-NC group; three asterisks (***) P < 0.001 vs. control. B–D Level of ROS, MDA and SOD activities were measured using corresponding commercial kits. Three asterisks (***) P < 0.001 vs. the control group; three number signs (^###) P < 0.001, two number signs (^##) P < 0.01 vs. the HG + si-NC group; three circumflex accent (^^^) P < 0.001, one circumflex accent (^) P < 0.05 vs. the HG + circCOL1A2 group. E NLRP3, Caspase-1, IL-1β, and GSDMD-N expression levels were assessed by RT-qPCR. Three asterisks (***) P < 0.001 vs. the control group; three number signs (^###) P < 0.001, two number signs (^##) P < 0.01 vs. the HG + si-NC group; three circumflex accent (^^^) P < 0.001, two circumflex accent (^^) P < 0.01, one circumflex accent (^) P < 0.05 vs. the HG + circCOL1A2 group. F The protein expression levels of NLRP3, Caspase-1, IL-1β, and GSDMD-N were evaluated by Western blot. Three asterisks (***) P < 0.001, two asterisks (**) P < 0.01 vs. the control group; three number signs (^###) P < 0.001, two number signs (^##) P < 0.01 vs. the HG + si-NC group; three circumflex accent (^^^) P < 0.001, two circumflex accent (^^) P < 0.01, one circumflex accent (^) P < 0.05 vs. the HG + circCOL1A2 group. G IL-18 and IL-1β expression was assessed through ELISA. Three asterisks (***) P < 0.001 vs. the control group; three number signs (^###) P < 0.001 vs. the HG + si-NC group; two circumflex accent (^^) P < 0.01, one circumflex accent (^) P < 0.05 vs. the HG + circCOL1A2 group