Interference of Hsa_circ_0003928 Alleviates High Glucose-Induced Cell Apoptosis and Inflammation in HK-2 Cells via miR-151-3p/Anxa2

Abstract

Background: Diabetic nephropathy (DN) is a severe end-stage kidney disease developed from diabetes mellitus. The involvement of circular RNA (circRNAs) in the regulation of DN pathogenesis has been implied, but the underlying mechanism of DN is still lacking. This study aimed to investigate the effect of hsa_circ_0003928 on the inflammation and apoptosis of high glucose (HG)-induced renal tubular cells.

Methods: The expression of hsa_circ_0003928, miR-151-3p and Anxa2 in blood samples from DN patients and healthy controls was detected by RT-qPCR. Human renal epithelial cells HK-2 were incubated with D-glucose (30 mmol/l) to establish DN model in vitro. RT-qPCR analysis confirmed the transfection effects and detected the expressions of TNF-α, IL-6 and IL-1β. Western blotting analysis determined the protein expression of Anxa2, Bcl-2, Bax, cleaved caspase-3 and caspase-3. The production of ROS was detected by DCF-DA method and production of inflammatory cytokines was verified by ELISA assay. CCK-8 assay and TUNEL assay were performed to determine cell viability and apoptosis, respectively. Dual-luciferase reporter assay was performed to confirm the relationship between miR-151-3p and hsa_circ_0003928 or Anxa2.

Results: Hsa_circ_0003928 and Anxa2 mRNA levels were increased, whereas miR-151-3p was decreased in both HG-induced HK-2 cells and patients with DN. Hsa_circ_0003928 knockdown could decrease cell viability loss and apoptosis, increase Bcl-2 expression, and decrease Bax and cleaved caspase-3 expression. Besides, hsa_circ_0003928 knockdown suppressed HG-induced overproduction of ROS, TNF-α, IL-6 and IL-1β. However, the effects made by miR-151-3p inhibition were opposite to those made by hsa_circ_0003928 knockdown. Furthermore, the binding sites between miR-151-3p and hsa_circ_0003928 or Anxa2 were predicted and verified. Protein expression of Anxa2 was suppressed by hsa_circ_0003928 knockdown, which was rescued by miR-151-3p inhibition.

Conclusion: These results demonstrated that hsa_circ_0003928 could act as a sponge of miR-151-3p and regulate HG-induced inflammation and apoptosis partly through regulating Anxa2.

Keywords: Anxa2; diabetic nephropathy; hsa_circ_0003928; miR-151-3p.

Figure 1

Hsa_circ_0003928 was upregulated in high D-glucose-induced HK-2 cells and serum of patients with DN. (A) HK-2 cells were subjected to different concentrations of D-glucose (5, 10, 15, 20, 25 and 30 mmol/l) for 48 h. Besides, mannitol (MA; 30 mmol/l) was used as negative control. The expression level of hsa_circ_0003928 was measured by RT-qPCR. **, ***p<0.01, 0.001 vs 5 mmol/l D-glucose. (B) HK-2 cells were subjected to 30 mmol/l D-glucose for 6, 12, 24 and 48 h, respectively. The expression level of hsa_circ_0003928 was measured by RT-qPCR. **, ***p<0.01, 0.001 vs 0 h. (C) The expression level of hsa_circ_0003928 in serum of healthy control and patients with DN was measured by RT-qPCR. ***p<0.001.

Figure 2

Hsa_circ_0003928 knockdown suppressed cell viability loss and apoptosis in HG-induced HK-2 cells. (A) HK-2 cells were transfected with siRNA-circ_0003928-1/2 and the siRNA negative control (siRNA-NC) using Lipofectamine 2000, and the transfection efficacy was determined using RT-qPCR. ***p<0.001 vs siRNA-NC. (B) HK-2 cells without transfection were treated with normal D-glucose (NG; 5 mmol/l) or high D-glucose (HG; 30 mmol/l) for 48 h. Besides, transfected HK-2 cells were inoculated into 96-well plates and treated with HG for 48 h. Then, CCK-8 assay was performed to determine cell viability of different groups. (C) TUNEL assay was conducted to determine HK-2 cell apoptosis. (D and E) The protein expression of Bcl-2, Bax, cleaved caspase-3 and GAPDH were detected using Western blotting, and the band intensities were quantified. GAPDH acted as the loading control, and the band intensity was normalized to GAPDH. ***p<0.001 vs normal D-glucose (NG); ^{##, ###}p<0.01, 0.001 vs HG+siRNA-NC.

Figure 3

Hsa_circ_0003928 knockdown suppressed production of ROS and inflammatory cytokines in HG-induced HK-2 cells. (A) HK-2 cells without transfection were treated with NG or HG for 48 h. Besides, HK-2 cells transfected with siRNA-circ_0003928 or siRNA-NC were inoculated into 96-well plates and treated with HG for 48 h. Production of ROS was detected by DCF-DA method. (B–D) The production of TNF-α, IL-6 and IL-1β was determined using corresponding ELISA kits. (E–G) The mRNA levels of TNF-α, IL-6 and IL-1β were detected using RT-qPCR. ***p<0.001 vs NG; ^{#, ##, ###}p<0.05, 0.01, 0.001 vs HG+siRNA-NC.

Figure 4

Hsa_circ_0003928 binds to miR-151-3p. (A) According to the bioinformatics assay (https://circinteractome.nia.nih.gov), a binding site between hsa_circ_0003928 and miR-151-3p was predicted. (B) HEK293 cells were co-transfected with wild-type (WT) hsa_circ_0003928 or mature (MUT) hsa_circ_0003928 and miR-151-3p mimic or miR-NC (mimic control) using Lipofectamine 2000. 48 h after transfection, the luciferase activities were tested by the Dual-Luciferase Reporter Assay system. **p<0.01 vs miR-NC. (C) HK-2 cells were transfected with siRNA-NC or siRNA-circ_0003928, and the expression level of miR-151-3p was detected by RT-qPCR. ***p<0.001 vs siRNA-NC. (D) HK-2 cells were treated with NG, HG and MA, respectively, and the expression level of miR-151-3p was detected by RT-qPCR. ***p<0.001 vs NG. (E) The expression level of miR-151-3p in serum from healthy control and patients with DN was measured by RT-qPCR. ***p<0.001.

Figure 5

Anxa2 is a direct target of miR-151-3p. (A) StarBase software (http://starbase.sysu.edu.cn) predicted a binding site between miR-151-3p and Annexin A2 (Anxa2). (B) HEK293 cells were co-transfected with wild-type (WT) Anxa2 or mature (MUT) Anxa2 and miR-151-3p mimic or miR-NC (mimic control) using Lipofectamine 2000. 48 h after transfection, the luciferase activities were tested by the Dual-Luciferase Reporter Assay system. **p<0.01 vs miR-NC. (C–E) HK-2 cells were transfected with miR-151-3p mimic and miR-NC, then the protein expression and mRNA level of Anxa2 were detected using Western blotting and RT-qPCR, respectively. ***p<0.001 vs miR-NC. (F–H) HK-2 cells were treated with NG, HG and MA, respectively, and the protein expression and mRNA level of Anxa2 were detected using Western blotting and RT-qPCR, respectively. GAPDH acted as the loading control, and the band intensity of Anxa2 was normalized to GAPDH. ***p<0.001 vs NG. (I) The mRNA level of Anxa2 in serum from healthy control and patients with DN was measured by RT-qPCR. ***p<0.001.

Figure 6

Hsa_circ_0003928/miR-151-3p axis regulates HG-induced viability loss and apoptosis in HK-2 cells. (A and B) HK-2 cells were transfected with miR-151-3p inhibitor or siRNA-circ_0003928, or co-transfected with miR-151-3p inhibitor and siRNA-circ_0003928, then the protein expression of Anxa2 was detected using Western blotting. *, **p<0.05, 0.01 vs control; ##p<0.01 vs miR-151-3p inhibitor; ΔΔp<0.01 vs siRNA-circ_0003928. (C) HK-2 cells were treated with NG or HG for 48 h. Besides, after transfection with miR-151-3p inhibitor or siRNA-circ_0003928 or the combination of iR-151-3p inhibitor and siRNA-circ_0003928, HK-2 cells were treated with HG for 48 h, then cell viability of different groups was assayed using CCK-8. (D) TUNEL assay was conducted to determine HK-2 cell apoptosis. (E) The protein expression of Bcl-2, Bax, cleaved caspase-3, and GAPDH were detected using Western blotting, and the band intensities were quantified. GAPDH acted as the loading control, and the band intensity was normalized to GAPDH. ***p<0.001 vs NG; ^{#, ##, ###}p<0.05, 0.01, 0.001 vs HG; ^{Δ, ΔΔ, ΔΔΔ}p<0.05, 0.01, 0.001 vs HG+miR-151-3p inhibitor; ^$,$$,$$$ p<0.05, 0.01, 0.001 vs HG+siRNA-circ_0003928.

Figure 7

Hsa_circ_0003928/miR-151-3p axis regulates HG-induced inflammatory response in HK-2 cells. (A) HK-2 cells were treated with NG or HG for 48 h. Besides, after transfection with miR-151-3p inhibitor or siRNA-circ_0003928 or the combination of iR-151-3p inhibitor and siRNA-circ_0003928, HK-2 cells were treated with HG for 48 h, then production of ROS was determined using DCF-DA method. (B–D) The mRNA levels of TNF-α, IL-6 and IL-1β were determined by RT-qPCR. ***p<0.001 vs NG; ^{#, ##}p<0.05, 0.01 vs HG; ^{Δ, ΔΔ}p<0.05, 0.01 vs HG+miR-151-3p inhibitor; ^{$, $$} p<0.05, 0.01 vs HG+siRNA-circ_0003928.

Figure 8

An illustration showing that high-glucose induced overexpression of has_circ_0003928 promoted cell viability loss, apoptosis and inflammation in HK-2 cells, possibly via regulating miR-151-3p/Anxa axis.