Hsa_circRNA_0008028 Deficiency Ameliorates High Glucose-Induced Proliferation, Calcification, and Autophagy of Vascular Smooth Muscle Cells via miR-182-5p/TRIB3 Axis

Abstract

Background: It is well-known that dysfunctions of vascular smooth muscle cells (VSMCs) act an essential part in vascular complications of diabetes. Studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) play a crucial role in regulating cell functions. However, their influence on the proliferation, calcification, and autophagy of VSMCs remains to be further explored. Therefore, this study elucidates the role and mechanism of hsa_circRNA_0008028 in high glucose- (HG-, 30 mM) treated VSMCs in vitro.

Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was chosen to detect the levels of hsa_circRNA_0008028, miR-182-5p, and tribble 3 (TRIB3). Then, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to predict and verify the binding relationship between miR-182-5p and hsa_circRNA_0008028 or TRIB3. Cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, corresponding commercial kits, and western blotting were used to measure indexes reflecting cell viability, proliferation, calcification, and autophagy of VSMCs, respectively.

Results: In HG-induced VSMCs, hsa_circRNA_0008028 and TRIB3 were highly expressed, whereas miR-182-5p decreased. Meanwhile, cell proliferation, calcification, and autophagy could be repressed by silencing of hsa_circRNA_0008028. However, these effects can be eliminated by miR-182-5p inhibition. Furthermore, it was demonstrated that hsa_circRNA_0008028 could promote the expression of TRIB3, a target of miR-182-5p, by directly sponging miR-182-5p. The expression of TRIB3 was suppressed by hsa_circRNA_0008028 knockout, which was rescued by miR-182-5p inhibition.

Conclusion: This study reveals that hsa_circRNA_0008028 can act as a sponge of miR-182-5p and promote HG-induced proliferation, calcification, and autophagy of VSMCs partly by regulating TRIB3.

Figure 1

hsa_circRNA_0008028 is highly expressed in HG-treated VSMCs. (a) hsa_circRNA_000802 expression level detected by qRT-PCR. VSMCs were exposed to different concentrations of D-glucose (5.5, 10, 20, and 30 mmol/L) for 48 hours. MA was used as a negative control. ^∗∗p < 0.05 vs. the 5.5 mmol/L group. (b) hsa_circRNA_000802 expression level in VSMCs under D-glucose (30 mmol/L) for 0, 12, 24, and 48 hours, respectively. ^∗∗p < 0.05 vs. the 0 hour group. (c) Expression levels of hsa_circRNA_000802 and linear GAPDH were examined by qRT-PCR in VSMCs treated with RNase R⁺ or RNase R^–. ^∗∗p < 0.05.

Figure 2

Silencing hsa_circRNA_0008028 inhibits HG-induced proliferation, calcification, and autophagy of VSMCs. VSMCs were treated with normal D-glucose (NG, 5.5 mmol/L) or high D-glucose (HG, 30 mmol/L) and then transfected with siRNA negative control (HG + si-NC) or siRNA-hsa_circRNA_0008028 (HG+si-hsa_circRNA_0008028) for 48 hours. (a) The relative expression level of hsa_circRNA_000802 determined by qRT-PCR. (b) Cell viability measured by CCK-8. (c) Cell proliferation analysis measured by EdU staining. (d) Percentage of EdU positive cells. (e) ALP activity. (f) The protein expression of cyclin D1, Runx2, OPN, and α-SMA. (g) The protein expression of LC3B-II/LC3B-I and SQSTM1/p62. ^∗∗p < 0.05 vs. the NG group; ^$$p < 0.05 vs. the HG+si-NC group.

Figure 3

hsa_circRNA_0008028 binds to miR-182-5p. (a) Binding sites between hsa_circRNA_0008028 and miR-182-5p predicated using starBase. The mutated (Mut) version of hsa_circRNA_0008028 was shown. (b) Dual-luciferase reporter assay was used to verify the binding relationship between hsa_circRNA_0008028 and miR-182-5p. ^∗∗p < 0.05 vs. the WT-hsa_circRNA_0008028 group. (c) RIP assay performed the endogenous correlation of hsa_circRNA_0008028 and miR-182-5p. ^{∗∗or $$}p < 0.05 vs. the anti-IgG group. (d) qRT-PCR detected the miR-182-5p expression regulated by hsa_circRNA_0008028 silencing or over-expression. ^∗∗p < 0.05 vs. the NG group; ^$$p < 0.05 vs. the HG+si-NC group; ^##p < 0.05 vs. the HG+Vector group.

Figure 4

miR-182-5p inhibition reverses the effect of hsa_circRNA_0008028 silence on HG-stimulated VSMCs. (a) qRT-PCR analysis of miR-182-5p expression transfected with miR-182-5p inhibitor. ^∗∗p < 0.05 vs. the miR-NC group. (b–h) VSMCs were cotransfected with si-circ-hsa_circRNA_0008028 and/or miR-182-5p inhibitor and then subjected to HG stimulation. (b) miR-182-5p expression detected by qRT-PCR. (c) Cell viability analysis using CCK-8. (d) Cell proliferation analysis with EdU staining. (e) Percentage of EdU positive cells. (f) ALP activity. (g) Western blotting detection of cell cycle and osteogenic differentiation-related protein expression. (h) Western blotting examination of autophagy-related protein expression. ^∗∗p < 0.05 vs. the NG group; ^$$p < 0.05 vs. the HG+si-NC group; ^##p < 0.05 vs. the HG +si-hsa_circ_00080+miR-NC group.

Figure 5

TRIB3 is a downstream target of miR-182-5p. (a) Binding sites between miR-182-5p and tribble 3 (TRIB3) 3′UTR predicated using the TargetScan software. The interaction between miR-182-5p and TRIB3 was measured by (b) dual luciferase reporter assay and (c) RIP assay. ^{∗∗or $$}p < 0.05. (d) qRT-PCR and (e) western blotting detection of TRIB3 expression in VSMCs transfected with miR-NC, miR-182-5p, or miR-182-5p inhibitor and then exposed to HG. ^∗∗p < 0.05 vs. the NG group; ^$$p < 0.05 vs. the HG+miR-NC group.

Figure 6

TRIB3 knockdown blocks the side effects of HG on VSMCs. VSMCs were transfected with TRIB3 siRNA upon HG condition. (a) TRIB3 protein expression detected by western blotting. (b) Cell viability detection using CCK-8. (c) Cell proliferation observation by EdU staining. (d) Percentage of EdU positive cells. (e) ALP activity. (f) Western blotting detection of cell cycle and osteogenic differentiation-related protein expression. (g) Western blotting examination of autophagy-related protein expression. ^∗∗p < 0.05 vs. the NG group; ^$$p < 0.05 vs. the HG+si-NC group.

Figure 7

hsa_circRNA_0008028/miR-182-5p regulates HG-induced VSMC dysfunctions through TRIB3. VSMCs were transfected with si-circ-hsa_circRNA_0008028 and/or miR-182-5p inhibitor and then subjected to HG stimulation. The expression level of TRIB3 was detected by (a) qRT-PCR and (b) western blotting. (c) Cell viability reflection using CCK-8. (d) Cell proliferation examination using EdU staining. (e) Percentage of EdU positive cells. (f) ALP activity. The protein expression of (g) cyclin D1, Runx2, OPN and α-SMA, and (h) LC3B-II/LC3B-I and SQSTM1/p62 was detected by western blotting. ^∗∗p < 0.05 vs. NG group; ^$$p < 0.05 vs. the HG group; ^##p < 0.05 vs. the HG+miR-182-5p inhibitor group; ^&&p < 0.05 vs. the HG+si-hsa_circ_00080 group.