CircRNA LRP6 promotes high-glucose induced proliferation and migration of vascular smooth muscle cells through regulating miR-545-3p/HMGA1 signaling axis

Abstract

Objective: The dysfunction of vascular smooth muscle cells (VSMCs) has been revealed to be closely linked with the pathogenesis of cardiovascular diseases in diabetes. Recently, circular RNAs (circRNAs) were found to regulate the behaviors of VSMCs. Here, we attempted to study the role of circLRP6 in VSMCs under diabetes condition.

Methods: Human VSMCs were cultured under the condition of normal glucose (NG) or high glucose (HG). VSMC viability and proliferation were estimated by CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. VSMC migration and invasion were assessed via wound-healing and transwell experiments. Protein expression of HMGA1 was measured in VSMCs using western blot and immunofluorescence analysis. Relative expressions of circLRP6, miR-545-3p, and HMGA1 mRNA were estimated in VSMCs using qRT-PCR. The co-localization of circLRP6 and miR-545-3p was verified by fluorescence in situ hybridization (FISH) analysis. Binding sequence of miR-545-3p in circLRP6 or HMGA1 was predicted using StarBase tool, and verified by RNA immunoprecipitation and dual-luciferase reporter experiments.

Results: HG exposure promoted VSMC proliferation, migration and invasion, upregulated the circLRP6 expression, and downregulated HMGA1 expression. Knockdown of circLRP6 or overexpression of miR-545-3p abrogated the HG-caused VSMC proliferation, migration and invasion. CircLRP6 severed as a miR-545-3p sponge, and HMGA1 was targeted by miR-545-3p. MiR-545-3p inhibitor blocked the suppressive effects of si-circLRP6 on VSMC in the presence of HG.

Conclusion: These findings suggest that circRNA LRP6 promotes HG-induced VSMC proliferation and migration through regulating miR-545-3p/HMGA1 signaling axis.

Keywords: HMGA1; Vascular smooth muscle cells; circLRP6; high glucose; miR-545-3p.

Figure 1

HG exposure increased circLRP6 expression while decreased HMGA1 expression in VSMCs. (A) CCK-8 assay and (B) EdU staining were employed to estimate proliferation of VSMCs under NG and HG condition. (C and D) Effects of HG exposure on the migratory and invasive abilities of VSMCs were assessed by wound-healing and transwell assays. (E) CircLRP6 level in NG- and HG-treated VSMCs was measured via qRT-PCR. (F) The subcellular localization of circLRP6 in VSMCs was determined by FISH assay.

Figure 2

Knockdown of circLRP6 repressed HG induced VSMCs proliferation, migration, and invasion. (A) Knockdown efficiency of si-circLRP6 in VSMCs was examined via qRT-PCR after 24 h of transfection. After transfection with si-NC (negative control) or si-circLRP6 for 48 h in culture medium containing NG or HG, VSMCs were subjected to the analysis of cell proliferation (B and C), migration (D), and invasion (E).

Figure 3

CircLRP6 served as a sponge of miR-545-3p. (A) StarBase showing the potential miR-545-3p sequences in circLRP6. (B) RIP and (C) dual-luciferase reporter assays were employed to test the interplay between circLRP6 and miR-545-3p in VSMCs. (D) The co-localization of circLRP6 and miR-545-3p in VSMCs. (E) miR-545-3p level in si-NC or si-circLRP6 treated VSMCs.

Figure 4

miR-545-3p negatively regulated HMGA1 expression by binding to it. (A) StarBase prediction showing the potential miR-545-3p binding sites in HMGA1. (B) Interplay between miR-545-3p and HMGA1 was tested by dual-luciferase reporter assay in VSMCs. (C and D) Relative mRNA and protein expression levels of HMGA1 in VSMCs treated with miR-545-3p mimic and inhibitor. NC mimic and NC inhibitor were used as negative control. (E) Western blot and (F) immunofluorescence analyses for HMGA1 in VSMCs treated with si-NC and si-circLRP6 under NG and HG condition for 24 h.

Figure 5

miR-545-3p overexpression repressed HG induced VSMCs proliferation, migration, and invasion. (A) Overexpression efficiency of miR-545-3p mimic in VSMCs. After 48 h of transfection with NC or miR-545-3p mimic under NG or HG condition, VSMCs were subjected to the analysis of cell proliferation (B and C), migration (D), and invasion (E). (F) Western blot and (G) immunofluorescence assays were carried out to measure the expression of HMGA1 in VSMCs transfected with NC mimic or miR-545-3p mimic under NG or HG condition.

Figure 6

CircLRP6/miR-545-3p/HMGA1 axis participated in the HG induced VSMCs proliferation, migration, and invasion. (A and B) After co-transfection with si-circLRP6 and miR-545-3p inhibitor, VSMCs were subjected to the detection of HMGA1 mRNA and protein. The treated VSMCs were also subjected to the assessment of proliferation (C and D), migration (E), and invasion (F).