lncRNA KCNQ1OT1 Suppresses the Inflammation and Proliferation of Vascular Smooth Muscle Cells through IκBa in Intimal Hyperplasia

Figures

• 

  Figure 1. KCNQ1OT1 Is Downregulated in the VSMCs of Mice with Intimal Hyperplasia and in the Process of VSMC Proliferation The vein graft model (VG) was constructed in mice (n = 25) to introduce intimal hyperplasia. (A and B) After 0, 1, 2, 3, and 4 weeks (n = 5 at each time point), (A) the intimal area was calculated by subtracting the luminal area from the area within the internal elastic lamina, and (B) the expression of KCNQ1OT1 in isolated VSMCs was detected using quantitative real-time PCR. VSMCs were isolated from the normal mice and stimulated with PDGF-BB with an increased concentration gradient (0, 5, 10, and 20 ng/mL) for 48 h. (C) The expression of KCNQ1OT1 in VSMCs was determined using quantitative real-time PCR. VSMCs were treated with PDGF-BB (10 ng/mL) for different durations (24, 48, and 72 h). (D) The expression of KCNQ1OT1 was examined by quantitative real-time PCR. *p < 0.05 and **p < 0.01 compared with the 0 time point or without PDGF-BB.

• 

  Figure 2. Overexpression of KCNQ1OT1 Suppresses VSMC Proliferation, Migration, and Secretion of Inflammatory Factors In VSMCs treated with PDGF-BB (10 ng/mL, 48 h), KCNQ1OT1 was overexpressed by transfecting the Ad-KCNQ1OT1 vector, with Ad-GFP acting as the negative control. (A) The proliferation of VSMCs was evaluated using the BrdU staining method. (B) VSMC migration was assessed by transwell assay. (C) The level of inflammatory factors, such as IL-1β, IL-6, and TNF-α, in VSMC culture supernatant was determined using ELISA. *p < 0.05 and **p < 0.01.

• 

  Figure 3. Hypermethylation Represses KCNQ1OT1 Expression and KCNQ1OT1 Binds to IκBa Protein to Impede Its Degradation (A) For mice undergoing vein graft (VG, n = 7) and those in the sham group (n = 7), the VSMCs were isolated and the methylation level of the KCNQ1OT1 promoter was detected. In the VSMCs isolated from normal mice, PDGF-BB (10 ng/mL, 48 h) was used, and (B) the methylation level in the promoter of KCNQ1OT1 was measured. (C) DNMT1, DNMT3a, or DNMT3b was silenced in PDGF-BB-induced VSMCs to detect the level of methylation in the promoter of KCNQ1OT1. (D and E) RNA pull-down (D) and RIP assay (E) were performed to assess the binding and interaction between KCNQ1OT1 and IκBa protein in VSMCs. VSMCs were divided into control, PDGF-BB, PDGF-BB+Ad-GFP, and PDGF-BB+Ad-KCNQ1OT1, and (F) the level of IκBa protein in VSMCs was analyzed by western blot. (G) Via transfecting si-KCNQ1OT1 or si-control in VSMCs, the expression level of the p-IκBa and IκBa proteins was determined by western blot. (H) VSMCs were transfected with pcDNA-KCNQ1OT1 or pcDNA, and the p-IκBa and IκBa protein levels were measured by western blot. (I and J) In VSMCs transfected with si-KCNQ1OT1 or si-control, (I) the…

• 

  Figure 4. KCNQ1OT1 Upregulates IκBa Levels by Sponging miR-221 (A) Via bioinformatics software, the binding sites of miR-221 (miR-221-3p) on the IκBa 3′ UTR were predicted (TargetScan), as were the complementary bases between KCNQ1OT1 and miR-221 (DIANA tools). (B) Luciferase reporter assay was performed to assess the binding between KCNQ1OT1 and miR-221. (C) AGO2-RIP assay was conducted to validate the interaction between KCNQ1OT1 and miR-221. (D and E) The expressions of (D) miR-221 and (E) IκBa protein were detected in VSMCs in which KCNQ1OT1 was overexpressed. (F and G) In VSMCs transfected with vector, si-KCNQ1OT1, si-KCNQ1OT1+miR-221 inhibitor (F), or vector + pre-miR negative control/miR-221 mimic, KCNQ1OT1 + pre-miR negative control/miR-221 mimic (G), the IκBa protein level was determined by western blot. **p < 0.01 compared with the pre-miR negative control or vector.

• 

  Figure 5. KCNQ1OT1 Suppresses VSMC Proliferation, Migration, and Secretion of Inflammatory Factors via Upregulating IκBa VSMCs were allocated into the following five groups: si-control, si-KCNQ1OT1-1, si-KCNQ1OT1-1+pcDNA-IκBa, si-KCNQ1OT1-2, and si-KCNQ1OT1-2+pcDNA-IκBa. (A) The proliferation of VSMCs was evaluated using the BrdU staining method. (B) VSMC migration was assessed via transwell assay. (C) The level of inflammatory factors, including IL-1β, IL-6, and TNF-α, in VSMC culture supernatant was determined by ELISA. *p < 0.05 and **p < 0.01.

• 

  Figure 6. Overexpression of KCNQ1OT1 Attenuated Intimal Hyperplasia in a VG Mouse Model The lentivirus vector expressing KCNQ1OT1 (Lv-KCNQ1OT1) or empty vector (vector, 108 pfu/mL) was subcutaneously injected into mice with VG (n = 7 in each group). (A) After 4 weeks, the graft vein was collected, and the intimal thickness was observed and intimal surface area was calculated. Scale bar represents 20 μm. (B and C) The VSMCs were isolated, and (B) the expressions of KCNQ1OT1 and (C) miR-221 were detected using quantitative real-time PCR. (D and E) p-IκBa and IκBa protein levels were determined using western blot (D) and mRNA levels of IL-1β, IL-6, and TNF-α were detected using quantitative real-time PCR (E). (F) KCNQ1OT1 upregulates IκBa, suppressing the inflammation and proliferation of VSMCs and attenuating intimal hyperplasia. *p < 0.05, **p < 0.01 compared with vector.