XIST knockdown suppresses vascular smooth muscle cell proliferation and induces apoptosis by regulating miR-1264/WNT5A/β-catenin signaling in aneurysm

Abstract

Long non-coding RNAs (lncRNAs) have been ascertained as vital modulators in abdominal aortic aneurysm (AAA) development. In this research, the function and molecular mechanisms of the lncRNA X-inactive specific transcript (XIST) in the evolution of vascular smooth muscle cells (VSMCs) were assessed. Results showed that XIST expression was increased but miR-1264 expression level was reduced in the serum of AAA patients. XIST depletion impeded human aorta VSMCs (HA-VSMCs') ability to proliferate and stimulate apoptosis, while repressing miR-1264 expression through an unmediated interaction. Additionally, the influence of XIST knockdown on apoptosis and proliferation could be rescued by an miR-1264 inhibitor. Subsequent molecular investigations indicated that WNT5A was miR-1264's target, and XIST functioned as a competing endogenous RNA (ceRNA) of miR-1264 to raise WNT5A expression. Further, an miR-1264 inhibitor stimulated the proliferation and suppressed the apoptosis of HA-VSMCs through the activation of WNT/β-catenin signaling. Taken together, XIST impeded the apoptosis and stimulated the proliferation of HA-VSMCs via the WNT/β-catenin signaling pathway through miR-1264, demonstrating XIST's underlying role in AAA.

Keywords: abdominal aortic aneurysm; lncRNA; miR-1264; proliferation.

Figure 1. XIST expression is elevated while miR-1264 expression is reduced in the serum of AAA patients due to a direct interaction

(A) Expression of XIST and (B) miR-1264 in serum from AAA patients (n=24) and healthy participants (n=24). (C) The predicted binding sites between miR-1264 and XIST, and the mutant sites in the XIST-MUT reporter are shown. (D,E) Luciferase activity in HA-VSMCs co-treated with XIST-WT or XIST-MUT reporters and miR-1264 or its scramble control (mimic NC or inhibitor NC). (F) A negative correlation between XIST and miR-1264 expression in the serum of 24 AAA patients. Data are presented as mean ± SEM (n=3) of at least three independent assays. *P<0.05.

Figure 2. Down-regulation of XIST inhibited the morbidity and development of AAA

(A) XIST was up-regulated in AAA mice model, but miR-1264 was down-regulated. (B) The incidence rate of AAA in each group was analyzed. (C) The maximum diameter of each aortic tissue was measured. (D) The elastin filament degradation score in each group (n=10 aorta/group). (E) Hematoxylin staining was performed to detect the pathological change in aortic tissues in each group. *P<0.05.

Figure 3. XIST knockdown represses HA-VSMCs proliferation and triggers apoptosis

HA-VSMCs transfected with si-NC, si-XIST#1 or si-XIST#2 were assessed for (A) XIST expression (B) proliferative capacity, (C) clone formation ability, (D) PCNA and Ki-67 expression, and (E) Bcl-2 and Bax expression and the relative cell apoptosis (F). Data are reflected as mean ± SEM (n=3) of at least three independent assays. *P<0.05.

Figure 4. miR-1264 inhibitor reverses the effect of XIST knockdown on HA-VSMC apoptosis and proliferation

HA-VSMCs treated with si-NC + inhibitor NC, si-NC + miR-1264 inhibitor, si-XIST + inhibitor NC, or si-XIST and miR-1264 inhibitor were assessed for (A) miR-1264 expression, (B) cell proliferation ability and (C) colony formation capacity. HA-VSMCs treated with si-NC + miR-1264 inhibitor, si-XIST + inhibitor NC or si-XIST and miR-1264 inhibitor, were assessed for (D) expression of Ki-67, PCNA, (E) Bax and Bcl-2 and the relative cell apoptosis (F). Data are reflected as mean ± SEM (n=3) of at least three independent assays. *,^#P<0.05 (*, compared with si-NC + inhibitor NC or si-NC + miR-1264 inhibitor; ^#, compared with si-XIST + inhibitor NC).

Figure 5. WNT5A is a miR-1264 target

(A) Predicted binding sequences between miR-1264 and WNT5A 3′ UTR region with mutant sites in the WNT5A-MUT reporter. (B,C) Luciferase activity test following co-treatment of HA-VSMCs with WNT5-WT or WNT5-MUT reporter and miR-1264 or miR-1264 inhibitor for 48 h. (D) WNT5A expression in the serum of healthy participants (n=24) and AAA patients (n=24). (E,F) Relationship between WNT5A and XIST or miR-1264 in the serum of 24 AAA patients is analyzed. Data are reflected as mean ± SEM (n=3) of at least three independent assays. *P<0.05.

Figure 6. miR-1264 modulates HA-VSMC proliferation and apoptosis through WNT/β-catenin signaling

(A,B) HA-VSMCs transfected with si-NC + inhibitor NC, siWNT5A + inhibitor NC, si-NC + miR-1264 inhibitor or si-WNT5A + miR-1264 inhibitor were transfected for 24 h and the expression of WNT5A, β-catenin, E-cadherin and C-myc were assessed. HA-VSMCs underwent transfection with NC inhibitor or miR-1264 inhibitor with or without XAV939 (10 μM) for 10 h, followed by tests for (C) cell proliferation capacity, (D) colony formation capacity and the expression of (E) Ki-67, PCNA, and (F) Bcl-2 and Bax. Data are reflected as mean ± SEM (n=3) of at least three independent assays. *,^#P<0.05 (*, compared with si-NC + inhibitor NC or control+ inhibitor NC; ^#, compared with siWNT5A + inhibitor NC or XAV939+ inhibitor NC).