Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension

Abstract

Background: In this study, we aimed to investigate whether and how lncRNA CASC2 was involved in hypoxia-induced pulmonary hypertension (PH)-related vascular remodeling.

Methods: The expression of lncRNAs or mRNAs was detected by qRT-PCR, and western blot analysis or immunochemistry was employed for detecting the protein expression. Cell number assay and EdU (5-ethynyl-2'-deoxyuridine) staining were performed to assess cell proliferation. Besides, flow cytometry and wound healing assay were employed for assessments of cell apoptosis and cell migration, respectively. Rat model of hypoxic PH was established and the hemodynamic measurements were performed. Hematoxylin and eosin (HE) and Masson's trichrome staining were carried out for pulmonary artery morphometric analysis.

Results: The expression of lncRNA CASC2 was decreased in hypoxia-induced rat pulmonary arterial tissues and pulmonary artery smooth muscle cells (PASMCs). Up-regulation of lncRNA CASC2 inhibited cell proliferation, migration yet enhanced apoptosis in vitro and in vivo in hypoxia-induced PH. Western blot analysis and immunochemistry showed that up-regulation of lncRNA CASC2 greatly decreased the expression of phenotype switch-related marker α-SMA in hypoxia-induced PH. Furthermore, it was indicated by the pulmonary artery morphometric analysis that lncRNA CASC2 suppressed vascular remodeling of hypoxia-induced rat pulmonary arterial tissues.

Conclusion: LncRNA CASC2 inhibited cell proliferation, migration and phenotypic switch of PASMCs to inhibit the vascular remodeling in hypoxia-induced PH.

Keywords: Pulmonary artery smooth muscle cells (PASMCs); Pulmonary hypertension; Vascular remodeling; phenotypic switch; lncRNA CASC2.

Fig. 1

Up-regulation of lncRNA CASC2 inhibited proliferation in hypoxia-induced PASMCs. (a) The expression of lncRNA CASC2 was detected by qRT-PCR in PASMCs. (b) Cell number of PASMCs was counted by CyQuant cell proliferation assay kit. (c) The mRNA expression of PCNA was detected by qRT-PCR in PASMCs. (d) The protein expression of PCNA was detected by western blot analysis in PASMCs. (e, f) Cell proliferation status of PASMCs was assessed by EdU staining assay. ^###p < 0.001, compared with normoxia group. ^## p < 0.01, compared with normoxia group. ^#p < 0.05, compared with normoxia group. ^**p < 0.01, ^*p < 0.05, compared with hypoxia + pcDNA3.1 group

Fig. 2

Up-regulation of lncRNA CASC2 enhanced apoptosis yet inhibited migration in hypoxia-induced PASMCs. (a, c) Cell apoptosis was detected using flow cytometry in PASMCs. (b, d) Cell migration was assessed by wound healing assay in PASMCs. ^##p < 0.01, compared with normoxia group. ^#p < 0.05, compared with normoxia group. **p < 0.01, ^*p < 0.05, compared with hypoxia + pcDNA3.1 group

Fig. 3

LncRNA CASC2 suppressed phenotype switch induced by hypoxia in PASMCs. (a-b) The expression of phenotype switch marker proteins including myocardin, tropomysin, syndecan-1 and α-SMA was detected by western blot analysis in PAMSCs. ^##p < 0.01. ^#p < 0.05, compared with normoxia group. ^###p < 0.001, compared with normoxia group. **p < 0.01, compared with hypoxia + pcDNA3.1 group

Fig. 4

LncRNA CASC2 was down-regulated in hypoxia-induced rats and reversed the increase of mPAP and progression of right ventricular hypertrophy caused by hypoxia. (a) The expression of lncRNA CASC2 was detected by qRT-PCR in rat pulmonary arterial tissues. (b) Systemic blood pressure (SBP) was assessed in normoxia and hypoxia-induced rats. (c) Mean pulmonary arterial pressure (mPAP) was detected in normoxia and hypoxia-induced rats. (d) The right ventricular hypertrophy index (RVI) was calculated in normoxia and hypoxia-induced rats. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with normoxia group. **p < 0.01, compared with hypoxia + pcDNA3.1 group

Fig. 5

LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. (a) The mRNA expression of PCNA was detected by qRT-PCR in rats′ PAs tissues. (b, c) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ^##p < 0.01, compared with normoxia group. ^###p < 0.001, compared with normoxia group. **p < 0.01, compared with hypoxia + pcDNA3.1 group

Fig. 6

LncRNA CASC2 suppressed phenotype switch of hypoxia-induced rat pulmonary arterial tissues. (a) Immunohistochemical detection of phenotype switch marker protein α-SMA was performed in rat pulmonary arterial tissues. (b) The expression of phenotype switch markers including myocardin, tropomysin, syndecan-1 and α-SMA was detected by western blot analysis in rat pulmonary arterial tissues. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with normoxia group. **p < 0.01, compared with hypoxia + pcDNA3.1 group

Fig. 7

LncRNA CASC2 suppressed vascular remodeling of hypoxia-induced rat pulmonary arterial tissues. (a) Tissue fibrosis was measured by Masson′s trichrome staining in rat pulmonary arterial tissues and the collagen fiber was stained with blue. (b) The morphometric analysis of rat pulmonary arterial tissues was performed by hematoxylin and eosin (HE) staining. The parameters of pulmonary vascular cross-section were calculated by measuring the medial wall thickness, total vessel wall thickness, cross-sectional vessel area and wall area. (c) The percentage of medial wall thickness to the external diameter (WT%) was calculated in rats′ PAs tissues. (d) The percentage of cross-sectional total vessel wall area to the total area (WA %) was calculated in rat pulmonary arterial tissues. ### p < 0.001, compared with normoxia group. ^** p < 0.01, compared with hypoxia + pcDNA3.1 group