Tanshinone ⅡA suppresses hypoxia-induced human pulmonary artery smooth muscle cell over-proliferation via the LINC01013/miR-548c-3p/IL6 pathway

Introduction

The changes in the structure or function of pulmonary blood vessels caused by diseases or other factors can cause increased vascular resistance, leading to the occurrence of PH ultimately. The right ventricle is responsible for pumping blood to the lungs, and continuous PH increases its burden. This prolonged high load may eventually progress to right heart failure [1], [2], [3]. Therefore, if PH is not treated promptly and effectively, its further deterioration may lead to the death of patients.

The pathogenesis of PH mainly includes pulmonary vasoconstriction, remodeling, and thrombosis. The disruption of the balance between vasoconstrictor/dilator substances and the changes in ion channel activity are important factors leading to pulmonary vasoconstriction [4]. The proliferation of endothelial cells, smooth muscle cells, and fibroblasts in the pulmonary artery is an important process of vascular remodeling, often accompanied by abnormal accumulation of collagen fibers and elastin, which is irreversible and will lead to the media thickening of the lung intima/blood vessel and lumen stenosis [5]. Moreover, inflammation, endothelium-mesenchymal transformation, dysregulation of proliferative/apoptotic signaling, or genetic changes can lead to vascular remodeling. Persistent inflammation is also a potential risk for pulmonary thrombosis, and other factors include platelet activation and endothelial dysfunction, etc. [6].

In the view of traditional Chinese medicine, Radix Salviae, the dried root and rhizome of the Labiatae plant “Salvia miltiorrhiza Bge.”, has the effect of promoting blood circulation and removing blood stasis, cooling blood to eliminate the carbuncle, and relieving pain. It can be used to treat heart disease, hypertension, and other diseases [7], 8]. Tanshinone IIA is the main active ingredient of Radix Salviae, which is a natural product for treating PH. Tanshinone IIA has been reported to be effective in alleviating monocrotaline-induced PH in rats, which can inhibit the intimamedia thickening of the pulmonary artery [9]. Sodium tanshinone IIA sulfonate is a water-soluble salt of tanshinone IIA which can improve hypoxia-induced PH in rats by inhibiting PI3K/AKT/mTOR pathway, promoting autophagy and reducing infiltration of inflammatory factors in the lung tissue, and it can suppress the proliferation of rats PASMCs by restoring PKG-PPAR-γ signaling [10], 11]. Here, we revealed a novel mechanism by which tanshinone IIA hindered PASMC proliferation by regulating the LINC01013/miR-548c-3p/IL6 pathway.

Materials and methods

Results

Tanshinone ⅡA inhibited PASMC proliferation

After treatment with different concentrations of tanshinone ⅡA, the viability of hypoxia-induced PASMCs was significantly inhibited in a concentration-dependent manner, but normoxic PASMCs were relatively insensitive to it (Figure 1A and B). Since 20 μg/mL tanshinone ⅡA significantly suppressed PASMC activity in the hypoxia group but hardly affected normoxic PASMCs, this concentration was used for subsequent mechanism studies. Tanshinone ⅡA treatment reduced the hypoxia-induced expressions of cell proliferation markers PCNA and Ki-67 (Figure 1C and D). PFK-1 is a rate-limiting enzyme in glycolysis. Tanshinone ⅡA attenuated the induction of PFK-1 activation and pyruvic acid production by hypoxia (Figure 1E and F). The expression of PFK-1 was significantly down-regulated after transfection of its small interfering RNA (si-PFK-1) (Figure 1G). The down-regulation of PFK-1 expression further enhanced the tanshinone ⅡA-mediated inhibition on cell viability and PCNA expression of hypoxic PASMCs (Figure 1H and I). These results demonstrated that tanshinone ⅡA hindered the propagation of hypoxic PASMCs by inhibiting glycolysis.

Figure 1:

Tanshinone ⅡA in the PASMC proliferation. (A–B) Sensitivity of hypoxic and normoxic PASMCs to different concentrations of tanshinone ⅡA. (C–D) The expressions of PCNA and Ki-67 in cells with hypoxia or tanshinone ⅡA treatment. (E–F) The influence of tanshinone ⅡA on PFK-1 activity and pyruvic acid level in hypoxic PASMCs. (G) Transfection with si-PFK-1 silenced PFK-1 expression. (H–I) PFK-1 silencing enhanced the inhibition of cell viability and PCNA expression by tanshinone ⅡA. *p<0.05, **p<0.01, ***p<0.001.

IL6 overexpression reversed the tanshinone ⅡA mediated-inhibition on PASMC growth

By making a VENN map using the target genes of tanshinone ⅡA and the DEGs in non-PH/PH arterial, 29 shared genes were obtained in the intersection (Figure S1A and Table S1). The GO analysis showed that the biological processes enriched by these genes included the proliferation of smooth muscle cells, etc. (Figure 2A). The KEGG analysis displayed that the enriched signaling pathways included TNF, HIF-1, and Toll-like receptor pathways, etc. (Figure 2B), which have been reported to be related to PH [12], [13], [14]. The key gene IL6 was screened through a VENN diagram of the genes involved in these biological processes and signaling pathways (Figure 2C). Hypoxia induced the expression of IL6 (Figure 2D). The role of IL-6 in PASMC proliferation was demonstrated by transfecting its overexpression vector (oe-IL6, Figure 2E). Overexpression of IL6 increased PASMC viability and PCNA expression in the tanshinone ⅡA-treated group (Figure 2F and G), which reversed the tanshinone ⅡA-induced decreases of PFK-1 activity and pyruvic acid level (Figure 2H and I), proving the acceleration of IL6 on PASMC growth.

Figure 2:

IL6 in the PASMC proliferation. (A–B) The GO and KEGG analysis of genes. (C) VENN diagram of the genes involved in smooth muscle cell proliferation biological processes and TNF, HIF-1, and toll-like receptor signaling pathways. (D) The expression of IL6 in PASMCs. (E) IL6 was overexpressed after oe-IL6 transfection. Influence of IL6 overexpression on the growth inhibition (F–G) and glycolysis suppression (H–I) of hypoxic PASMCs by tanshinone ⅡA. *p<0.05, **p<0.01, ***p<0.001.

LINC01013 promoted IL6 expression by inhibiting miR-548c-3p

LINC01013 is a high-expressed lncRNA in both the blood and the thrombus fibroblasts of CTEPH patients (Figure S1B and Table S1). We then predicted the downstream miRNAs of LINC01013 and the upstream miRNAs of IL6 using the relevant database, and finally, miR-548c-3p was identified by a VENN diagram (Figure S1C and Table S1). Figure S1D shows the binding sequences of miR-548c-3p to LINC01013 and IL6 mRNA predicted by the database. After transfection of the mimics, miR-548c-3p was significantly upregulated (Figure 3A), which weakened the luciferase intensity in cells transfected with wild-type (wt)-LINC01013/-IL6 reporter vector but didn’t affect the mutant (mut) transfection group (Figure 3B and C), verifying the target relationship among them. The overexpression vector of LINC01013 (oe-LINC01013) was introduced to explore its regulation on miR-548c-3p/IL6 (Figure 3D), whose transfection down-regulated miR-548c-3p and increased IL6 expression (Figure 3E and F). Moreover, the miR-548c-3p mimics hindered IL6 expression, and its co-transfection reversed the facilitation of LINC01013 on IL6 expression (Figure 3F). All these experiments certify that LINC01013 enhanced IL6 expression by down-regulating miR-548c-3p.

Figure 3:

Regulation of LINC01013 on the expression of miR-548c-3p/IL6 axis. (A) The efficiency of the mimics in up-regulating miR-548c-3p. (B–C) Dual-luciferase assay demonstrated the targeting effect of LINC01013 on miR-548c-3p and miR-548c-3p on IL6. (D) Effect of oe-LINC01013 on LINC01013 expression. (E) Regulation of miR-548c-3p expression by LINC01013. (F) The regulatory effect of LINC01013 on miR-548c-3p/IL6 expression. *p<0.05, **p<0.01, ***p<0.001.

Tanshinone ⅡA inhibited hypoxic PASMC proliferation by regulating the LINC01013/miR-548c-3p/IL6 axis

As shown in Figure 4A–D, overexpression of LINC01013 further enhanced the glycolysis-mediated growth of hypoxic PASMCs, manifesting increased cell viability, PCNA expression, PFK-1 activity, and pyruvic acid level, which were reversed by co-transfection of the miR-548c-3p mimics. Overexpression of IL6 partially eliminated the suppression of miR-548c-3p mimics on cell proliferation and glycolysis process in hypoxic PASMCs (Figure 4A–D). Tanshinone ⅡA inhibited LINC01013/IL6 expression and up-regulated miR-548c-3p in hypoxia-induced PASMCs (Figure 4E–G). These results hint that tanshinone ⅡA can restrain hypoxic PASMC propagation by regulating the LINC01013/miR-548c-3p/IL6 axis.

Figure 4:

The LINC01013/miR-548c-3p/IL6 axis mediated the suppression of tanshinone ⅡA on hypoxic PASMC growth. The roles of LINC01013/miR-548c-3p/IL6 axis in hypoxic PASMC proliferation (A–B) and glycolysis (C–D). (E–G) Effects of hypoxia and tanshinone ⅡA on the expressions of LINC01013, miR-548c-3p, and IL6. *p<0.05, **p<0.01, ***p<0.001.

Discussion

The potential of tanshinone IIA for the treatment of PH is already self-evident. In the PH rat model, treatment of tanshinone IIA intraperitoneal injection can reduce pulmonary artery muscularization and intimal-media thickening, and improve pulmonary vascular morphology [9]. Given the cardiac burden caused by PH, Zhang et al. revealed the role of tanshinone IIA in reducing right ventricular systolic pressure and improving right ventricular fat index in PH rats. It can also inhibit hypoxia-induced proliferation of pulmonary artery smooth muscle cells by reducing transforming growth factor β-induced smad3 phosphorylation [15]. Another study revealed that targeting SOCE regulated by the PGG-PPAR-γ pathway is a potential strategy for the treatment of PH with sodium tanshinone IIA sulfonate [10]. However, its specific regulatory mechanisms remain to be explored. We report that tanshinone IIA inhibited the viability of hypoxic PASMCs in a concentration-dependent manner, but its low concentrations were relatively safe for normoxic cells.

It has been reported that the competitive endogenous RNA (ceRNA) network mediates the progression of PH. Simply put, many types of RNA (such as mRNA, lncRNA, circRNA, etc.) contain the miRNA response elements. LncRNA or circRNA can compete with mRNA to bind miRNA, thereby mediating the gene expression regulation by miRNA. Under hypoxia conditions, lncRNA HOXA-AS3 is up-regulated but miR-675-3p was down-regulated in PASMCs. HOXA-AS3 can promote PDE5A expression by sponging miR-675-3p, facilitating the progression of PH [16]. LncRNA Tug1 is overexpressed in hypoxic PH mice. Its silencing down-regulates FOXC1 expression by releasing miR-374c, thereby impeding pulmonary vascular remodeling [17]. LncRNA PAHRF is down-regulated in PH patients and hypoxic PASMCs. It can reduce the proliferation of PASMCs under hypoxia conditions by regulating the miR-23a-3p/MST1 axis [18]. CircGSAP levels are significantly reduced in the plasma of patients with idiopathic PH, and its overexpression is associated with high survival. CircGSAP can reduce the inhibition of miR-27a-3p on BMPR2 expression by binding it, thus hindering pulmonary microvascular endothelial cells from proliferating and migrating induced by hypoxia and inducing their death [19]. Here, we report a novel ceRNA network affecting PH. We found that hypoxia treatment induced the expressions of LINC01013 and IL6 and inhibited miR-548c-3p expression. Tanshinone IIA restrained IL6 by alleviating the targeting effect of LINC01013 on miR-548c-3p, thereby reducing the glycolysis-mediated PASMC proliferation.

Myocardial fibrosis greatly increases the risk of death from heart disease. Silencing of LINC01013 decreased the expression of pro-fibrotic markers and reduced the activation of fibroblasts [20]. Silencing LINC01013 in endothelial cells damages its ability to migrate and angiogenesis [21]. LINC01013 is upregulated in calcified aortic valve disease and can control the transcription of CCN2 by inducing NELF-E expression to mediate the fibrocalcific process of the disease [22]. MiR-548c-3p is an inhibitor of tumor cell growth. Circ_0002395 can enhance the expression of PDK1 by down-regulating miR-548c-3p and promoting the aerobic glycolysis-mediated proliferation of pancreatic cancer cells [23]. Another study showed that miR-548c-3p may be an important player in heart-related diseases. In the myocardium of rats with myocardial infarction, miR-548c-3p is absent. Its upregulation reduces myocardial fibrosis and improves heart function [24]. Notably, miR-548c-3p was shown to be a maladjusted miRNA in a PH pig model [25], but its specific role in PH remains unknown. As we mentioned in the “Introduction”, inflammation is an important factor leading to pulmonary vascular remodeling and thrombosis. Therefore, IL6, a pleiotropic proinflammatory cytokine, has a potential position in PH development. Cai et al. reported that overexpression of miR-125a-5p can inhibit the proliferation and induce apoptosis in PASMCs by reducing the production of IL6 and TGF-β1 [26].

In conclusion, we highlighted a new ceRNA network of which the LINC01013/miR-548c-3p/IL6 axis may be involved in the progression of PH and revealed the reliable suppression of tanshinone ⅡA on PASMC proliferation. Our study provides new insight into the understanding of PH pathogenesis and the use of tanshinone ⅡA in PH treatment.