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Introduction

Pulmonary arterial hypertension (PAH) typically originates from hyperplasia of pulmonary arterial smooth muscle cells (PASMCs) (1,2). Under normal physiological conditions, PASMCs are quiescent and contractile (3). However, under hypoxia, the phenotype of PASMCs can switch, leading to excessive proliferation and migration (4,5), eventually resulting in pulmonary vasoconstriction and vascular remodelling (6). Therefore, exploring the mechanism by which hypoxia induces the proliferation and migration of PASMCs is vital for treating PAH in the clinic.

Various long non-coding RNAs (lncRNAs) have been revealed to promote the development of PAH (7–9). For example, Zhang et al reported that HOXA-AS3 was overexpressed in both the lung vasculature of monocrotaline (MCT)-treated mice and PASMCs from patients with PAH (7). In addition, Wang et al detected high MALAT1 expression in PAH tissues and PASMCs (8). Yang et al demonstrated that hypoxia increased the expression of taurine upregulated gene 1 (TUG1) in the pulmonary artery (PA) of a PAH mouse model, whereas silencing of TUG1 markedly decreased the hyperproliferation of PASMCs (9). Additionally, inhibition of NEAT1 has been reported to suppress the proliferation and migration of numerous human cancers, including pancreatic (10), breast (11), non-small cell lung (12) and colorectal cancers (13). More relevant to the present study, Ahmed et al (14) demonstrated that knockout of NEAT1 increased the levels of contractile proteins in vascular smooth muscle (VSM) cells (VSMCs), allowing them to maintain their contractile status and ultimately inhibit vascular hyperplasia. However, the possible role of NEAT1 in hypoxia-treated PASMCs has yet to be elucidated.

Notably, the expression levels of some microRNAs (miRNAs) are reduced in lung vascular tissues and/or PASMCs, which are likely involved in the progression of PAH (15–17). For instance, downregulation of miR-98 was revealed to enhance the proliferation-promoting effect of hypoxia on PASMCs (15). In an MCT-induced rat model, low miR-140-5p expression was observed, which suppressed PAH by targeting TNF-α (16). MiR-182-3p was downregulated in patients with PAH as well as in hypoxia-stimulated model mice/rats, and miR-182-3p upregulation had a distinct antiproliferative effect on PASMCs (17). Notably, a recent study revealed that miR-34a was expressed at low levels in the PA of rats, and it inhibited the proliferation of PASMCs by targeting PDGFRA (18). lncRNAs can modulate the function of miRNAs by sponging them and thus acting as competing endogenous RNAs. Whether miR-34a-5p interacts with NEAT1 and whether this interaction is involved in the pathogenesis of PAH needs to be further investigated.

Krüppel-like factor 4 (KLF4), a member of the KLF family of zinc-finger transcription factors (19), was reported to be associated with the progression of PAH (20,21). Sun et al (20) revealed that KLF4 expression was significantly increased in remodelled pulmonary arteries from a rat model of pulmonary vascular remodelling, and downregulation of KLF4 suppressed the proliferation and migration of PASMCs. Liang et al (21) demonstrated that increased KLF4 expression promoted the migration and cell cycle progression of pulmonary artery endothelial cells. In addition, KLF4 was revealed to be an oncogene that is regulated by miRNAs to promote tumour progression in various human cancers, such as miR-148-3p/miR-152-3p in prostate cancer (22), miR-32 in gastric cancer (23), and miR-543 in colorectal cancer (24). Most relevant to our research, a recent study revealed that miR-182-3p mediated vascular remodelling in PAH by targeting KLF4 (17). However, whether regulation of KLF4 by miR-34a-5p is involved in PAH and the underlying mechanism have yet to be elucidated.

In the present study, the possible roles of NEAT1, miR-34a-5p, and KLF4 in the pathogenesis of PAH in vitro were investigated, which will provide novel insights into the diagnosis and treatment of PAH.

Materials and methods

Serum samples

From January 2018 to December 2019, serum samples were collected from 25 patients (19 females and 6 males; age range, 21–48 years old; mean age, 35.04±8.44 years old) with PAH and 25 healthy volunteers (19 females and 6 males; age range, 21–50 years old; mean age, 34.96±8.58 years old) in The People's Hospital of Rizhao (Shandong, China). Patients with ≥1 of the following conditions were excluded: i) Other types of PAH, including familial PAH; ii) heart diseases, including left ventricular diseases and acute heart failure; iii) chronic respiratory disorders, including chronic obstructive pulmonary disease; iv) diabetes mellitus; and v) prior targeted therapy. No patients had received prior medical treatment. The inclusion criteria for healthy controls were that subjects must be age- and sex-matched with patients and absent of any diseases when enrolled. The study was approved by the Ethics Committee of The People's Hospital of Rizhao, and written informed consent was obtained from all study participants. The collected serum samples were centrifuged at 450 × g for 20 min at 20°C and immediately stored at −80°C until use.

Cell culture, hypoxia stimulation, and transfection

Human PASMCs (BioVector NTCC, Inc.) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 0.1% penicillin/streptomycin (ScienCell Research Laboratories, Inc.) at 37°C with 5% CO2. The cells were divided into two groups: The hypoxia group, which was treated with 5% CO2 and 95% N2 at 37°C for 48 h, and the normoxia group, which was treated with 5% CO2, 21% O2, and 74% N2 at 37°C for 48 h. The short hairpin (sh) RNA targeting NEAT1 (sh-NEAT1-1, 5′-GCCAUCAGCUUUGAAUAAAUU-3′; shNEAT1-2, 5′-GGUGUUAUCAAGUGAAUUAUU-3′) and negative control (sh-NC, 5′-UUCUCCGAACGUGUCACGU-3′) were obtained from Shanghai Transheep Bio-Tech Co., Ltd. The KLF4 overexpression vector (pcDNA-KLF4), overexpression negative control (pcDNA-NC), miR-34a-5p mimics (5′-GAUGGACGUGCUUGUCGUGAAAC-3′) and non-targeting mimics control (miR-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′), miR-34a-5p inhibitor (5′-CUACCUGCACCAACAGCACUU-3′), and non-targeting inhibitor control (inhibitor NC, 5′-CAGUACUUUUGUGUAGUACAA-3′) were obtained from Beina Biology. All the above agents (all at 20 nM) were co-transfected into hypoxia-treated PASMCs using the Lipofectamine® RNAiMAX kit (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. Subsequently, 48 h after transfection, PASMCs were harvested to perform further experiments.

Reverse transcription-quantitative (RT-q)PCR

Total RNA was extracted from the serum of patients with PAH, healthy volunteers, and PASMCs using the Total RNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd.). According to the manufacturer's instructions, cDNA was synthesized using the First-Strand cDNA Synthesis Kit (APExBIO Technology), and RT-qPCR was performed with SYBR Green FAST Mastermix (Qiagen GmbH). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 3 min followed by 40 cycles at 95°C for 15 sec, annealing at 60°C for 30 sec, elongation at 72°C for 1 min, and a final extension at 72°C for 5 min. Experimental results were quantified by the 2−ΔΔCq method (25). The expression levels of NEAT1 and KLF4 were normalised to GAPDH, and the expression level of miR-34a-5p was normalised to U6. The respective sequences of primers were as follows: NEAT1 forward, 5′-GGGGCCACATTAATCACAAC-3′ and reverse, 5′-CAGGGTGTCCTCCACCTTTA-3′; miR-34a-5p forward, 5′-GGGGTGGCAGTGTCTTAGC-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; KLF4 forward, 5′-TTCCCATCTCAAGGCACACC-3′ and reverse, 5′-CATGTGTAAGGCGAGGTGGT-3′; GAPDH forward, 5′-CCAGGTGGTCTCCTCTGA-3′ and reverse, 5′-GCTGTAGCCAAATCGTTGT-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide (MTT) assay

Anoxic and transfected PASMCs (2×103) were cultured in 96-well plates for 48 h at 37°C. Then, MTT (Nanjing Keygen Biotech Co., Ltd.) was added, and the cells were incubated for 2 h at 37°C. Thereafter, 100 µl dimethyl sulfoxide was added to dissolve the formazan. Cell viability was assessed by measuring the absorbance at an optical density of 450 nm using a microplate reader (Molecular Devices, LLC).

Transwell migration assay

The hypoxia-treated and transfected PASMCs (2×104) were resuspended in 200 µl of serum-free medium and then plated into the upper chambers of each Transwell apparatus (8 µm pore size; BD Biosciences). A total of 600 µl of medium containing 10% FBS was added to the lower chambers followed by incubation at 37°C for 48 h. Subsequently, the cells in the upper chambers were wiped off using a cotton swab and those adhering to the lower chambers were fixed with 4% paraformaldehyde for 1 h and stained with 0.5% crystal violet [TCI (Shanghai) Development Co., Ltd.] at 37°C for 30 min. Stained cells were imaged using an inverted light microscope (magnification, ×400; Olympus Corporation) and analysed with ImageJ software (version 1.46, National Institutes of Health).

Western blotting

Hypoxia-treated and transfected PASMCs were lysed with RIPA buffer (Beyotime Institute of Biotechnology) to extract total protein. The protein concentration was detected by the BCA Protein Assay kit. Then, a total of 50 µg of protein/lane was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. Following blocking with 5% skimmed milk for 2 h at 25°C, the membranes were incubated with the following primary antibodies: PCNA (1:1,000; Abcam; cat. no. ab92552), MMP2 (1:1,000; Abcam; cat. no. ab92536), α-smooth muscle actin (α-SMA; 1:1,000; Abcam; cat. no. ab5694), total caspase-3 (t-caspase-3; 1:1,000; Abcam; cat. no. ab32351), cleaved caspase-3 (c-caspase-3; 1:1,000; Abcam; cat. no. ab2302), KLF4 (1:1,000; Abcam; cat. no. ab215036), and GAPDH (1:1,000; Abcam; cat. no. ab215036) at 4°C overnight, and then with the secondary antibody, HRP-conjugated anti-rabbit IgG (1:3,000; Abcam; cat. no. ab6721) for 1 h at 37°C. The immunoblots were visualised using an ECL detection kit (Thermo Fisher Scientific, Inc.) under Gel-Pro analyzer (version 4.0; Media Cybernetics, Inc.).

Dual luciferase reporter (DLR) assay

The targeting relationship between NEAT1 and miR-34a-5p was analysed using the StarBase software (version 2.0; http://starbase.sysu.edu.cn). Additionally, the targeting relationship between miR-34a-5p and KLF4 was predicted using TargetScan software (release 7.2; http://www.targetscan.org/vert_72/). Fragments of NEAT1/KLF4 containing the miR-34a-5p binding site [wild-type (wt) and mutated (mut)] were inserted into the pGL3 vector (Promega Corporation) to generate recombinant luciferase reporter plasmids. PASMCs in 96-well plates (2,000 cells/well) were co-transfected with either miR-34a-5p mimics or miR-NC and either wt or mut NEAT1/KLF4 using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 48 h at 37°C, luciferase activity was analysed using the DLR gene assay system (Promega Corporation). The activity of firefly luciferase was normalized to that of Renilla luciferase.

Statistical analysis

All experiments were performed in triplicate, and each experiment was repeated three times. Data were analysed using SPSS 23.0 software (IBM Corp.) and presented as the mean ± SD. Comparisons between two groups were performed using unpaired t-test, and comparisons among multiple groups were performed using ANOVA, followed by Tukey's multiple comparison test. Pearson's correlation analysis was used to assess the linear correlation of serum concentrations in patients with PAH. P<0.05 was considered to indicate a statistically significant difference.

Results

Low expression of lncRNA NEAT1 protects against PAH in vitro

It was initially observed that the expression of NEAT1 was significantly increased in hypoxia-treated PASMCs when compared to its expression levels in normoxic PASMCs (Fig. 1A; P<0.01). Given that NEAT1 was overexpressed in hypoxia-treated PASMCs, NEAT1 was knocked down in hypoxia-treated PASMCs by transfecting sh-NEAT1-1/-2 to further explore the effects of NEAT1 on PAH in vitro. An analysis of transfection efficiency revealed a decrease in NEAT1 expression in transfected cells (Fig. 1B; P<0.01). sh-NEAT1-1-transfected cells were selected for further study due to their relatively low NEAT1 expression. An MTT assay was performed to measure cell viability, which indicated that transfection of sh-NEAT1 reversed the positive effect of hypoxia on the viability of PASMCs (Fig. 1C; P<0.01). The Transwell assay produced similar data, revealing that hypoxia treatment significantly promoted the migration of PASMCs, which was reversed by transfection with sh-NEAT1 (Fig. 1D; P<0.01). To further verify the effects of sh-NEAT1 on the proliferation and migration of hypoxia-treated PASMCs, a western blot assay was performed, which demonstrated that knock down of NEAT1 expression markedly reduced the hypoxia-induced increases in the protein levels of PCNA (a proliferation-related protein) (26) and MMP2 (a migration-related protein) (27) (Fig. 1E; P<0.01). It was also determined that hypoxia treatment significantly reduced the level of α-SMA (a marker of VSM-specific contraction) (28), and the ratio of c-caspase-3/t-caspase-3 (a pro-apoptosis-related protein) (29) compared to the normoxic group, indicating that more contractile PASMCs were converted to a proliferative phenotype. However, these inhibitory effects were reversed by transfection with sh-NEAT1-1 (Fig. 1F; P<0.01).

Figure 1. Low expression of lncRNA NEAT1 protects against PAH in vitro. (A) The expression of NEAT1 in hypoxia-induced PASMCs was detected by RT-qPCR. **P<0.01 vs. the normoxia PASMC group. (B) The expression of NEAT1 after transfection of sh-NEAT1-1/-2/NC in hypoxia-induced PASMCs was detected by RT-qPCR. **P<0.01 vs. the sh-NC group. (C) The viability (OD450) of hypoxia-induced PASMCs was measured by MTT assay. (D) The migration of hypoxia-induced PASMCs was measured by Transwell assay. Scale bar, 50 µm. (E) The protein levels of PCNA and MMP2 in hypoxia-induced PASMCs after transfection of sh-NEAT1-1/NC were detected by western blotting. (F) The protein levels of α-SMA, and c-caspase-3/t-caspase-3 ratio in hypoxia-induced PASMCs after transfection of sh-NEAT1-1/NC was detected by western blotting. **P<0.01 vs. the normoxia PASMC group; ##P<0.01 vs. the hypoxia + sh-NC group. lncRNA, long non-coding RNA; NEAT1, nuclear paraspeckle assembly transcript 1; PAH, pulmonary arterial hypertension; PASMCs, pulmonary arterial smooth muscle cells; sh-, short hairpin; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide.

NEAT1 sponges miR-34a-5p

An underlying binding site between NEAT1 and miR-34a-5p was revealed using StarBase (Fig. 2A). Then a DLR assay was conducted to assess the role of this binding site and miR-34a-5p on NEAT1 expression, which yielded an interesting result; the luciferase activity of the wt NEAT1 reporter was decreased by transfection of miR-34a-5p mimics, whereas the luciferase activity of the NEAT1 mut reporter was unaffected (Fig. 2B; P<0.01). This result confirmed the relationship between NEAT1 and miR-34a-5p. Additionally, the expression of miR-34a-5p in PASMCs was increased by transfection of sh-NEAT1 (Fig. 2C; P<0.01), indicating an inverse relationship between them.

Figure 2. NEAT1 sponges miR-34a-5p. (A) The complementary binding site of NEAT1 and miR-34a-5p was predicted by StarBase software. (B) The luciferase activity in PASMCs co-transfected with pGL3-NEAT1 wt/pGL3-NEAT1 mut and miR-34a-5p mimics/NC was determined by dual luciferase reporter assay. **P<0.01 vs. the miR-NC group. (C) The expression of miR-34a-5p after transfection of sh-NEAT1-1/NC in hypoxia-induced PASMCs was detected by reverse transcription-quantitative PCR. **P<0.01 vs. the sh-NC group. NEAT1, nuclear paraspeckle assembly transcript 1; miR-34a-5p, microRNA-34a-5p; PASMCs, pulmonary arterial smooth muscle cells; wt, wild-type; mut, mutated; NC, negative control; sh-, short hairpin.

High expression of miR-34a-5p has suppressive effects on PAH in vitro

The expression of miR-34a-5p in PASMCs under hypoxia was evaluated by RT-qPCR. The results of RT-qPCR revealed that, when compared to PASMCs under normoxia, the expression of miR-34a-5p was reduced in hypoxia-treated PASMCs (Fig. 3A; P<0.01). To explore the possible functions of miR-34a-5p in the pathogenesis of PAH in vitro, miR-34a-5p mimics or inhibitor was transfected into hypoxia-treated PASMCs. As anticipated, miR-34a-5p expression was increased by miR-34a-5p mimics, whereas it was reduced by the miR-34a-5p inhibitor (Fig. 3B; P<0.01). The increases in proliferation (Fig. 3C; P<0.01) and migration (Fig. 3D; P<0.01) of PASMCs induced by hypoxia were partly reversed by overexpression of miR-34a-5p. Western blotting revealed that transfection of miR-34a-5p mimics significantly reduced the hypoxia-induced increases in PCNA and MMP2 protein levels (Fig. 3E; P<0.01), which further confirmed the results of the MTT and Transwell assays. Concurrently, overexpression of miR-34a-5p increased the protein levels of α-SMA, and the c-caspase-3/t-caspase-3 ratio (Fig. 3F; P<0.01).

Figure 3. High expression of miR-34a-5p plays a suppressive role in PAH in vitro. (A) The expression of miR-34a-5p in hypoxia-induced PASMCs was detected by RT-qPCR. **P<0.01 vs. the normoxia PASMC group. (B) The expression of miR-34a-5p after transfection of miR-34a-5p mimics/inhibitor in hypoxia-induced PASMCs was detected by RT-qPCR. **P<0.01 vs. the miR-NC group. (C) The viability (OD450) of hypoxia-induced PASMCs was measured by MTT assay. (D) The migration of hypoxia-induced PASMCs was measured by Transwell assay. Scale bar, 50 µm. (E) The protein levels of PCNA and MMP2 in hypoxia-induced PASMCs after transfection of miR-34a-5p mimics/NC were detected by western blotting. (F) The protein levels of α-SMA, and c-caspase-3/t-caspase-3 ratio in hypoxia-induced PASMCs after transfection of miR-34a-5p mimics/NC were detected by western blotting. **P<0.01 vs. the normoxia PASMC group; ##P<0.01 vs. the hypoxia + miR-NC group. miR-34a-5p, microRNA-34a-5p; PAH, pulmonary arterial hypertension; PASMCs, pulmonary arterial smooth muscle cells; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide; c-, cleaved; t-, total.

MiR-34a-5p targets KLF4

TargetScan predicted a potential binding site for miR-34a-5p in KLF4 (Fig. 4A; P<0.01). In addition, a DLR assay revealed lower luciferase activity in KLF4-wt/miR-34a-5p-mimic-transfected cells than in KLF4-wt/miR-NC-transfected cells (Fig. 4B; P<0.01), indicating a strong association between KLF4 and miR-34a-5p. Western blotting was performed to further examine the interactions among NEAT1, miR-34a, and KLF4 in hypoxia-treated PASMCs. It was determined that KLF4 protein levels were reduced by transfection of miR-34a-5p mimics (Fig. 4C; P<0.01) as well as by transfection of sh-NEAT1-1, whereas transfection of a miR-34a-5p inhibitor reversed the suppressive effect of sh-NEAT-1 on KLF4 levels (Fig. 4D; P<0.05).

Figure 4. MiR-34a-5p targets KLF4. (A) The complementary binding site of NEAT1 and miR-34a-5p was predicted by TargetScan software. (B) The luciferase activity in PASMCs co-transfected with pGL3-KLF4 wt/pGL3-KLF4 mut and miR-34a-5p mimics/NC was determined by dual luciferase reporter assay. **P<0.01 vs. the miR-NC group. (C) The protein level of KLF4 in hypoxia-induced PASMCs after transfection of miR-34a-5p mimics/NC was detected by western blotting. **P<0.01 vs. the miR-NC group. (D) The protein level of KLF4 in hypoxia-induced PASMCs after transfection of sh-NEAT1-1/sh-NEAT1-1 + miR-34a-5p inhibitor was detected by western blotting. **P<0.01 vs. the sh-NC group; #P<0.01 vs. the sh-NEAT1-1 group. miR-34a-5p, microRNA-34a-5p; KLF4, Krüppel-like factor 4; NEAT1, nuclear paraspeckle assembly transcript 1; PASMCs, pulmonary arterial smooth muscle cells; wt, wild-type; mut, mutated; NC, negative control; sh-, short hairpin.

Knockdown of NEAT1 slows the process of PAH by sponging miR-34a-5p and downregulating KLF4

As presented in Fig. 5A, KLF4 protein levels were increased in hypoxia-treated PASMCs but not in cells under normoxia (P<0.01). pcDNA-KLF4 was transfected into hypoxia-treated PASMCs and the transfection efficiency was first evaluated. Transfection of pcDNA-KLF4 was determined to be successful due to the significant increase in KLF4 levels (Fig. 5B; P<0.01). Using these transfected cells, feedback verification experiments were performed to further explore the regulatory mechanism involving NEAT1, miR-34a-5p, and KLF4 in PAH progression in vitro. The MTT, Transwell, and western blot assays revealed that under hypoxia, transfection of miR-34a-5p inhibitor and pcDNA-KLF4 partly eliminated the inhibitory effects of sh-NEAT1-1 on the proliferation and migration of PASMCs (Fig. 5C-E; P<0.05). The role of the NEAT1/miR-34a-5p/KLF4 axis in PASMC phenotype conversion and apoptosis was then further explored. As revealed in Fig. 5F, both low miR-34a-5p expression and high KLF4 expression reversed the promoting effects of NEAT1 downregulation on α-SMA level and the ratio of c-caspase-3/t-caspase-3 (P<0.01).

Figure 5. Knockdown of NEAT1 slows the process of PAH via sponging miR-34a-5p and downregulating KLF4. (A) The protein level of KLF4 in hypoxia-induced PASMCs was determined by western blotting. **P<0.01 vs. the normoxia PASMC group. (B) The protein level of KLF4 in hypoxia-induced PASMCs after transfection of pcDNA-KLF4/NC was determined by western blotting. **P<0.01 vs. the pcDNA-NC group. (C) The viability (OD450) of hypoxia-induced PASMCs was measured by MTT assay. (D) The migration of hypoxia-induced PASMCs was measured by Transwell assay. Scale bar, 50 µm. (E) The protein levels of PCNA and MMP2 in hypoxia-induced PASMCs after transfection of sh-NEAT1-1/sh-NEAT1-1 + miR-34a-5p inhibitor/sh-NEAT1-1 + pcDNA-KLF4 were detected by western blotting. (F) The protein levels of α-SMA, and c-caspase-3/t-caspase-3 ratio in hypoxia-induced PASMCs after transfection of sh-NEAT1-1/sh-NEAT1-1 + miR-34a-5p inhibitor/sh-NEAT1-1 + pcDNA-KLF4 were detected by western blotting. **P<0.01 vs. the hypoxia + sh-NC group; #P<0.05, ##P<0.01 vs. the hypoxia + sh-NEAT1-1 group. NEAT1, nuclear paraspeckle assembly transcript 1; PAH, pulmonary arterial hypertension; KLF4, Krüppel-like factor 4; PASMCs, pulmonary arterial smooth muscle cells; NC, negative control; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide; sh-, short hairpin; miR-34a-5p, microRNA-34a-5p; c-, cleaved; t-, total.

NEAT1 expression is upregulated in the serum of patients with PAH

To investigate the roles of NEAT1, miR-34a-5p, and KLF4 in PAH, their expression levels were assessed in the serum of patients with PAH. As revealed in Fig. 6A-C, in comparison to healthy volunteers, the expression levels of NEAT1 and KLF4 were significantly increased in the serum of patients with PAH, whereas miR-34a-5p expression was decreased (P<0.01). Additionally, there was a significant negative correlation between miR-34a-5p and NEAT1 (Fig. 6D; P=0.0052, r=−0.5408) as well as between miR-34a-5p and KLF4 (Fig. 6E; P=0.0041, r=−0.5536), whereas a significant positive correlation was observed between NEAT1 and KLF4 (Fig. 6F; P=0.0077, r=0.5197).

Figure 6. Upregulation of NEAT1 is revealed in the serum of patients with PAH. (A) The expression of NEAT1 in the serum of PAH patients (n=25) and the serum of healthy volunteers (n=25) was detected by RT-qPCR. **P<0.01. (B) The expression of miR-34a-5p in the serum of PAH patients (n=25) and the serum of healthy volunteers (n=25) was detected by RT-qPCR. **P<0.01. (C) The expression of KLF4 in the serum of PAH patients (n=25) and the serum of healthy volunteers (n=25) was detected by RT-qPCR. **P<0.01. (D) The correlation between the expression of NEAT1 and miR-34a-5p in the serum of PAH patients. P=0.0052, r=−0.5408. (E) The correlation between the expression of miR-34a-5p and KLF4 in the serum of PAH patients. P=0.0041, r=−0.5536. (F) The correlation between the expression of NEAT1 and KLF4 in the serum of PAH patients. P=0.0077, r=0.5197. NEAT1, nuclear paraspeckle assembly transcript 1; PAH, pulmonary arterial hypertension; miR-34a-5p, microRNA-34a-5p; KLF4, Krüppel-like factor 4; RT-qPCR, reverse transcription-quantitative PCR.

Discussion

PAH is a cardiovascular disease with a high mortality rate that is characterised by aberrant function of PASMCs (18,30). Sun et al analysed a microarray of lncRNAs in a rat model of PAH, and revealed that several lncRNAs were overexpressed (31). In addition, numerous previous studies have revealed several lncRNAs that are highly expressed in PAH, such as HOXA-AS3 (7), MALAT1 (8) and TUG1 (9). Consistently, in the present study it was revealed that NEAT1 expression was increased not only in hypoxia-treated PASMCs but also in the serum of patients with PAH. This result suggested that NEAT1 may be a pathogenetic lncRNA involved in the progression of PAH.

sh-NEAT1 was transfected into hypoxia-treated PASMCs to explore the possible role of NEAT1 in PAH progression in vitro. Notably, it was revealed that under hypoxic conditions, downregulation of NEAT1 markedly inhibited the proliferation and migration of PASMCs. Our results are in line with data from previous studies showing that aberrantly expressed NEAT1 had significant effects on lung-related diseases (32–36). In addition, it has been revealed that downregulation of NEAT1 suppressed cell proliferation, migration, and invasion in lung cancer (32–34), and NEAT1 overexpression aggravated inflammation and decreased lung function in asthma (35) and chronic obstructive pulmonary disease (36). It was hypothesized that knockdown of NEAT1 may also inhibit hyperproliferation of the PA, thereby accelerating the progression of PAH. Transfection of si-NEAT1 was shown to inhibit the proliferation and migration of vascular smooth muscle cells (14). Given the inhibitory effects of NEAT1 silencing on the proliferation and migration of several types of human cancers (10–13), it was concluded that NEAT1 downregulation attenuates the progression of PAH in vitro.

Over the past decade, several researchers have identified miRNAs with decreased expression levels in a rat model of PAH and PASMCs, including miR-98 (16), miR-140-5p (16), miR-106b-5p (37) and miR-124-3p (8). Similarly, in the present study, a reduction in the expression of miR-34a-5p in hypoxia-treated PASMCs and PAH patient serum was also revealed. Aberrant expression (especially overexpression) of miR-34a-5p has been shown to inhibit the malignant behaviours of pulmonary cancers and acute lung injury (38,39), and high miR-34a-5p expression inhibited cell proliferation, migration, and invasion in lung adenocarcinoma (38) and non-small cell lung cancer (39). In the present study, it was revealed that cell viability was reduced by transfection with miR-34a-5p mimics. Data from a recent study conducted by Wang et al are in line with our results: That upregulation of miR-34a reduced the proliferation of PASMCs (40). It was further demonstrated that miR-34a-5p plays an important role in suppressing cell migration and decreasing PCNA and MMP2 protein levels. Binding of NEAT1 and miR-34a-5p was predicted and confirmed in the present study. Therefore, it was predicted that the effect of miR-34a-5p on hyperplasia of PASMCs is modulated by NEAT1. To verify this, it was assessed whether NEAT1 can interact with miR-34a-5p, and the results revealed that transfection of a miR-34a-5p inhibitor reversed the suppressive effects of sh-NEAT1 on cell proliferation and migration. It was also revealed that miR-34a-5p expression was inversely correlated with NEAT1 expression. These results indicated that NEAT1 knockdown alleviates the development of PAH by upregulating miR-34a.

Deletion of KLF4 in smooth muscle protects against vascular diseases, such as aortic aneurysm and atherosclerosis (41,42). Notably, expression of KLF4 was shown to be elevated in both PAH model rats and patients (43,44). Consistently, it was determined that KLF4 was not only highly expressed in the serum of PAH patients but also in hypoxia-treated PASMCs. Given the positive correlation between KLF4 and NEAT1 in the serum of PAH patients and the targeting relationship between KLF4 and miR-34a-5p, it was hypothesized that KLF4 is also involved in PAH. The feedback verification experiments demonstrated that in hypoxia-treated PASMCs, transfection of pcDNA-KLF4 markedly reversed the inhibitory effects of sh-NEAT1 on cell proliferation and migration, which supported our hypothesis. In conclusion, the present results indicated that NEAT1 silencing alleviates PAH by regulating miR-34a-5p/KLF4.

However, there may be some limitations in this study. First, the expression of NEAT1 was only determined in PAH patients, and further detection of NEAT1 expression in patients with remission of PAH may be more scientific. Second, this study only focused on the cellular level, and more in vivo experiments are required. These issues will be elucidated in future studies.

In summary, the present results revealed that NEAT1 expression was increased in the serum of PAH patients and hypoxia-treated PASMCs. NEAT1 silencing attenuated PAH progression by upregulating miR-34a-5p and targeting KLF4. Our data provide a potential novel target and biomarker for the treatment or diagnosis of PAH.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XD and YM designed and analysed the data obtained from the experiments and were major contributors in writing the manuscript. XD and YM confirm the authenticity of all the raw data. YQ and QD performed the experiments and helped draft the manuscript. SZ, RT, and MP assisted in the experiments and revision of the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of The People's Hospital of Rizhao (Rizhao, China). Written informed consent was obtained from all subjects.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References