Long non-coding RNA PVT1 promotes proliferation and migration of pulmonary smooth muscle cells in pulmonary arterial hypertension through miR-140-5p/Fxr1 axis


 Background: Pulmonary arterial hypertension (PAH) is a devastating disease and long non-coding RNAs (lncRNAs) are essential for PAH progression. Also, lncRNA plasmacytoma variant translocation 1 (PVT1) plays important roles in cell proliferation and migration, however, the effect of PVT1 on pulmonary artery smooth muscle cells (HPASMCs) in PAH remains unknown. This study aimed to determine the role and underlying mechanisms of PVT1 in HPASMCs.Methods: The results demonstrated that the expression of PVT1 was upregulated in pulmonary arterial tissues and HPASMCs and was positively correlated with pulmonary arterial pressure.Results: Knockdown and overexpression of PVT1 suppressed and promoted HPASMCs proliferation and migration, respectively. Mechanical and functional studies revealed that PVT1 functioned as a competing endogenous RNA of miR-140-5p and their expressions were negatively correlated.Conclusions: Knockdown of PVT1 did not suppress proliferation and migration in HPASMCs transfected with the miR-140-5p inhibitor. Moreover, fragile X-related proteins 1 (Fxr1) was identified as the target gene of miR-140-5p. Downregulation of PVT1 decreased the level of Fxr1 while this inhibitory effect was reversed by the miR-140-5p inhibitor. In conclusion, the upregulation of PVT1 promoted proliferation and migration in HPASMCs though the miR-140-5p/Fxr1 signaling pathway, indicating PVT1 may serve as a promising diagnostic biomarker for PAH.


Introduction
Pulmonary arterial hypertension (PAH) is a fatal disease lacking effective early diagnosis and treatments [1]. Also, PAH is characterized by aberrant functions and/or structures of the pulmonary artery smooth muscle cells (PASMCs), endothelial cells, vascular broblasts, and in ammatory cells, leading to vascular stiffening, vasoconstriction, and vascular lumen loss [2]. This devastating vascular remodeling results in pulmonary vascular resistance and promotes right ventricular failure and death [3]. Although PAH is a rare pulmonary disorder, PAH has been widely regarded as a severe and poor prognosis disease. Therefore, it is worthwhile to explore the mechanisms underlying the pathological progression of PAH, particularly in vascular remodeling, which would facilitate the development of novel diagnostic and treatment strategies for PAH.
In PAH, pulmonary vessel wall remodeling, vasoconstriction, and thrombosis causes elevated pulmonary vascular resistance and narrowing [4]. Pulmonary vascular remodeling is a complicated process associated with multiple layers of the vessel wall and cellular heterogeneity in the pulmonary arterial wall [5]. In general, all types of PAH remodeling are characterized by the presence of a layer of smooth muscle cells in small peripheral, nonmuscular pulmonary arteries within the respiratory acinus [6]. Accordingly, such aberrant smooth muscle cell proliferation ultimately results in chronic obstruction of the small pulmonary arteries [7]. In addition, it has been demonstrated that vascular remodeling is the consequence of an imbalance between apoptosis and proliferation in smooth muscle cells, manifesting upregulated proliferation [8]. Collectively, the cellular activity of smooth muscles is tightly associated with pulmonary vascular remodeling and the progression of PAH.
Long non-coding RNAs (lncRNAs) is de ned as a group of non-protein-coding RNAs over 200 nucleotides in length [9]. It has been well-documented that lncRNAs play essential roles in gene expression through multiple mechanisms, such as transcription modulation, post-transcriptional processing, and chromosome remodeling [10]; thus, lncRNAs are crucial regulators for diverse molecular and cellular activities, impacting many physiological and pathological processes [11]. The lncRNA plasmacytoma variant translocation 1 (PVT1) is encoded by the human PVT1 gene and is involved in several important molecular mechanisms, including encoding microRNAs, regulating transcription factor myelocytomatosis (MYC), and DNA rearrangement [12]. In addition, the aberrant expression of PVT1 is a promising marker for predicting progression and prognosis of several tumor types [13], such as, gallbladder cancer [14], colorectal cancer [15], and lung cancer [16,17]. Furthermore, PVT1 promotes angiogenesis of vascular endothelial cells by in uencing cell proliferation and migration [18]. Together, PVT1 is an important regulator contributing to various cellular activities, however, its role in pulmonary vascular remodeling, to date, has not been reported. Therefore, the objective of this study was to determine the role of PVT1 in proliferation and migration of human PASMCs (HPASMCs) in PAH and its related mechanisms. The observations of this study may provide novel therapeutic biomarkers for predicting the progression and prognosis of PAH.

Patient and sample information
In the study, all patients were informed before their inclusion and written consents of patients were obtained. All experiment protocols were approved by the Ethics Committee of Cangzhou Central Hospital (Cangzhou, China) and experimental procedures were conducted according to the Declaration of Helsinki Principles [19]. Fourteen patients (22- [20]. Patients with the following conditions were excluded from this study, including psychosis, severe obstructive pulmonary disease, chronic liver disease, chronic kidney disease, amyloidosis, portal hypertension, drug addiction history and so on. Meanwhile, patients who had taken prostacyclin, L-arginine, sildena l, and endothelin receptor antagonist were also excluded. In this study, healthy lung tissue samples were donored by patients who were not suitable for transplantation. The inclusion criteria for the donors of healthy lung tissues were as follows: <55 years old; <20 packs of cigarattes consumption per year; no chest trauma; no sustained mechanical ventilation; positive end-expiratory pressure (PEEP), 5 cm; the fraction of inspired oxygen (FiO2), 1.0; partial pressure of oxygen (PaO2), > 300 mmHg; clear lung elds shown in the chest lms; and a clear trachea conformed by bronchoscopy.

Preparation of lung tissues and HPASMCs
The PAH lung tissues were collected from patients who had received lung transplantations at Cangzhou Central Hospital (Cangzhou, China). Healthy lung tissues were collected from participants who were not suitable for lung transplantation and were used as the control in this study. The pulmonary artery (PA) tissues preparation procedure was conducted as previously described [21,22]. Brie y, a small piece of distal PA (< 1,000 µm diameter) were microdissected from explanted PAH from 4 patients and healthy lung tissues were collected from 3 health donors. PAs were rst digested in Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scienti c, Shanghai, China) supplemeted with collagenase (type II) and deoxyribonuclease (DNase). After isolating epithelial layer, the smooth muscles were further diested in Hematoxylin and eosin (H&E) staining The histopathological features of lung tissue samples were assessed by H&E staining. Brie y, lung tissues were sectioned into blocks (5 µm thickness) and xed in 10% (w/v) neutral-buffered formalin at 4 °C overnight. Next, tissue sections were dehydrated with a graded ethanol series, cleaned, and embedded in para n. Sections were stained with hematoxylin for 10 minutes and then eosin for 5 minutes. Lung tissue slices were imaged by light microscopy.

Immuno uorescence assay
The purity of HPASMCs was assessed by immuno uorescence assays which detect the expression of the smooth muscle myosin heavy chain. The HPASMCs were placed on glass cover slips and xed with 4% polyformaldehyde for 15 minutes. Slices were then incubated with smooth muscle myosin heavy chain (SM-MHC) antibody (1:400; Abcam, Cambridge, USA). Slices were imaged by confocal laser scanning microscopy (Leica, Solms, Germany).

Real-time PCR
Total RNA was extracted from lung tissues and HPASMCs using the TRIzol reagent (Invitrogen, CA, USA). First strand cDNAs were synthesized using the M-MLV Reverse Transcriptase (RNase H) kit (GeneCopoeia, Rockville, USA). Real-time PCR was performed using the ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, USA) with recommended reaction conditions in the manufacturer's instructions. The primers were synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China). The ampli cation e ciency of primers was validated before use and the e ciency were 95-105%. Data were analyzed using the 2 −ΔΔCt method [23]. GAPDH and U6 were used as reference genes.

CCK-8 assay
Wound-healing migration assay After transfection, HPASMCs were subjected to the wound-healing assay and the detailed procedure was previously described [24].

Statistical analysis
Data were presented as mean ± standard error of mean. Statistical analysis was performed using SPSS v. 19.0 software (SPSS, IL, USA). The correlations between PVT1 expression and clinicopathological features, PVT1 expression, and miR-140-5p level were analyzed with the Pearson's correlation. Differences between groups were analyzed with Student's t-test or one-way ANOVA. Differences were regarded to be statistically signi cant at P < 0.05. In this study, each treatment group consisted of 3-5 replicates.

Upregulation of PVT1 in PA tissues and HPASMCs
In this study, the vascular wall morphology of healthy and PAH lung tissues was assessed. The H&E staining assay revealed that the vascular wall thickness of PAH patients was larger than those of healthy individuals ( Fig. 1.A). The immuno uorescence assay was used to verify the purity of HPASMCs by detecting the smooth muscle myosin heavy chain ( Fig. 1.B). Moreover, PVT1 exerts essential roles in various tumor types, promoting proliferation and inhibiting apoptosis [12]. Some common cellular properties, such as high proliferative levels, are observed in both HPASMCs and tumor cells [22,23]. Therefore, PVT1 may also participate in the progression of PAH, particularly vascular remodeling. To verify this hypothesis, the expression of PVT1 in both PA tissue samples and HPASMCs were detected.
The results showed that the level of PVT1 was higher in PA tissue samples ( Fig. 1.C) and HPASMCs ( Fig. 1.D) compared with those in the control group. As one of primary pathological features of PAH, the pulmonary artery pressure of PAH patients was signi cantly higher than those of healthy participants ( Fig. 1.E). Furthermore, the expression of PVT1 was positively correlated with pulmonary artery pressure ( Fig. 1.F), suggesting that PVT1 may serve as a diagnostic marker for PAH. Taken together, upregulated expression of PVT1 may be involved in the development and progression of PAH.
PVT1 modulated the viability, proliferation, and migration of HPASMCs To further determine the roles of PVT1 in HPASMCs, sh-PVT1 and pcDNA-PVT1 were applied to knockdown and overexpress PVT1 in HPASMCs, respectively. The transfection e ciencies were assessed using real-time PCR (Fig. 2.A and Fig. 3.A). In CCK-8 assays, HPASMCs transfected sh-PVT1 displayed lower cell viability whereas the higher cell viability was found in HPASMCs transfected with pcDNA-PVT1 compared to the control group (Fig. 2.B and Fig. 3.B). In addition, the ow cytometry assay revealed that downregulation and upregulation of PVT1 decreased and increased the proportion of cells in the S and G 2 phases, respectively (Fig. 2.C and Fig. 3.C). To con rm the role of PVT1 in cell proliferation, protein expressions of several cell proliferation protein markers were detected, including PCNA, Cyclin A1, Cyclin D1, and Cyclin E1 [27,28]. The results showed that reduced PVT1 level was associated with decreased expressions of PCNA, Cyclin A1, Cyclin D1, and Cyclin E1 while elevated PVT1 exerted opposite roles to these cell proliferation markers compared to the control group (Fig. 2.D and Fig. 3.D). Furthermore, using the wound-healing migration assay, the knockdown and overexpression of PVT1 inhibited and promoted HPASMCs migration ability, respectively ( Fig. 2.E and Fig. 3.E). Collectively, PVT1 plays important regulator roles in HPASMCs viability, proliferation, and migration, which suggests that there is an essential involvement of PVT1 in PAH progression.

PVT1 regulated HPASMCs activities through sponging miR-140-5p
To further determine the mechanisms underlying the effect of PVT1 in PAH, the miRNA associated with the function of PVT1 in the progression of PAH was identi ed. Bioinformatics analysis revealed that a putative binding site of miR-140-5p was found in the 3′UTR of PVT1 (Fig. 4. H). Thus, the expression of miR-140-5p by real-time PCR was detected and the results showed that miR-140-5p was downregulated in PA tissues and HPASMCs (Fig. 4.A and B). Also, the downregulation and upregulation of PVT1 increased and decreased the expression of miR-140-5p in HPASMCs, respectively (Fig. 4.C and D). The miR-140-5p mimic and inhibitor inhibited and elevated the level of PVT1, respectively (Fig. 4. E). The expression of PVT1 was negatively correlated with miR-140-5p expression (Fig. 4. F). These results together revealed that PVT1 may act as a competing endogenous RNA (ceRNA) to miR-140-5p. To further verify the interaction between PVT1 and miR-140-5p, luciferase reporter assays showed that HPASMCs cotransfected with the wide-type PVT1 plasmid and miR-140-5p mimic reduced the luciferase activity while the luciferase activity was not affected by co-transfection of the mutant PVT1 plasmid (Fig. 4.I).
Moreover, the physical interaction between PVT1 and miR-140-5p was also found in the RIP assay where both PVT1 and miR-140-5p were enriched in AGO2 immunoprecipitates compared to the control (Fig. 4.J). Thus, these results suggest that PVT1 functions as a ceRNA for miR-140-5p in HPASMCs.
MiR-140-5p directly targeted Fxr1 in PA tissues and HPASMCs To determine the downstream regulator of miR-140-5p in PAH, bioinformatics analysis was applied to predict the putative target gene of miR-140-5p. The results showed that a binding site of miR-140-5p was observed in 3′UTR of Fxr1 (Fig. 5.G). Subsequently, the luciferase reporter assay con rmed this prediction, where HPASMCs co-transfected with wild-type plasmid of Fxr1 and miR-140-5p mimic had reduced luciferase activity while the luciferase activity did not alter in HPASMCs transfected with the mutant Fxr1containing plasmid (Fig. 5.H). Also, functional studies demonstrated that the miR-140-5p mimic and inhibitor inhibited and enhanced the protein expression of Fxr1 in HPASMCs, respectively (Fig. 5. A and  B). The transfection e cacies of the miR-140-5p mimic and inhibitor were veri ed by real-time PCR ( Fig. 4.G). To determine the role of Fxr1 in PAH, the Fxr1 mRNA and protein expressions were detected in PA tissues and HPASMCs which demonstrated that Fxr1 was signi cantly upregulated ( Fig. 5. C-F). Together, Fxr1 may be a target gene of miR-140-5p and may be involved in PAH progression.

PVT1/ miR-140-5p/Fxr1 signaling axis regulated PAH progression
Based on the observations, PVT1, miR-140-5p, and Fxr1 may form a signaling axis to regulate PAH progression. Thus, mRNA and protein levels of PVT1 in sh-PVT1-trasnfected HPASMCs were detected which showed that PVT1 was downregulated at both mRNA and protein levels (Fig. 6.A and B).
Furthermore, sh-PVT1-induced downregulation of PVT1 was enhanced and reversed by the miR-140-5p mimic and inhibitor, respectively ( Fig. 6.C and D). Functionally, sh-PVT1-induced inhibitory effect on HPASMCs proliferation and migration was enhanced and offset by miR-140-5p mimic and inhibitor, respectively (Fig. 7.A and B). The similar effects of the miR-140-5p mimic and inhibitor on cell proliferation was found in sh-PVT1-treated HPASMCs, as detected by the ow cytometer ( Fig. 7.C).
Western blots revealed that cell proliferation marker expressions were decreased in HPASMCs treated with the sh-PVT1 and miR-140-5p mimic whereas HPASMCs transfected with the sh-PVT1 and miR-140-5p inhibitor did not alter (Fig. 7.D). Collectively, the PVT1/ miR-140-5p/Fxr1 signaling axis may be essential for the regulation of cell proliferation and migration in HPASMCs.

Discussion
Pulmonary arterial hypertension (PAH) is de ned as a devastating disease of the small pulmonary arteries manifesting aberrant vascular proliferation and remodeling [29,30]. Pulmonary vascular remodeling, along with vasoconstriction and thrombosis in situ, are three primary factors leading to the elveated pulmonary vascular resistance [4]. As a complex pathological process, pulmonary vascular remodeling is characterized by the disrupted HPASMC proliferation and migration, as well as the inhibited apoptosis. Therefore, exploring the molecular and cellular mechanism underlying HPASMC proliferation and apoptosis in PAH would greatly contribute to the understanding of PAH pathology. In the present study, the results demonstrated that the expression of PVT1 was increased in PA tissues and HPASMCs of patients with PAH and its expression was positively correlated with pulmonary artery pressure. In addition, the knockdown and overexpression of PVT1 inhibited and promoted HPASMC proliferation and migration, respectively. Furthermore, bioinformatics analysis and functional studies showed that the effect of PVT1 on HPASMC cellular activities in PAH may be mediated by the miR-140-5p/Fxr1 signaling axis.
The lncRNA PVT1 is 1716 nucleotides in length and is encoded by an oncogene PVT1 [31]. It has been reported that PVT1 exerts essential roles in various types of tumors, including hepatocellular carcinoma [32], malignant pleural mesothelioma [33], bladder cancer [34], and gastric cancer [13], where PVT1 suppresses apoptosis while promoting cell proliferation and migration, eventually facilitating cancer progression [12]. Since PASMCs and cancer cells share some common cellular properties, such as hypermetabolism, aberrant mitochondrial function, and high proliferative activity [35,36], PVT1 may also function as a stimulator for PASMC proliferation and migration. In this study, PVT1 was upregulated in both PA tissues and HPASMCs. Functional studies revealed that overexpression of PVT1 signi cantly elevated viability and proliferation of HPASMCs. The results are consistent with the hypothesis that PVT1 promotes pulmonary vascular remodeling by enhancing proliferation and inhibiting apoptosis, as its roles in several tumor types [12]. However, since the effect of PVT1 displays the MYC copy-number-dependent pattern, the mechanism underlying its effect may vary in each pathological process [37].
In addition to its role in cellular activities, this study found that the expression of PVT1 was positively correlated with pulmonary artery pressure, indicating PVT1 may be a promising diagnostic biomarker for patients with PAH. Similarly, PVT1 has been identi ed as an ideal prognostic biomarker in several cancer types [32,38,39], in which the upregulation of PVT1 is signi cantly correlated with poor overall survival, distant metastasis, and poor differentiation grade [19]. Therefore, more studies are needed to further determine the predictive value of PVT1 in PAH.
It has been well-documented that lncRNAs can function as ceRNA to regulate the expression of microRNAs (miRNAs), primarily through sponging and silencing miRNAs [40,41]. Therefore, to further determine the mechanism underlying the effect of PVT1 on PAH, bioinformatics analysis was performed to screen the putative miRNAs interacting with PVT1. Combining with the mechanic and functional studies, the results revealed that the expression of PVT1 was negatively correlated with the level of miR-140-5p and their physical and functional interactions played important roles in the regulation of PAH progression, suggesting that PVT1 may act as a ceRNA of miR-140-5p in PAH. As a multifunctional factor, miR-140-5p exerts essential roles in various pathological processes, such as osteogenesis [42], epithelial mesenchymal transition [43], and autophagy [44]. A comprehensive bioinformatics analysis also demonstrated that miR-140-5p was associated with 23 target genes and seven signaling pathways in multiple biological processes, such as signal transduction and cell proliferation [45]. The expression of miR-140-5p is downregulated in PAH and the suppression of miR-140-5p facilitates proliferation and migration in HPASMCs [46], which agrees with the observation in the present study. As such, like PVT1, miR-140-5p may also serve as a potential biomarker for PAH.
In this study, the results also demonstrated that Fxr1 was upregulated in both PA tissues and HPASMCs and its effect on vascular remodeling was opposite to those of miR-140-5p. These results together indicated that miR-140-5p may act as a sponge of Fxr1 in PAH, which was con rmed by bioinformatics analysis and luciferase reporter assays. The Fxr1 is an RNA-binding protein and its sequence is highly conservative across many species [47,48]. Like the roles of RNA-binding proteins, Fxr1 is associated with gene translation, mRNA transport, and mRNA binding through G4 RNA structures or AU-rich elements [48,49]. On the basis of modulation of cellular activities, Fxr1 participates in the regulation of p21 expression via p53-dependent patterns to regulate cell proliferation in the head and neck squamous cell carcinoma and non-small cell lung cancer cells [50]. Also, Fxr1 functions as an oncogene promoting proliferation, invasion, as well as migration in cancer cells [51]. Together, Fxr1 may be an important regulator of cell proliferation and migration.
In this study, we have demonstrated that the important role of PVT1 in the HPASMCs of PAH. However, some limitations in this study should not be ignored. For example, the animal experiment should be perfomed to validate the effect of PVT1 in vivo. Also, the pathogenesis of PAH is heterogeneous [52,53] and there are several subtypes of PAH, such as idiopathic PAH (IPAH), heritable PAH (HPAH), and associated PAH (APAH) [54]. Of which, we only included HPASMCs of HPAH in the present study. Thus, more subtype of PAH should be covered in future study.
In conclusion, PVT1 was upregulated in PA tissues and HPASMCs and the expression of PVT1 was positively correlated with pulmonary artery pressure. Also, PVT1 could promote proliferation while inhibiting apoptosis in HPASMCs though the miR-140-5p/Fxr1 signaling axis. Understanding the molecular mechnisms of PVT1 in PAH may provide novel insight into developing therapy for PAH.

Conclusions
In conclusion, PVT1 was upregulated in PA tissues and HPASMCs and the expression of PVT1 was positively correlated with pulmonary artery pressure. Also, PVT1 could promote proliferation while inhibiting apoptosis in HPASMCs though the miR-140-5p/Fxr1 signaling axis. Understanding the molecular mechnisms of PVT1 in PAH may provide novel insight into developing therapy for PAH.