Overexpressed lncRNA AC068039.4 contributes to proliferation and cell cycle progression of pulmonary artery smooth muscle cells via sponging miR-26a-5p/TRPC6 in hypoxic pulmonary arterial hypertension CURRENT STATUS: POSTED

Background: Hypoxic pulmonary hypertension (HPH) is a devastating and incurable disease characterized by pulmonary vascular remodeling, resulting to right heart failure and even death. Accumulated evidence has confirmed long coding RNAs (lncRNAs) are involved in hypoxia induced pulmonary vascular remodeling in HPH. The exact mechanism of lncRNA in hypoxic pulmonary hypertension remains unclear. Methods Microarray analysis was applied to investigate the profiles of lncRNA expression in pulmonary artery smooth muscle cells (PASMCs) cultured under hypoxia and normoxia condition. qRT-PCR was performed for the expression of lncRNAs, miRNA and mRNAs, western blot analysis was employed for detection the expression of proteins. CCK-8 and transwell chamber assay were applied for assessment of PASMC proliferation and migration, respectively. Besides, flow cytometry was performed for assessments of cell cycle progression. The binding between AC068039.4 and miR-26a-5p, miR-26a-5p and TRPC6 3’UTR were detected by dual luciferase reporter assay. Results A total of 1211 lncRNAs (698 up-regulated and 513 down-regulated) were differently expressed in hypoxia induced PASMCs. Consistent with microarray analysis, quantitative PCR verified that AC068039.4 was obviously up-regulated in hypoxia induced PASMCs. Knocking down AC068039.4 alleviated proliferation and migration of PASMCs and regulated cell cycle progression through inhibiting cells entering the G0/G1 cell cycle phase. Further experiment indicated AC068039.4 promoted hypoxic PASMCs proliferation via sponging miR-26-5p. In addition, transient receptor potential canonical 6 (TRPC6) was confirmed to be a target gene of miR-26a-5p. Conclusion: In conclusion, downregulation of lncRNA AC068039.4 inhibited pulmonary vascular remodeling through AC068039.4/miR-26a-5p/TRPC6 axis, providing new therapeutic insights for the treatment of HPH.


Introduction
Pulmonary hypertension (PH) is an incurable disease with complex pathogenesis and characterized by over-proliferation, apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs), pulmonary vascular remodeling (PVR) and elevated pulmonary vascular resistance, resulting in increased pulmonary arterial pressure, right heart failure and even death [1,2]. It is convincing that over-proliferation of PASMCs is the pathophysiological basis of PAR and plays a vital role in the occurrence and development of PH [3]. Hypoxic pulmonary hypertension (HPH) is a common cause of PH in multiple hypoxia and lung diseases, such as chronic obstructive pulmonary disease, interstitial lung disease, sleep disordered breathing and chronic exposure to high altitude [4]. Although clinical symptoms could be relieved under current treatments, no specifically effective treatments for HPH have been developed and disease progression remains inevitable [5]. Besides, no specific symptoms usually lead to delay in diagnosis and poor prognosis [6]. Therefore, further exploration of the pathogenesis of HPH is of great significance for finding novel effective treatments.
Based on the development of next-generation sequencing technology, a large amount of lncRNAs, longer than 200 nucleotides and incapable of protein transcription, have been identified exert their role in diverse biological process [7][8][9]. The "competitive endogenous RNA (ceRNA) hypothesis" indicates that lncRNA could sequestrate targeted miRNA and participate in miRNA regulation, indirectly involves in the regulation of miRNA-targeted genes [10]. The interaction between lncRNA and miRNA has been demonstrated in modulation of cell proliferation and apoptosis [11]. Previous studies have identified the role of several emerging lncRNAs, such as MEG3 [12], LnRPT [13], Tug1 [14], MALAT1 [15], Hoxaas3 [16], CASC2 [17], are involved in the regulation of PASMCs proliferation, cell cycle progression and apoptosis of PASMCs and modulation of PVR. However, the role of lncRNA in HPH remains largely unknown. The current microarray analysis identified a significantly expressed novel LncAC068039.4.
Previous study demonstrated that miR-26a was down-regulated in both plasma from PAH patients and lung tissues of monocrotaline (MCT)-induced rats [18]. Store-operated calcium channels (SOCE) induced store-operated calcium entry (SOCE) is the main cause of calcium imbalance in PASMCs under hypoxia, transient receptor potential canonical 6 (TRPC6) is the main component of SOCC, upregulated TRPC6 is related to PASMCs proliferation and PVR [19,20]. TRPC6 also involves in PASMCs phenotype from contractile phenotype to the proliferative phenotype [21]. Previous study demonstrated lncRNAs may participate in regulation of TRPC6 expression [22]. Therefore, the current research aims to explore the role of lncRNA AC068039.4 in regulation of pathogenesis of HPH. AC068039.4 was found up-regulated in hypoxia induced PASMCs, AC068039.4 interacted with and sponged miR-26a-5p, resulting to decreased degradation of TRPC6 and increased TRPC6 expression, ultimately contributing to over-proliferation of PASMCs. AC068039.4/miR-26a-5p/TRPC6 axis might provide a novel therapeutic target for HPH treatment.

Methods And Materials Cells culture and ethics statement
The Human PASMCs used in the experiment were isolated from the distal pulmonary arterioles in patients undergoing pulmonary lobectomy. This study was approved by the Medical Ethics Committee for Clinical Research of Zhongda Hospital (Nanjing, China). All subjects signed informed consent before the research. After removing the adventitia and intima of the pulmonary artery, the pulmonary artery smooth muscle was cut into small pieces of 1 × 1 mm 2 , and then transferred to a culture flask.
RNA interference, construction of the lentiviral vector and cell transfection Lentivirus encoding full length of AC068039.4 sequence, specific short hairpin RNA targeting AC068039.4 (sh-AC068039.4) tagged with Green fluorescent protein (GFP) and the negative control lentiviral vectors tagged with GFP were constructed by Genechem Co. (Shanghai, China).
Recombinant lentiviral vectors noncoding miR-26a-5p mimic, mimic negative control (NC), miR-26a-5p inhibitor and inhibitor NC were also designed and synthesized by Genechem Co. to regulate the expression of miR-26a-5p. The sequences for each were listed in Table 1. The PASMCs were cultured in 6-well plates 24 h before transfection with a cell density of 70-80% and PASMCs were transfected following the manufacturer's introductions. Transfection reagents were removed after 24 h, and the cells were further cultured in DMEM containing 5% FBS. 3-4 days after transfection, fluorescence expression was observed using fluorescence microscopy and transduction efficiency was assessed by GFP expression > 80%. PASMCs were collected used for subsequent experiments and shRNA with the best silencing effect tested by qPCR was used to perform the following experiment.   (ab53699), anti-Cyclin E1 (ab133266) and anti-ACTIN antibody (ab8226) were purchased from Abcam (Cambridge, UK), the next day the PVDF membrane was incubated with a secondary antibody (Beijing TDY Biotech Co., Ltd., Beijing, China) for 1 hour. An ECL detection kit (GE Healthcare, UK) was used for blots visualization using and bolts were exposed with X-ray film.
Dual luciferase reporter assay Statistical Analysis SPSS 22.0 software (SPSS, Chicago, IL, USA) was used for analysis. Data were expressed as mean ± standard deviation. Independent sample student's t test was used for the comparison between two groups, and one-way ANOVA followed by Dunnett 's test was performed to evaluate differences among groups. P < 0.05(*) was considered statistically significant, P < 0.01 (**) was regarded as highly significant difference. All experiments were performed at least three times.

Differentially expressed profiles of lncRNAs in normoxia and hypoxia induced PASMCs
As shown in the schematic diagram in Fig    was significantly downregulated compared to healthy controls. In addition, circulating miR-26a was identified as a novel biomarker candidate for PAH and closely correlated with 6-minute walk distance [18]. qPCR analysis was conducted to detect the expression of miR-21 in PASMCs and demonstrated that miR-26a-5p significantly decreased as the hypoxia exposure time increased (Fig. 4A). Therefore, we investigated the effect of AC068039.4 on miR-26a-5p in hypoxia. qPCR analysis showed reduced miR-26a-5p expression caused by hypoxia can be partially reversed after PASMCs was transfected with sh-AC068039.4 and miR-26a-5p was further downregulated in PASMCs transfected with OE-AC068039.4 compared with negative control, suggesting that miR-26a-5p could be regulated by AC068039.4 (Fig. 4B). Dual luciferase reporter gene assay was performed after co-  [25]. Figure 5A and 5B illustrated that both mRNA and protein level of TRPC6 significantly up-regulated in PASMCs exposed to hypoxia condition. As shown in Fig. 5C (Fig. 5D). Based on that AC068039.4 was involved in hypoxic PASMCs proliferation through interaction with miR-26a-5p, TRPC6 could be regulated by AC068039.4, we wonder whether miR-26a-5p exerted its role in regulation of hypoxia induced PASMCs proliferation through targeting TRPC6. Western blotting indicated the expression of TRPC6 protein decreased in PASMCs transfected with miR-26a-5p mimic compared with mimic NC, miR-26a-5p inhibitor could further up-regulated the expression of TRPC6 protein (Fig. 5E), which is consistent with qPCR (Fig. 5F). Dual luciferase report analysis was performed to confirm the interaction between miR-26a-5p and TRPC6. Bioinformatics prediction software showed that miR-425-5p had a potential binding site to the 3'-UTR of TRPC6 mRNA (Fig. 5G).

Discussion
In this present research, microarray analysis was performed to search for differentially expressed The neointimal and medial hypertrophy of the small and medium pulmonary arteries plays an essential role in PVR, mainly due to the excessive proliferation of PASMCs [26]. LncRNAs are endogenously RNAs lacking protein-encoding function with more than 200 nucleotide length, and were considered to be transcription junk before [8]. Multiple lncRNAs has been confirmed participating in the development of PH through the regulation of the proliferation of PASMCs and pulmonary vascular remodeling [27][28][29][30]. Benefit from the rapid development of next-generation sequencing technology, emerging lncRNAs have been discovered involved in the regulation of cardiovascular disease [31,32]. Gu [34]. Different from previous studies, in the current research, microarray analysis was conducted to find out the differently expressed lncRNAs in hypoxia and normoxia induced PASMCs. Significantly differential lncRNAs were selected for PCR verification, AC068039. LncRNAs function as miRNAs sponges or bait to isolate miRNAs through complementary base pairing, leading to the loss or reduction of miRNA function, further involves in post-transcriptional regulation [35]. CHRF was identified as endogenous sponge through downregulation of miR-489 to participate in the regulation of cardiac hypertrophy [36]. Furthermore, MALAT1 served as ceRNA through sponging miR-124-3p. 1 and promoted proliferation and migration of PASMCs. MEG3 and H19 acted as miRNA sponges of miR-328 and miR-675 respectively in PH [37,12]. In the current research,  [39]. SOCC is mainly composed of transient receptor potential canonical channel (TRPC) proteins [40], of which TRPC6 is a major protein constituting SOCC [41]. Previous research indicated that chronic hypoxia upregulate the expression of TRPC6 in pulmonary vein smooth muscle cells (PVSMCs) and [Ca 2+ ] i , and thereby promoted the proliferation and migration of PVSMC [19]. Ying Yu et al. demonstrated up-regulated TRPC6 expression promoted PASMCs proliferation isolated from IPAH patients [42]. Bosentan exerts a medicinal effect on PASMCs involves the downregulation of TRPC6 and inhibits the proliferation of PASMCs [43]. Gene sequencing revealed higher frequency of -254 (C→G) of single nucleotide polymorphism (SNP) in TRPC6 promoter region in idiopathic PAH patients [44]. TRPC6 expression was significantly increased in hypoxia induced PASMCs, but the upstream regulatory mechanism of TRPC6 remains largely unknown. microRNAs (miRNAs) are a class of highly conserved non-coding RNAs with 21 to 23 nucleotides, miRNAs exert their role through inhibition of downstream targeted mRNA or promotion of the degradation of targeted mRNAs via RNA interference [45]. miRNAs are essential in a variety of pathophysiological processes [46]. miR-26a is regarded as a tumor suppressor, it could inhibit lung cancer cell proliferation, migration and invasion [47]. The current research demonstrated knocking down AC068039.4 significantly down-regulated the expression of TRPC6 in PASMCs under hypoxia condition. Besides, PASMCs transfected with miR-26a-5p mimic could reverse the up-regulated TRPC6 induced by hypoxia, luciferase reports assay confirmed that miR-26a could bind to TRPC6 3'UTR, suggesting that miR-26a may be involved in the upstream regulation of TRPC6. Consistently, Schlosser K and his colleagues found circulating miR-26 decreased in plasma from PAH patients and monocrotaline (MCT)-induced PH rats, indicating that miR-26a may be a reliable potential biomarker of PH [18]. Interestingly, studies have shown that overexpression of miR-26a can inhibit TRPC6 expression and related downstream apoptotic pathway activation, thereby participating in reversing ox-LDL-induced apoptosis [48]. Besides, miR-26a was downregulated in experimental atherosclerosis model both in vivo and in vitro via targeting TRPC3 [49]. These data indicated that AC068039.4/miR-26a-5p/TRPC6 axis might participate in the development of hypoxic pulmonary hypertension.

Conclusion 15
In conclusion, the current research demonstrated that AC068039.4 is up-regulated in PASMCs induced by hypoxia, AC068039.4 promoted PASMCs proliferation, migration and cell cycle progression through competitively combining with miR-26a-5p through ceRNA pattern. Decreased free miR-26a-5p binded to the downstream target gene TRPC6, resulting in decreased degraded TRPC6 and increased TRPC6 expression and contributing to increased SOCE and [Ca 2 + ] i level, ultimately promoting the proliferation and migration of PASMCs, eventually contributing to pulmonary vascular remodeling.

Acknowledgements
We thanked all subjects who participated in this study.

Funding
This work was supported by National Natural Science Foundation of China under Grant No. 81600227 and 81970237.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions
YQ, BZ, GY, DW,YQ contributed to the conception and design of the work. YQ, BZ and LL performed the experiments, analyzed the data, and made the figures; YQ, BL, EL, JH analyzed the results; YQ drafted the paper. CT revised the work critically. All authors read and approved the final manuscript.

Ethics approval and consent to participate
This study was authorized by the Zhongda Hospital, and obtained written informed consents from all the participants.

Consent for publication
Not applicable.