MicroRNA-222 Promotes the Proliferation of Pulmonary Arterial Smooth Muscle Cells by Targeting P27 and TIMP3

Pulmonary arterial hypertension (PAH), a progressive disorder characterized by pulmonary vascular remodeling, can lead to an increase in pulmonary vascular resistance, right heart failure, and ultimately death [1, 2]. Excessive pulmonary artery smooth muscle cell (PASMC) proliferation is one of the most prominent features of PAH, which can lead to the narrowing or occlusion of pulmonary vessels and therefore plays an important role in the occurrence and development of PAH [3].

MicroRNAs (miRNAs, miRs) are a class of endogenous, small, non-coding RNAs that can regulate gene expressions by targeting the 3’untranslational regions (UTRs) of mRNAs [4, 5]. Being the central regulators of gene expressions, miRNAs participate in many essential biological processes, such as cell metabolism, development, proliferation, and death [6-8]. Of note, miRNAs have also been implicated in the development of PAH [9-11]. In fact, multiple miRNAs such as miR-21, -17, -30c, -125a, -126, -143, -145, -206, -130/301, and -328 have been identified to be responsible for vascular remodeling in PAH [12-24]. miR-222 has been reported to promote cardiomyocyte hypertrophy and proliferation in exercise-induced cardiac growth [25]. However, chronic cardiac-specific overexpression of miR-222 might lead to cardiac remodeling and heart failure [26]. miR-222 also plays important roles in multiple cancer types [27, 28]. Moreover, miR-222 was found to regulate essential pathophysiological vascular processes [29]. For example, miR-222 has a pro-proliferation effect on vascular smooth muscle cells (VSMCs) [30], while it has an anti-proliferation effect on vascular endothelial cells (ECs) [31]. As the biological function of a single miRNA could be cell-type specific, the role of miR-222 in PASMC proliferation is not clear and deserves to be explored.

This study was approved by the ethical committees of the Nanjing Medical University and all animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by National Institutes of Health (No. 85-23, revised 1996).

The primary PASMCs were isolated from the pulmonary arteries of male Sprague-Dawley rats (5 weeks old) by using a tissue-sticking method. After the dissection of adventitia and endothelia, pulmonary arteries were quickly cut into small pieces and stuck to cell culture bottle. The primary PASMCs were confirmed by immunofluorescent stainings for SM α-actin (α-SMA) and Desmin (Sigma-Aldrich, St. Louis, MO, USA). The basic culture medium was consisted of DMEM-F12 supplemented with 5% fetal bovine serum (FBS) while the starvation medium was with 1% FBS. The primary PASMCs between passage 3 and 6 were used for experiments.

Hypoxic condition was created in a hypoxic (1% O₂ and 5% CO₂, 37°C) incubator (Billups-Rothenberg, INC. California, United States). Experiments were performed after 24 hours of hypoxic incubation, at which stage cell lysate and total RNA were collected.

Before transfection, PASMCs were starved for 8 hours for cycle synchronization. Lipofectamine 2000 reagent was used to transfect miR-222 mimic (50 nM), miR-222 inhibitor (100 nM), and their negative control (NC) into the different groups according to the instructions. siRNA-P27(Kip1) and/or siRNA-TIMP3 were transfected to PASMCs to knock-down P27 and/or TIMP3. After incubation for 48 hours, the effect of siRNAs was confirmed by real-time polymerase chain reactions (PCRs).

P27 siRNA sequence: sense (5'-3'): GCGGCAGAAGAUUCUUCUUTT, anti-sense (5'-3'): AAGAAGAAUCUUCUGCCGCTT.

TIMP3 siRNA sequence: sense (5'-3'): GCUAUCAGUCCAAACACUATT, anti-sense (5'-3'): UAGUGUUUGGACUGAUAGCTT.

NC siRNA sequence: sense (5'-3'): UUCUCCGAACGUGUCACGUTT, anti-sense (5'-3'): ACGUGACACGUUCGGAGAATT.

Briefly, total RNA was extracted from cells with miRNeasy Mini Kit (Qiagen, Hilden, Germany). For the evaluation of miR-222 level, mature miRNA was reverse transcribed with Bulge-Loop^TM miRNA qPCR Primers (Ribobio, Guangzhou, China) prior to qPCR according to the manufacturer's instructions. qRT-PCR for miR-222 cDNA synthesis was performed with Bio-Rad iScript^TM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). For quantitative miRNA analysis, a template equivalent to 400 ng of total RNA was subjected to 40 cycles of quantitative PCR using the Takara SYBR Premix Ex TaqTM (Tli RNaseH Plus, Takara, Tokyo, Japan) in the 7900HT Fast Real-Time PCR System.

For immunofluorescence, PASMCs were determined by SM α-actin (α-SMA) and Desmin. The apoptosis was examined by TdT-mediated dUTP nick end labeling (TUNEL) assay using In Situ Cell Death Detection Kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). The proliferation of PASMCs was determined by 5-ethynyl-2’-deoxyuridine (EdU) assay, Ki-67 staining, and cell number counting. For EdU assay, EdU was added to the culture medium for 8 hours in order to incorporate into replicating cells’ DNA. Then cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. 0.2% Triton X-100 was used to permeabilize the nuclear membrane and PBS containing 10% goat serum was used for blocking for 1 hour at room temperature. Ultimately, PASMCs were stained by Cell-Light^TM EdU Apollo®488 In Vitro Imaging Kit (Life Technologies, New York, USA) according to the instructions. For Ki-67 staining, after fixation, permeabilization, and blocking, PASMCs were incubated with Ki-67 antibody (1: 500 dilution; Abcam, Cambridge, MA, USA) at 4°C overnight and then stained with Alexa Fluor 488 goat anti-rabbit IgG antibody for 2 hours at room temperature. For cell number counting, at least 200 cells or 10 images were quantified in each well to get accurate numbers for each group. Nuclei were stained with DAPI. Finally, cells were detected with a fluorescence microscope.

The total protein was extracted from lysed PASMCs. After determined by BCA protein assays, equal quantities of protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the PVDF membranes. The membranes were blocked by Tris-buffered saline Tween-20 (TBST) containing 5% BSA for 2 hours at room temperature and then incubated on a rotary shaker (80 rpm/min) with antibodies against B-cell lymphoma 2 (Bcl-2, 1: 1000 dilution; Cell Signaling Technology, Boston, Massachusetts, USA), Bcl-2-associated X protein (Bax, 1: 1000 dilution; Cell Signaling Technology, Boston, Massachusetts, USA), Proliferating Cell Nuclear Antigen (PCNA, 1: 1000 dilution; Cell Signaling Technology, Boston, Massachusetts, USA), P27 (1: 1000 dilution; Abcam, Cambridge, UK), Tissue Inhibitor of Metalloproteinase 3 (TIMP3, 1: 1000 dilution, Cell Signaling Technology, Boston, Massachusetts, USA), respectively. β-actin (1: 1000 dilution; Cell Signaling Technology, Boston, Massachusetts, USA) and glyceraldehyde-3-phosphate dehydrogenase (1: 1000 dilution; Cell Signaling Technology, Boston, Massachusetts, USA) were used as a loading control. After incubation with the HRP-conjugated rabbit anti-goat IgG at room temperature for 1 hour, the protein bands were quantified with ECL Plus Western blotting detection reagents (Bio-Rad) and ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA).

A fragment of the 3’-UTR of TIMP3 mRNA containing the putative miR-222 binding sequences was cloned into the firefly luciferase reporter construct pmiR-RB-Report (Ribobio, Guangzhou, China). For the reporter assay, human embryonic kidney 293T cells (HEK293T cells) were seeded onto 96-well plates and co-transfected with TIMP3-3’UTR-Luc (1 µg) and miR-222 mimic (50 nM) by Lipofectamine 2000. In addition, the construct with mutated fragment of the 3′-UTR of TIMP3 without putative miR-222 binding sequences was used as a mutated control. Following 48 hour incubation, luciferase activity was measured on a scintillation counter by a dual luciferase reporter system (GeneCopoeia^TM, Luc-Pair^TM Duo-Luciferase Assay kit 2.0, USA).

All data were expressed as mean ± SE. For relative gene expression, proliferation rate, and cell counting, the mean value of negative control group was defined as 100% or 1.0. Two group samples were compared by an independent-samples T test. Three or more group samples were compared by one-way ANOVA followed by Bonferroni’s post-hoc test. A P value < 0.05 was considered to be statistically significant.

The primary PASMCs were specified by immunofluorescent stainings for α-SMA and Desmin (Fig. 1A). To explore the effect of miR-222 on PASMC proliferation, three different methods were used including EdU assay, Ki-67 staining, and cell number counting. As shown in Fig. 1B, miR-222 mimic increased whereas miR-222 inhibitor decreased the expression level of miR-222 in cultured PASMCs 48 hours after transfection. In addition, miR-222 mimic up-regulated PCNA expression while miR-222 inhibitor down-regulated PCNA expression (Fig. 1C). The Bax/Bcl2 ratio and PASMC apoptosis were not significantly influenced after transfection with miR-222 mimic compared with the negative control mimic (Fig. 1D and E). In accordance with the Western blot result of PCNA, miR-222 mimic increased the percentage of EdU positive cells, Ki-67 positive cells, and the total number of PASMCs (Fig. 1F-H). In contrast, miR-222 inhibitor decreased PASMC proliferation (Fig. 1F-H). These results suggested that miR-222 had a pro-proliferation effect on PASMCs.

P27 is the most well-known target gene of miR-222 [32, 33]. To determine whether P27 was putative target gene of miR-222 in PASMCs, we detected the P27 protein level by Western blot after up- or down-regulation of miR-222. As expected, P27 was up-regulated by miR-222 inhibitor and down-regulated by miR-222 mimic (Fig. 2A). These data indicate that P27 is a target gene of miR-222 in PASMCs.

The effect of P27 siRNA used in the study was confirmed by Western blot (Fig. 2B). We found that P27 knockdown by P27 siRNA could significantly increase the proliferation of PASMCs as determined by Ki-67 staining, EdU staining, and cell number counting, while co-transfection of miR-222 mimic and P27 siRNA did not further increase the proliferation of PASMCs (Fig. 2C-E), indicating that P27 inhibition might mediate the pro-proliferation effect of miR-222 in PASMCs. Besides that, P27 siRNA could attenuate the anti-proliferation effect of miR-222 inhibitor in PASMCs (Fig. 2F-H), supporting that P27 is at least partially responsible for the regulatory effect of miR-222 on PASMC proliferation.

TIMP3 has also been reported to be a target gene of miR-222 in several types of cells [34, 35]. However, if TIMP3 is a target gene of miR-222 in PASMCs remains undetermined. We cloned the 3’UTR of TIMP3 mRNA containing the putative miR-222 binding sequences into the firefly luciferase reporter construct pmiR-RB-Report. Luciferase assay showed that exogenous miR-222 led to the reduction of luciferase activity in cells transfected with the construct with 3’UTR of TIMP3, but had no effect when the putative miR-222 binding site in TIMP3 was mutated (Fig. 3A and B). This suggests that TIMP3 is a direct target of miR-222. Besides that, we also determined the expression level of TIMP3 in the presence of miR-222 mimic or inhibitor and found that miR-222 could negatively regulate TIMP3 protein level in PASMCs (Fig. 3C).

The effect of TIMP3 siRNA used in this study was confirmed by Western blot (Fig. 4A). TIMP3 knockdown by siRNA could significantly enhance the proliferation of PASMCs as determined by Ki-67 staining, EdU staining, and cell number counting, while co-transfection of miR-222 mimic and TIMP3 siRNA did not further enhance the proliferation of PASMCs (Fig. 4B-D), indicating that TIMP3 inhibition might also be responsible for the pro-proliferation effect of miR-222 in PASMCs. Moreover, TIMP3 siRNA could attenuate the anti-proliferation effect of miR-222 inhibitor in PASMCs (Fig. 4E-G), supporting that TIMP3 at least in part mediates the regulatory effect of miR-222 on PASMC proliferation.

To further clarify if concomitant silencing of P27 and TIMP3 has additive effect on PASMC proliferation, the P27 and TIMP3 were simultaneously knocked-down by co-transfection of P27 siRNA and TIMP3 siRNA. The data showed that co-silencing of P27 and TIMP3 did not have additive effect on PASMC proliferation (Fig. 5).

To reveal the physiological relevance of miR-222 for PASMC proliferation and eventually PAH, experiments under hypoxic condition were performed. The data showed that miR-222 was upregulated in PASMCs under hypoxic condition for 24 hours (Fig. 6A). Hypoxia promoted PASMC proliferation which was attenuated by miR-222 inhibitor (Fig. 6B and C). In addition, P27 and TIMP3 were both downregulated in PASMCs under hypoxia, which could be reversed by miR-222 inhibitor (Fig. 6D).

It is well known that aberrant vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of many proliferative cardiovascular diseases, such as atherosclerosis, vein graft failure, restenosis after angioplasty, as well as PAH [1, 3]. Inhibition of VSMC proliferation could lead to better prevention and treatment of these diseases.

Increasing evidence shows that miRNAs play pivotal roles in many biological activities, including VSMC proliferation. It has been reported that miR-222 could promote proliferation and migration, while inhibit apoptosis of aortic SMCs [31]. However, the role of miR-222 in PASMCs is unclear. In this study, we demonstrate that miR-222 has a pro-proliferation effect on SMCs derived from pulmonary artery as determined by PCNA expression, Ki-67 and EdU staining, and total cell number counting.

A single miRNA can target several target genes while a single gene can also be regulated by several miRNAs. P27 is a well-known target gene of miR-222 in several types of cells including VSMCs. As a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, P27 has been found to negatively regulate cell proliferation including cancer cells and VSMCs [36]. In the present study, we found a negative regulation of P27 by miR-222 in PASMCs and P27 inhibition was responsible for the pro-proliferation effect of miR-222 in PASMCs. In addition, TIMP3 is a newly found target gene of miR-222 [28, 35]. As a member of the TIMP family, TIMP3 can regulate many physiological effects including cell proliferation, apoptosis, and migration through MMP-dependent or MMP-independent manner [34, 35]. It has been proved that miR-222 could induce neuronal apoptosis by targeting TIMP3 in vitro [34]. However, if TIMP3 is a target gene of miR-222 in PASMCs has not been determined. In the current study, we identified that TIMP3 was a target gene of miR-222 in PASMCs, as confirmed by luciferase assay and the negative regulation of TIMP3 by miR-222 in PASMCs. Importantly, co-transfection of miR-222 mimic and TIMP3 siRNA could not further enhance the proliferation of PASMCs while TIMP3 siRNA could attenuate the anti-proliferation effect of miR-222 inhibitor in PASMCs, indicating that TIMP3 inhibition is also responsible for the pro-proliferation effect of miR-222 in PASMCs. In addition, simultaneous knock-down of P27 and TIMP3 did not show an additive effect on PASMC proliferation, indicating that the two genes might be regulated and act in a similar way. Collectively, P27 and TIMP3 were identified as two target genes of miR-222 in regulating PASMC proliferation.

Chronic hypoxia is one of the major causes of pulmonary hypertension. Persistent hypoxia can result in endothelial dysfunction, uncontrolled cell proliferation, and pulmonary vascular remodeling [37]. As hypoxia is a common symptom of pulmonary hypertension which can promote the proliferation of PASMCs, we also examined whether miR-222 could regulate PASMC proliferation under hypoxic condition. We found an upregulation of miR-222 in hypoxic PASMCs. More importantly, hypoxia-induced PASMC proliferation can be reduced by miR-222 inhibition. In addition, P27 and TIMP3 expressions were both down-regulated in PASMCs under hypoxic condition and were reversed by miR-222 inhibitor. These data provide evidence indicating that miR-222 inhibition may exert beneficial effect in reducing PASMC proliferation in pulmonary hypertension related to hypoxic conditions such as chronic obstructive pulmonary disease and interstitial lung disease [38, 39].

In conclusion, miR-222 promotes PASMC proliferation at least partially through targeting P27 and TIMP3. Therefore, inhibition of miR-222 in PASMCs may be a potential therapy for PAH.

This work was supported by the grants from National Natural Science Foundation of China (91639101 and 81570362 to JJ Xiao, 81370332 and 81170201 to XL Li, 81400647 to Y Bei), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-09-E00042 to JJ Xiao), the grant from Science and Technology Commission of Shanghai Municipality (17010500100), the development fund for Shanghai talents (to JJ Xiao), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD20102013 to XL Li), and the development fund for Shanghai talents (to JJ Xiao). Dr XL Li is an Associate Fellow at the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.

The authors declare there are no conflicts of interest.

Y. Xu and Y. Bei contributed equally to this work.