miR-185/ P2Y6 Axis Inhibits Angiotensin II-Induced Human Aortic Vascular Smooth Muscle Cell Proliferation

Abstract

The abnormal proliferation and apoptosis of human aortic vascular smooth muscle cells (HAVSMCs) play an important role in the pathogenesis of hypertension. Recent study revealed that angiotensin II (Ang II) could elicit HAVSMC dysfunction, to induce or aggravate hypertension. Purinergic receptor P2Y6, an inflammation-inducible G protein-coupled receptor, promoted Ang II-induced hypertension. In the present study, we revealed that Ang II induced HAVSMC proliferation and upregulated P2Y6 protein levels. After knockdown of P2Y6, the promotive effect of Ang II on HAVSMC proliferation was restored. microRNAs (miRNAs) involve in most biological processes. In this study, we scanned out seven candidate miRNAs, which were predicted to contain binding site of P2Y6's 3′-UTR by online tools. Among them, miR-185 was significantly downregulated by Ang II treatment. miR-185 reduced P2Y6 protein levels by direct binding to the 3′UTR of P2Y6. miR-185 overexpression suppressed HAVSMC proliferation; P2Y6 overexpression or Ang II treatment promoted HAVSMC proliferation, and restored the suppressive effect of miR-185 on HAVSMC proliferation. Besides, miR-185/P2Y6 axis also affected pERK1/2 protein levels. Taken together, the present study indicated that miR-185/P2Y6 axis might inhibit Ang II-induced HAVSMC proliferation through miR-185 negatively regulating P2Y6 expression and the downstream ERK pathway; rescuing miR-185 expression to inhibit P2Y6 may represent a therapeutic strategy against HAVSMC dysfunction and hypertension.

Introduction

Human aortic vascular smooth muscle cells (HAVSMCs), which are present under the vascular endothelial cells, are the main cellular components that form the structure of the vascular wall and maintain the blood vessel. In normal adults, vascular smooth muscle cells maintain the tension of the blood vessel wall by slow and mild contraction. Blood pressure is largely maintained by vascular tension. Abnormal proliferation of vascular smooth muscle cells plays an important role in the pathogenesis of hypertension (Voelkel and Tuder, 1997). Overproliferation of vascular smooth muscle cells lead to smaller vascular lumen, thickening of the vascular wall, and plays an important role in pathogenesis of hypertension (Voelkel and Tuder, 1997).

The rennin–angiotensin system (RAS) plays an important role in the pathogenesis of hypertension. The system includes renin, angiotensin (Ang II), angiotensin-converting enzyme and so on. Ang II has strong capability of vasoconstriction, and can promote vascular smooth muscle cell proliferation, hardens the vessel wall, and causes the lumen to narrow (Touyz, 2005). Ang II could increase blood pressure and promote arterial reconstruction through the strong contraction of blood vessels; on the other hand, Ang II can directly promote vascular smooth muscle cell hypertrophy and collagen fiber proliferation, leading to vascular remodeling (Pueyo and Michel, 1998; Takemoto and Egashira, 1999). Ang II can also promote many processes of cardiovascular tissue, including cell growth, migration, differentiation, and apoptosis. Therefore, Ang II may be one of the important factors affecting the cardiovascular remodeling (Komuro and Kudoh, 1999). However, how HAVSMCs determine the responsiveness to Ang II under different developmental and region-specific conditions is mostly unclear.

Purinergic P2Y receptors are a family of inflammation-inducible G protein-coupled receptors that respond to extracellular mononucleotides (Abbracchio et al., 2006). Multiple P2Y receptor subtypes, including P2Y6 (Pediani et al., 1999; Wang et al., 2002), are present in blood vessels. P2Y6 activation induces a Ca²⁺ response in vascular smooth muscle cells that results in contraction of isolated blood vessels (Abbracchio et al., 2006). These pharmacological studies suggest that P2Y6 plays an important role in cardiovascular function. In addition, P2Y6 ablation attenuates vascular inflammation in vivo under pathological conditions (Riegel et al., 2011; Stachon et al., 2014). These results suggest that P2Y6 participates in the development of pathological vascular remodeling. However, the functional role of P2Y6 on HAVSMCs' proliferation still remains to be validated.

The discovery of microRNAs (miRNAs) has opened new avenues for studying and understanding hypertension and hypertension-associated endothelial dysfunction, featuring a postgenomic era of biomedical research (Nemecz et al., 2016). These noncoding regulatory RNA molecules of ∼22 nucleotides have emerged as potential biomarkers, effectors, and targets for diagnosis, prognosis, and therapy in hypertension. Lately, many studies have been done on understanding miRNA biology and function. It seems that miRNAs play an important role in the regulation of almost every cellular process. Usually, a single miRNA can interact with hundreds of mRNA molecules and a specific mRNA molecule may be the target of multiple miRNAs. Thus, miRNA–mRNA interactions may delineate the complex regulatory networks with consequence on the target gene expression and hence on some biological processes. Additionally, the disruptions of miRNA regulation are frequently associated with some pathological states, including hypertension-associated endothelial dysfunction (Nemecz et al., 2016).

In the present study, we validated the functional role of Ang II in HAVSMCs proliferation and P2Y6 expression, and the effect of Ang II-induced P2Y6 expression on HAVSMC proliferation. By using the online tools, we scanned out some candidate miRNAs that contain the binding site in 3′UTR of P2Y6 and were associated with cell proliferation. Among them, miR-185 could be downregulated by Ang II treatment. As verified by performing a series of functional and mechanistic assays, we revealed that miR-185/P2Y6 axis could suppress HAVSMCs proliferation through ERK signaling.

Materials and Methods

Cell lines and cell transfection

HAVSMCs were obtained from ATCC, cultured in RPMI-1640 medium (Invitrogen, CA) adding 10% fetal calf serum (FBS) (Gibco, CA) as the supplement, and incubated at 37°C in a humidified atmosphere with 5% CO₂.

P2Y6-siRNA1 and P2Y6-siRNA2 were used to achieve P2Y6 silence (GeneCopoecia, Guangzhou, China). pCMV-P2Y6 vector was used to achieve exogenous P2Y6 expression (GeneCopoecia, Guangzhou, China). miR-185 mimics or miR-185 inhibitor (GeneCopoecia, Guangzhou, China) was used to achieve miR-185 overexpression or inhibition. After being plated in 96- or 6-well plates, HAVSMCs were then transfected with indicated siRNAs, mimics or inhibitor with lipo2000 (Invitrogen), cultured for 24 h, and consequently used for the next experiments.

RNA extraction and real-time PCR assays

We extracted total RNA from target cells by using TRIzol reagent (Invitrogen). Mature miR-185, miR-483, miR-20a, miR-1182, miR-3151, miR-149, and miR-877 expression in cells was detected using the Hairpin-it™ miRNAs qPCR Kit (GenePharma, Shanghai, China). P2Y6 mRNA expression was monitored by using SYBR Green qPCR assay. RNU6B expression was used as an endogenous normalization. Data were analyzed using 2^−ΔΔCT method.

Cell Counting Kit-8 cell proliferation assay

Cell Counting Kit-8 (CCK-8) (Beyotime, Hangzhou, China) was used to measure HAVSMCs proliferation rates. We seeded 0.5 × 10⁴ cells in each 96-well plate for 24 h, transfected them with the indicated siRNA, miRNA mimics or inhibitor, and further incubated cells for 24, 48, 72, 96, and 120 h, respectively. At 1 h before the endpoint of incubation, we added 10 μL CCK-8 reagents to each well. A microplate reader was used to determine OD_570nm value in each well.

5-Bromo-2-deoxyUridine cell proliferation assay

By measuring 5-Bromo-2-deoxyUridine (BrdU) incorporation, the DNA synthesis in proliferating cells was determined. BrdU assays were conducted at 24 and 48 h after HAVSMCs were transfected with the indicated vectors. Cells were seeded in 96-well culture plates at a density of 2 × 10³ cells/well, cultured for 48 h, then incubated with a final concentration of 10 μM BrdU (BD Pharmingen, San Diego, CA) for 2 h. When the incubation period ended, the medium was removed, the cells were fixed for 30 min at RT, incubated with peroxidase-coupled anti-BrdU-antibody (Sigma-Aldrich) for 60 min at RT, washed three times with PBS, incubated with peroxidase substrate (tetramethylbenzidine) for 30 min, and the 450 nm absorbance values were measured for each well. Background BrdU immunofluorescence was determined in cells not exposed to BrdU, but stained with the BrdU antibody.

Western blotting

RIPA buffer (Cell-Signaling Tech.) was used to homogenize the cells. The expression of P2Y6, ERK1/2, and pERK1/2 in HAVSMC was detected by performing immunoblotting. After treatments, HAVSMCs were lysed in 1% PMSF supplemented RIPA buffer. Protein were loaded onto SDS-PAGE minigel, and then transferred onto PVDF membrane. The blots were probed with 1:1000 diluted rabbit polyclonal P2Y6, ERK1/2, and pERK1/2 antibody (Cat no. ab92504, ab17942, ab50011; Abcam) at 4°C overnight, and incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibody (1:5000) (Cat nos. A0208 and A0216; Beyotime, China). Signals were visualized using enhance chemiluminescence (ECL) Substrates (Millipore). The protein expression was normalized to endogenous GAPDH (AF0006; Beyotime, China).

Luciferase reporter assay

UTR luciferase reporter assays were performed in 293T cells (ATCC). After being cultured overnight, cells were cotransfected with the wild-type (wt-P2Y6)/mutated-type (mut-P2Y6 3′UTR1 and mut-P2Y6 3′UTR2) reporter plasmids (purchased from Yrbio Tech, China) and miR-185 mimics or miR-185 inhibitor. Luciferase assays were performed 48 h after transfection using the Dual Luciferase Reporter Assay System (Promega, WI).

Statistical analysis

Data from at least three independent experiments were exhibited as mean ± standard deviation, analyzed by SPSS 17.0 statistical software (SPSS, Chicago). Paired Student's t-test was used to compare the differences between two groups. One-way ANOVA was used to analyze the differences between groups in the proliferation assays. A p value of <0.05 was considered statistically significant.

Results

Ang II induced HAVSMCs proliferation and upregulated P2Y6 protein level

Ang II has been reported to induce vascular smooth muscle cells' proliferation. In this study, we treated HAVSMC with different concentrations of Ang II (0, 10 nM, 100 nM, 1 μM), or for different durations (0, 12, 24, 48 h), and determined the cell proliferation by using CCK-8 assays, determined the protein levels of P2Y6 by using western blot assays. Results showed that the proliferation of HAVSMC was promoted by Ang II in a concentration- or time-dependent manner (Fig. 1A, B). P2Y6 protein levels were also upregulated by Ang II in a concentration- or time-dependent manner (Fig. 1C, D). These data indicated that Ang II promoted HAVSMC proliferation and P2Y6 protein levels in HAVSMC in a concentration- and time-dependent manner.

FIG. 1.

Ang II induced HAVSMCs proliferation and upregulated P2Y6 protein level (A) The cell proliferation of HAVSMCs in response to different concentrations of Ang II treatment for 24 h (0, 10 nM, 100 nM, 1 μM) determined by using CCK-8 assays. (B) The cell proliferation of HAVSMC in response to 100 nM Ang II treatment for 0, 12, 24, and 48 h determined by using CCK-8 assays. (C) P2Y6 protein levels in response to different concentrations of Ang II treatment for 24 h (0, 10 nM, 100 nM, 1 μM) determined by using western blot assays. (D) P2Y6 protein levels in response to100 nM Ang II treatment for 0, 12, 24, and 48 h determined by using western blot assays. Data are presented as mean ± SD, n = 8. *p < 0.05, **p < 0.01. Ang II, angiotensin II; CCK-8, Cell Counting Kit-8; HAVSMC, human aortic vascular smooth muscle cell; SD, standard deviation.

P2Y6 silence could partially restore the promotive effect of Ang II on HAVSMCs proliferation

To validate the role of P2Y6 in Ang II-induced HAVSMC proliferation, two siRNAs were used to achieve P2Y6 silence. Inhibitory efficiency of two siRNAs was verified by using real-time PCR assays, and P2Y6-siRNA1 reduced P2Y6 expression more effectively (Fig. 2A). Protein levels of P2Y6 were determined by using western blot assays, and similar results were observed: P2Y6-siRNA1 reduced P2Y6 protein level more effectively (Fig. 2B). Then P2Y6-siRNA1 was transfected into HAVSMC with the presence of Ang II, and the cell proliferation was determined by using CCK-8 and BrdU assays. Results showed that Ang II significantly promoted HAVSMCs proliferation, whereas P2Y6 silence suppressed HAVSMCs proliferation, compared with the control group (Fig. 2C, D); after being transfected with P2Y6-siRNA1, the promotive effect of Ang II on HAVSMCs proliferation was partially restored (Fig. 2C, D). These data indicated that P2Y6 is involved in Ang II-induced HAVSMC proliferation; P2Y6 silence by P2Y6-siRNA could partially restore the promotive effect of Ang II on HAVSMC proliferation.

FIG. 2.

P2Y6 silence could partially restore the promotive effect of Ang II on HAVSMC proliferation (A) P2Y6-siRNA1 and P2Y6-siRNA2 were constructed to achieve P2Y6 silence. The mRNA levels of P2Y6 were determined by using real-time PCR assays. (B) The protein levels of P2Y6 after P2Y6-siRNA1 or P2Y6-siRNA2 transfection were determined by using western blot assays. (C) The cell viability of HAVSMC in response to P2Y6-siRNA1 transfection with the presence of Ang II treatment (100 nM) was determined by using CCK-8 assays. (D) The cell proliferation of HAVSMC in response to P2Y6-siRNA1 transfection with the presence of Ang II treatment was determined by using BrdU assays. Data are presented as mean ± SD, n = 6. *p < 0.05 and **p < 0.01 versus si-NC group, ^## p < 0.01 versus P2Y6-siRNA1 group, ^ΔΔ p < 0.01 versus AngII + si-NC group.

Prediction and screening of upstream miRNAs of P2Y6

The disruptions of miRNA regulation are frequently associated with some pathological states including hypertension-associated endothelial dysfunction. Ang II has previously been reported to regulate the expression of miR-29b, miR-129-3p, and miR-212 through mechanisms depending on Gαq/11 and ERK1/2 activation (Jeppesen et al., 2011). Likewise, recent data suggested that miR-132 and miR-212 are involved in Ang II-induced hypertension (Eskildsen et al., 2013). In this study, we used online tools, including miRwalk, miRanda, RNA22, TargetScan, and microT-CDS to scan out several candidate miRNAs that could potentially target P2Y6 (Fig. 3A). According to the previous literature, we chose six miRNAs (miR-185, miR-483, miR-20a, miR-1182, miR-149, and miR-877) to determine their expression levels in response to Ang II treatment (100 nM, 24 h). By using real-time PCR, we observed that the expression of miR-185, miR-483, miR-20a, miR-1182, and miR-877 was modulated by Ang II treatment, except miR149, among which miR-185 expression was significantly downregulated (Fig. 3B). These data suggested that miR-185 might be involved in Ang II-induced HAVSMC proliferation and P2Y6 expression.

FIG. 3.

Prediction and screening of upstream miRNAs of P2Y6 (A) Six candidate miRNAs (miR-185, miR-483, miR-20a, miR-1182, miR-149, and miR-877), which had the potential of targeting P2Y6 were screened out by using online tools, including miRwalk, miRanda, RNA22, TargetScan, and microT-CDS, and referring to the previous studies. (B) The expression levels of the indicated candidate miRNAs in response to Ang II treatment (100 nM, 24 h) were determined by using real-time PCR assays. Data are presented as mean ± SD, n = 6. *p < 0.05, **p < 0.01 versus DMSO group. miRNAs, microRNAs.

miR-185 inhibited P2Y6 expression by direct binding to the 3′UTR of P2Y6

To verify the association between miR-185 and P2Y6, miR-185 mimics or inhibitor was transfected into HAVSMCs to achieve miR-185 overexpression or inhibition (Fig. 4A). The protein levels of P2Y6 were determined by using western blot assays after transfection with miR-185 mimics or inhibitor. Results showed that P2Y6 protein was downregulated by miR-185 overexpression, whereas upregulated by miR-185 inhibition (Fig. 4B). We constructed a wt-P2Y6 3′UTR luciferase reporter gene vector, as well as a mut-P2Y6 3′UTR vector by mutating the two predicted binding sites of miR-185 in the 3′UTR of P2Y6 (Fig. 4C). The indicated vectors were cotransfected into 293T cells with miR-185 mimics or inhibitor, and the luciferase activity was determined by using dual luciferase assays. Results showed that the luciferase activity of wt-P2Y6 3′UTR vectors was reduced by miR-185 mimics, whereas promoted by miR-185 inhibitor, compared with the NC (negative control) group; the effect of miR-185 mimics or inhibitor on luciferase activity was offset by either mutation in the 3′UTR of P2Y6 (Fig. 4D). These data indicated that miR-185 could reduce P2Y6 protein level by direct binding to the 3′UTR of P2Y6.

FIG. 4.

miR-185 inhibited P2Y6 expression by direct binding to the 3′UTR of P2Y6 (A) miR-185 mimics or inhibitor was used to achieve miR-185 overexpression or inhibition. The expression efficiency was verified by using real-time PCR. (B) The protein levels of P2Y6 in response to miR-185 overexpression or inhibition were determined by using western blot assays. (C) A wt-P2Y6 3′UTR luciferase reporter gene vector, as well as a mut-P2Y6 3′UTR vector was constructed by mutating the two predicted binding sites of miR-185 in the 3′UTR of P2Y6. (D) The indicated luciferase vectors were cotransfected into 293T cells with miR-185 mimics or inhibitor. The luciferase activity was then determined by using dual luciferase assays. Data are presented as mean ± SD, n = 6. *p < 0.05, **p < 0.01.

miR-185 regulated HAVSMC proliferation and downstream ERK pathway through targeting P2Y6

We revealed that miR-185 inhibits P2Y6 protein level through direct binding to its 3′UTR; next we validated the functional role of miR-185 in regulating HAVSMCs proliferation. As determined by CCK-8 and BrdU assays, miR-185 overexpression significantly inhibited HAVSMCs proliferation, P2Y6 overexpression promoted HAVSMCs proliferation; P2Y6 could partially restore the inhibitory effect of miR-185 on HAVSMCs proliferation (Fig. 5A, B). According to previous studies, ERK pathway is involved in the pathophysiology of hypertension (Roberts, 2012), Moreover, P2Y receptors, including P2Y6, could mediate ERK activation to prevent cell apoptosis (Lenz et al., 2000; Kim et al., 2003). In this study, we further monitored the protein levels of total ERK1/2 and p-ERK1/2, as well as P2Y6 protein level in response to cotransfection of miR-185 mimics and pCMV-P2Y6 by using western blot assays. Results showed that miR-185 overexpression significantly downregulated P2Y6 and pERK1/2 protein levels without affecting total ERK1/2 protein. pCMV-P2Y6 transfection exerted an opposite function: upregulated both P2Y6 and pERK1/2 protein levels without affecting total ERK1/2 protein. In addition, P2Y6 partially restored the effect of miR-185 on the indicated factors (Fig. 5C). These data indicated that miR-185 inhibits HAVSMC proliferation through inhibiting P2Y6 expression and regulating the downstream ERK pathway.

FIG. 5.

miR-185 regulated HAVSMCs proliferation and downstream ERK pathway through targeting P2Y6. (A) The cell viability of HAVSMCs in response to cotransfection of miR-185 mimics and pCMV-P2Y6 was determined by using CCK-8 assays. (B) The cell proliferation of HAVSMC in response to cotransfection of miR-185 mimics and pCMV-P2Y6 was determined by using BrdU assays. (C) The protein levels of ERK1/2 and pERK1/2 in HAVSMC in response to cotransfection of miR-185 mimics and pCMV-P2Y6 was determined by using western blot assays. Data are presented as mean ± SD, n = 6. *p < 0.05, **p < 0.01 versus NC mimics + Vector group; ^## p < 0.01 versus miR-185 mimics + Vector group; ^ΔΔ p < 0.01 versus NC mimics + pCMV-P2Y6 group. BrdU, 5-Bromo-2-deoxyUridine.

miR-185 restored the promotive effect of Ang II on P2Y6 expression and HAVSMCs proliferation

We have revealed that miR-185 inhibits HAVSMC proliferation through inhibiting P2Y6 expression; we further investigated whether ectopic miR-185 expression could restore the effect of Ang II on P2Y6 and HAVSMC proliferation. In HAVSMCs, miR-185 expression was significantly upregulated by miR-185 mimics; when Ang II treatment was conducted, miR-185 mimics-induced miR-185 expression was partially reduced (Fig. 6A). Then the protein levels of P2Y6 were monitored. As shown by western blot assays, P2Y6 protein was reduced by ectopic miR-185 expression, increased by Ang II treatment; the promotive effect of Ang II treatment on P2Y6 protein could be partially reversed by ectopic miR-185 expression (Fig. 6B). Furthermore, the cell proliferation of HAVSMC was monitored by using CCK-8 and BrdU assays. Similarly, the promotive effect of Ang II treatment on HAVSMC proliferation could be partially reversed by miR-185 (Fig. 6C, D). These data further indicated that miR-185 restores the promotive effect of Ang II on P2Y6 expression; miR-185/P2Y6 axis regulates Ang II-induced HAVSMC proliferation.

FIG. 6.

miR-185 restored the promotive effect of Ang II on P2Y6 expression and HAVSMCs proliferation (A) miR-185 mimics was transfected into HAVSMC under Ang II treatment. The expression levels of miR-185 were determined by using real-time PCR assays. (B) The protein levels of P2Y6 in response to ectopic miR-185 expression under Ang II treatment were determined by using western blot assays. (C) and (D) The cell proliferation of HAVSMC in response to ectopic miR-185 expression under Ang II treatment was determined by using CCK-8 and BrdU assays. Data are presented as mean ± SD, n = 6. *p < 0.05, **p < 0.01 versus NC mimics group; ^# p < 0.05, ^## p < 0.01, versus miR-185 mimics group, ^ΔΔ p < 0.01, versus Ang II + NC mimics group.

Discussion

Excessive proliferation of vascular smooth muscle cells is a crucial event in the pathogenesis of several cardiovascular diseases, including hypertension. In the present study, we found that Ang II induced HAVSMC proliferation and P2Y6 protein levels in a concentration- or time-dependent manner. After silence of P2Y6 by P2Y6-siRNA, HAVSMC proliferation was downregulated; in addition, P2Y6 silence could also partially restore the effect of Ang II on HAVSMC proliferation. Through online tools and previous literatures, we chose several candidate miRNAs that might potentially target P2Y6 and were associated with cell proliferation. Among them, miR-185 expression could be significantly downregulated by Ang II treatment. By direct binding to the 3′UTR of P2Y6, miR-185 inhibited P2Y6 expression and HAVSMCs proliferation. Moreover, downstream ERK pathway might be involved in this process.

The excessive proliferation of vascular smooth muscle cell can be induced by various cytokines and growth factors, such as Ang II, insulin, and platelet-derived growth factors (Touyz and Schiffrin, 1996). Ang II has been frequently reported to be associated with cardiovascular smooth muscle cell proliferation and apoptosis (Pueyo and Michel, 1998; Takemoto and Egashira, 1999). Ang II could increase human brain vascular smooth muscle cell migration, proliferation, and apoptosis, and increased the blood pressure, neurological deficit score, middle cerebral artery (MCA) remodeling, and hemorrhage volume in intracerebral hemorrhage (ICH) mice (Bihl et al., 2015). One micromole of Ang II significantly promoted the proliferation of A7r5 cell, an aortic smooth muscle cell line, from 100% to 131% (Li et al., 2015). In the present study, we treated HAVSMCs with different concentrations of Ang II, for different durations. HAVSMCs proliferation was promoted by Ang II in a concentration- or time-dependent manner. Besides, Ang II treatment could also upregulate P2Y6 protein levels in a concentration- or time-dependent manner.

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y6 receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. Nishimura et al. reported that through heterodimerization with the angiotensin type 1 receptor, P2Y6 could promote Ang II-induced hypertension (Nishimura et al., 2016). By generating P2Y6 ^−/− mice, Bar et al. (2008) revealed that the P2Y6 receptor is indeed responsible for the contractile action of both UDP and UTP in aorta, and P2Y6 is expressed and functional in vascular smooth muscle cells. In the present study, we investigated the detailed role of P2Y6 in HAVSMCs proliferation. After P2Y6-siRNA transfection, HAVSMCs proliferation was suppressed. Moreover, Ang II-induced HAVSMC proliferation could be partially restored by P2Y6 silence. However, the mechanism by which P2Y6 affects Ang II-induced HAVSMC proliferation and Ang II-upregulated P2Y6 expression still remains to be validated.

The RAS has an essential role in blood pressure regulation by affecting cardiac contractility, vascular resistance, and blood volume. RAS overactivation is a major pathogenetic factor in hypertension. Several miRNAs have been shown to interact with the RAS. miR-155 has been found to regulate AGTR1 expression (Martin et al., 2006). miR-124 and miR-135a independently repressed the translation of NR3C2, a ligand-dependent transcription factor that regulates water and ion transporter expression, without affecting mRNA levels. The authors proposed that miR-124 and miR-135a may contribute to the modulation of the RAAS and thereby to blood pressure regulation (Sober et al., 2010). In this study, we used online tools to screen out several candidate miRNAs, which were predicted to have the potential of binding to P2Y6. These candidate miRNAs were also reported to be associated with cell proliferation: miR-185, miR-483, miR-20a, miR-1182, miR-149, and miR-877 (Bertero et al., 2011; Fan et al., 2015; Zhang et al., 2015; Chen et al., 2016; Li et al., 2016; Zhu et al., 2016). After Ang II treatment, the expression of all candidate miRNAs was modulated by Ang II, except miR-149, among which miR-185 expression was more strongly downregulated. We then verified that miR-185 could bind to the 3′UTR of P2Y6 on the two predicted binding sites, and inhibit P2Y6 expression through direct targeting. These data suggested the involvement of miR-185 in regulation of HAVSMCs proliferation; so we performed a series of functional assays to investigate whether miR-185 and P2Y6 constitute a regulatory axis to affect HAVSMC proliferation together.

We observed a significant inhibitory effect of miR-185 on HAVSMC proliferation by CCK-8 and BrdU assays; in addition, exogenous P2Y6 expression could partially restore the inhibitory effect of miR-185 on HAVSMCs proliferation, indicating that miR-185/P2Y6 axis affected HAVSMC proliferation. A similar result has been reported by Shan et al. that miR-185-5p inhibitor transfection increased the viability and proliferation of HUVECs; lncRNA-RNCR3, miR-185-5p, and KLF2 constitute a regulatory network to regulate the viability and proliferation of HUVECs (Shan et al., 2016). Recent studies reported that miRNA–mRNA interactions may delineate the complex regulatory networks with consequence on the target gene expression and hence on some biological processes. Due to its major role in pathophysiology of hypertension (Guo et al., 2014; Li et al., 2017), we then validated whether ERK pathway was involved in regulation of HAVSMC proliferation by miR-185. As shown by western blot, miR-185 reduced pERK1/2 protein level; P2Y6 increased pERK1/2 protein level, and partially restored the effect of miR-185 on pERK protein. These data suggested that ERK pathway was involved in the process of miR-185/P2Y6 axis inhibiting Ang II-induced HAVSMC proliferation.

Taken together, we indicated that miR-185/P2Y6 axis inhibits Ang II-induced HAVSMC proliferation through the downstream ERK pathway; rescuing miR-185 expression to inhibit P2Y6 expression may thus represent a therapeutic strategy against HAVSMCs dysfunction and hypertension.

Footnotes

Acknowledgments

This study was supported by the National Natural Science Foundation of China (program no.: 81473616), Science and Technology Program Major Project of China Hunan Provincial Science & Technology Department (2016DK2002), and Hunan Traditional Chinese Medicine Scientific Research Key Project (201753).

Disclosure Statement

No competing financial interests exist.

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