Tan IIA mitigates vascular smooth muscle cell proliferation and migration induced by ox-LDL through the miR-137/TRPC3 axis
Abstract
Tanshinone IIA (Tan IIA) has an important role in treatment of cardiovascular diseases, including atherosclerosis. The vascular smooth muscle cells (VSMCs) are a major part of the atherosclerotic plaque. However, the biological functions of Tan IIA in regulating VSMCs function remain mostly unclear. This research aimed at identifying the explicit molecular mechanism that Tan IIA regulates oxidized low-density lipoprotein (ox-LDL)-mediated VSMC proliferation and migration. VSMCs challenged by ox-LDL were adopted as cellular model of atherosclerosis, and suffered from Tan IIA treatment. After that, cells proliferation, apoptosis or migration were measured. The expression levels of microRNA (miR)-137, transient receptor potential cation channel subfamily C member 3 (TRPC3) and proliferating cell nuclear antigen (PCNA) were measured. The targeting relationship between miR-137 and TRPC3 was determined. It was found that Tan IIA blunted VSMC proliferation, PCNA expression and migration mediated by ox-LDL. Tan IIA promoted miR-137 level, and miR-137 knockdown reversed the influences of Tan IIA on VSMC proliferation, PCNA expression and migration in the presence of ox-LDL. TRPC3 was verified to be targeted by miR-137. Moreover, TRPC3 silencing exacerbated the influences of Tan IIA on VSMC proliferation, apoptosis and migration, and it mitigated the inhibitive effects of miR-137 knockdown on function of Tan IIA. We confirmed for the first time that Tan IIA constrained ox-LDL-stimulated VSMC proliferation and migration via regulating the miR-137/TRPC3 axis, which provided a theoretical basis for the research and promotion of Tan IIA as a therapeutic drug.
1 INTRODUCTION
Atherosclerosis is a class of cardiovascular diseases with high incidence and leading causes of death worldwide.1 Vascular smooth muscle cells (VSMCs) are an important cell type associated with atherosclerosis, and VSMC proliferation and migration facilitate plaque formation and development in atherosclerosis.2, 3 The oxidized low-density lipoprotein (ox-LDL) is an important signaling molecule in vascular system, and acts as the prime mover of atherosclerosis which could induce VSMC dysfunction.4, 5 Hence, exploring novel drug targeting VSMC proliferation and migration might find promising therapeutic strategies of atherosclerosis.
Tanshinone IIA (Tan IIA; C19H18O3) is an abietane diterpene compound derived from the Chinese herbal medicine Danshen. Tan IIA has a vital role in cardiovascular diseases and inflammatory disorders by its anti-inflammatory and anti-oxidative roles.6, 7 Furthermore, increasing reports suggest that Tan IIA could alleviate atherosclerosis progression by inhibiting inflammation and oxidative stress in animal model.8, 9 However, how Tan IIA modulating ox-LDL-stimulated VSMC proliferation and migration is largely unknown.
MicroRNAs (miRNAs) could mediate gene level through binding to the 3′-untranslated regions (3′-UTR) of mRNA to participate in regulation of VMSC function in atherosclerosis.10 MiR-137 is an important miRNA which is associated with multiple cardiovascular disorders.11, 12 Moreover, up-regulation of miR-137 is reported to be involved in VSMC proliferation and migration.13 Furthermore, decreased miR-137 is found in serum of atherosclerosis patients.14 However, the downstream pathways of miR-137 in atherosclerosis progression remains largely unknown. There is an available evidence indicates that Tan IIA could promote miR-137 expression in lung cancer cells.15 And whether miR-137 is required for Tan IIA-mediated regulation of VSMC function is worth further studying.
Transient receptor potential cation channel subfamily C member 3 (TRPC3) is widely expressed in many cell types including smooth muscle cells of vascular tissues,16 and it appears to regulate functions of cardiovascular system.17 Furthermore, TRPC3 could promote atherosclerosis progression through increasing ox-LDL-induced aortic endothelial cell damage.18 Our study predicted TRPC3 might act as a target of miR-137 by TargetScan online tool. Nevertheless, little is known about if miR-137 could regulate TRPC3 to control VSMC proliferation and migration.
This study focused on the effects of Tan IIA in VSMCs challenged by ox-LDL through detecting proliferation, migration and proliferation-related marker proliferating cell nuclear antigen (PCNA) expression levels.19 Furthermore, we analyzed whether the mechanism of Tan IIA was correlated with miR-137/TRPC3 axis. This might implicate a novel avenue for prevention and therapy of atherosclerosis.
2 MATERIALS AND METHODS
2.1 Cell culture
Human VSMCs were provided by ATCC (Manassas, VA, USA) and grown in F-12 K medium (Thermo Fisher, Wilmington, DE, USA) with 10% FBS (Zhejiang Tianhang Biotechnology, Huzhou, China) and 1% penicillin–streptomycin (Solarbio, Beijing, China) at 37°C with 5% CO2.
2.2 Cell treatment
For treatment with different concentrations (2.5, 5, and 10 μg/mL) of Tan IIA, VSMCs were treated with indicated concentrations of Tan IIA (Selleck, Shanghai, China) for different time points. For ox-LDL exposure, cells were cultured in medium containing 100 μg/mL of ox-LDL (Thermo Fisher) for 24 h.
2.3 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
Cell proliferation was examined through MTT method. VSMCs (1 × 104 cells/well) were placed in 96-well plates and challenged by ox-LDL and Tan IIA for 12, 24, or 48 h. After that, cells were incubated with 0.5 mg/mL MTT (Beyotime, Shanghai, China) for 4 h. Then the formed crystal was resolved in dimethyl sulfoxide (Beyotime). The value of optical density was measured through microplate reader (Biotek, Winooski, VT, USA) at 490 nm.
2.4 Transwell analysis
The migrated ability of VSMCs was analyzed by transwell insert chambers (Costar, Corning, NY, USA). VSMCs were resuspended in medium without serum and dispersed in the upper chambers. Meanwhile, the lower chambers were filled with 600 μL medium containing 10% FBS. Following 12 h, the cells transferred the membranes were stained by use of 0.1% crystal violet (Beyotime), followed by observation with a microscope (Olympus, Tokyo, Japan).
2.5 Flow cytometry
Cell apoptosis was analyzed through flow cytometry. VSMCs (1 × 105 cells/well) were placed in 12-well plates and treated with ox-LDL and Tan IIA for 24 h. After that, cells were collected and incubated with an Annexin V-FITC/PI apoptosis detection kit (Solarbio). Apoptotic cells were detected with a flow cytometry (Countstar, Shanghai, China).
2.6 Quantitative real-time polymerase chain reaction
VSMCs were collected and treated with Trizol reagent (Beyotime) for extraction of total RNA. The cDNA was synthesized from 500 ng RNA via use of a TaqMan cDNA synthesis kit (Thermo Fisher) and was utilized for quantitative real-time polymerase chain reaction (qRT-PCR) by using SYBR (Vazyme, Nanjing, China). GAPDH or U6 was used as internal reference, respectively. The primer sequences were displayed as: TRPC3: sense, 5′-CTCCCTTCTGACCCTCAGATATT-3′; antisense, 5’-GGGGGCCAAAGCTCTCATTT-3′; miR-137: sense, 5′-GCCGAGTTATTGCTTAAGAA-3′; antisense, 5′-GCTGTCAACGATACGCTACGTAAC-3′; GAPDH (sense, 5′-GGAGCGAGATCCCTCCAAAAT-3′; antisense, 5′-GGCTGTTGTCATACTTCTCATGG-3′); U6 (sense, 5′-CAGGTCTCGGGAGAGAGATCG-3′; antisense, 5′-TGTCGTCTTGGAGATCGGGAG-3′). The relative expression abundances of RNAs were calculated by 2−ΔΔCt method.20
2.7 Western blot
VSMCs were incubated with the protein extraction buffer (Solarbio) for total protein isolation. Protein concentration was detected via use of bicinchoninic acid (BCA) assay kit (Beyotime). Next, the protein was denatured, and 30 μg samples were loaded on SDS-PAGE and transfected on PVDF membranes (Millipore, Billerica, MA, USA). Five percent of skim milk was used to immerse the membranes. The membranes were incubated with primary and secondary antibodies, followed by incubation using enhanced chemiluminescence kit (Thermo Fisher). The antibodies were listed as: anti-TRPC3 (96 kDa, ab241343, 1:2000 dilution, Abcam, Cambridge, UK), anti-PCNA (29 kDa, ab18197, 1:2000 dilution, Abcam) or anti-GAPDH (36 kDa, ab181602, 1:10000 dilution, Abcam) and IgG labeled with horseradish peroxidase (ab6721, 1:20,000 dilution, Abcam). GAPDH was regarded as reference. Relative protein level was determined referring to the gray levels of bands detected via use of Image J software (NIH, Bethesda, MD, USA).
2.8 Cell transfection
MiR-137 mimics (5′-UUAUUGCUUAAGAAUACGCGUAG-3′), mimics negative control (NC) (5′-CGAUCGCAUCAGCAUCGAUUGC-3′), miR-137 inhibitor (5′-CUACGCGUAUUCUUAAGCAAUAA-3′), inhibitor NC (5′-CUAACGCAUGCACAGUCGUACG-3′), shRNA for TRPC3 (sh-TRPC3, 5′-AUACUUAAUGGCAAGUUUGAC-3′), and shRNA NC (sh-NC, 5′-AAGACAUUGUGUGUCCGCCTT-3′) were generated via Genomeditech (Shanghai, China). Cell transfection was performed in VSMCs through use of Lipofectamine 2000 (Thermo Fisher) for 24 h.
2.9 Dual-luciferase reporter analysis
The complementary sites between miR-137 and 3′-UTR of TRPC3 were analyzed using TargetScan. The wild-type (WT) 3′-UTR sites of TRPC3 with miR-137 binding sites were inserted in the psiCHECK2 vector (Promega, Madison, WI, USA) to generate the WT vectors. The corresponding mutant (MUT) vectors were obtained through mutating the binding sites. WT or MUT luciferase reporter vectors were transfected in VSMCs together with inhibitor NC, miR-137 inhibitor, mimics NC or miR-137 mimics for 24 h. Luciferase activity was determined via use of luciferase assay kit (Promega).
2.10 Statistical analysis
SPSS 20.0 (SPSS, Chicago, IL, USA) was utilized to statistical analysis. The experiments were performed more than three times. The data are presented as mean ± standard deviation (SD). The difference was assessed via Student's t-test or one-way ANOVA with Tukey's post-hoc test. It was considered statistically significant when p < 0.05.
3 RESULTS
3.1 Tan IIA mitigates VSMC proliferation and migration induced via ox-LDL.
ox-LDL-treated VSMCs were stimulated with indicated concentrations (2.5, 5, and 10 μg/mL) of Tan IIA for 12, 24, or 48 h. As shown in Figure 1A, VSMC proliferation was promoted due to ox-LDL stimulation, which was relieved because of treatment with Tan IIA. Moreover, Tan IIA notably blunted VSMC migration induced due to ox-LDL for 12 h (Figure 1B). Furthermore, ox-LDL stimulation remarkably decreased miR-137 level, and augmented TRPC3 and PCNA expression in VSMCs, and Tan IIA treatment reversed this effect (Figure 1C,D). Together, Tan IIA constrained VSMC proliferation and migration mediated via ox-LDL. VSMCs exposed to 10 μg/mL of Tan IIA were selected for further study.
3.2 Tan IIA enhances miR-137 expression to reduce proliferation and migration of VSMCs treated with ox-LDL
ox-LDL-treated VSMCs were stimulated with indicated concentrations (2.5, 5, and 10 μg/mL) of Tan IIA for 24 h. MiR-137 has been regarded as a target of Tan IIA. miR-137 levels were declined in VSMCs by ox-LDL treatment, which was rescued by introduction of Tan IIA (Figure 1C,D). Moreover, miR-137 levels were downregulated because of miR-137 inhibitor in the presence or absence of ox-LDL and Tan IIA (Figure 2A,B). In order to further analyze if Tan IIA-mediated VSMC processes under ox-LDL stimulation was associated with miR-137, VSMCs were transfected with inhibitor NC or miR-137 inhibitor and then suffered from ox-LDL and 10 μg/mL Tan IIA for 24 h. The suppressive effects of proliferation, migration and PCNA expression induced by Tan IIA were attenuated after miR-137 silencing (Figure 2C–E). These findings indicated Tan IIA constrained VSMC proliferation and migration caused because of ox-LDL through increasing miR-137.
3.3 TRPC3 is a target of miR-137
TRPC3 was suggested as a target of miR-137 by TargetScan webserver. TRPC3 expression was significantly increased in VSMCs after ox-LDL stimulation, and Tan IIA treatment reduced TRPC3 level, which was rescued because of miR-137 knockdown (Figure 3A). The complementary sequences between miR-137 and TRPC3 were presented in Figure 3B. In order to confirm their association, the WT and MUT TRPC3 vectors were conducted and transfected in VSMCs. Dual-luciferase reporter analysis displayed luciferase activity was increased after miR-137 inhibition and declined due to miR-137 mimics in WT group, but it was not changed in MUT group (Figure 3C). In addition, the influence of miR-137 on TRPC3 level was assessed in VSMCs. Results showed TRPC3 level was negatively regulated by miR-137 (Figure 3D). These results indicated TRPC3 was targeted via miR-137 in VSMCs.
3.4 Tan IIA reduces TRPC3 expression by enhancing miR-137 to inhibit proliferation and migration of VSMCs treated with ox-LDL
The following experiments were adopted to study the function of TRPC3 in VSMCs under ox-LDL and Tan IIA treatment. VSMCs were transfected with inhibitor NC + sh-NC, miR-137 inhibitor, sh-TRPC3 or miR-137 inhibitor+sh-TRPC3 and then incubated with ox-LDL and 10 μg/mL Tan IIA for 24 h. As a result, miR-137 expression up-regulated by treatment with Tan IIA was significantly decreased in VSMCs treated with ox-LDL because of transfection with miR-137 inhibitor, while it was not affected by transfection with sh-TRPC3 (Figure 4A). Moreover, miR-137 silencing led to significant elevation of TRPC3 and PCNA levels in ox-LDL- and Tan IIA-treated VSMCs, and this effect was reversed because of transfection with sh-TRPC3 (Figure 4A,B). Additionally, TRPC3 silencing by sh-TRPC3 aggravated Tan IIA-induced inhibition of VSMC proliferation and migration, and exacerbated Tan IIA-induced VSMC apoptosis (Figure 4C–E). Furthermore, TRPC3 attenuated the influences of miR-137 inhibitor (Figure 4C–E). These results suggested Tan IIA inhibited VSMC proliferation and migration but promoted VSMC apoptosis caused because of ox-LDL through regulating miR-137/TRPC3 axis.
4 DISCUSSION
Atherosclerosis is a vascular disease characterized via lipid-laden plaques formation in arterial wall.21 VSMCs are the main component of the vessel wall, and VSMC function is linked to atherosclerosis progression.21, 22 The traditional Chinese medicine has a therapeutic potential for treatment of atherosclerosis.23 This study wanted to explore a drug for therapy of atherosclerosis. ox-LDL could function on multiple cell types including VSMCs in atherosclerosis, and the ox-LDL-stimulated VSMCs could be regarded as a cellular model for studying atherosclerosis.24, 25 In this study, we first found Tan IIA could attenuate VSMC proliferation and migration through regulating miR-137 and TRPC3 in ox-LDL-induced atherosclerotic model.
Tan IIA is a class of abietane diterpene compound exhibiting anti-cancer activity by inhibiting proliferation, migration, metastasis and inflammatory response.26 Moreover, Tan IIA exhibited the anti-atherosclerosis role via inhibiting oxidative stress and inflammation.8, 9 Furthermore, this anti-atherosclerosis role might be associated with the regulation of macrophages and endothelial cells.27, 28 Here we mainly investigated the roles of Tan IIA in VSMC function. ox-LDL stimulation could induce VSMC proliferation and migration, which promotes plaque formation in atherosclerosis.25, 29
Similarly, we also established ox-LDL-induced cellular model, and found Tan IIA could inhibit ox-LDL-induced hyperproliferation and migration of VSMCs. It was also consistent to that under high glucose or homocysteine condition.30, 31 In the current work, we were the first to document that Tan IIA could play the pharmacological effect on atherosclerosis by attenuating VSMC proliferation and migration.
Previous studies demonstrated that deregulated miRNAs were associated with the mechanism mediated by Tan IIA in atherosclerosis.32 Many miRNAs are reported to be linked to VSMC proliferation and migration after ox-LDL treatment, such as miR-129-5p, miR-539-5p, and miR-192-5p.25, 33, 34 MiR-137 has been suggested to be reduced in atherosclerosis patients.14 Similarly, in this research, we validated miR-137 level was also reduced in VSMCs treated with ox-LDL. Moreover, it has been reported that miR-137 could repress proliferation and migration of VSMCs.13 This indicated the anti-proliferative and anti-migratory roles of miR-137 in VSMCs. Consistent to this report, our study also confirmed this effect, and firstly found miR-137 knockdown attenuated the protective function of Tan IIA in VSMCs stimulated via ox-LDL, which was in agreement with that in lung cancer cell as a previous report.15 This uncovered that the effect of Tan IIA was associated with miR-137.
In addition, we explored the downstream target of miR-137, and validated Tan IIA could reduce TRPC3 expression by directly targeting miR-137 in VSMCs. TRPC3 is linked to progression of cardiovascular diseases, including atherosclerosis.35 Our work found TRPC3 silencing promoted the suppressive effects of Tan IIA on VSMC proliferation and migration, indicating the promoting roles of TRPC3, which was also consistent to that in ovarian cancer and melanoma.36, 37 Furthermore, we found TRPC3 silencing partly attenuated the effects of miR-137 knockdown on Tan IIA-mediated inhibition of VSMC proliferation and migration after ox-LDL stimulation, uncovering Tan IIA up-regulated miR-137 to downregulate TRPC3, leading to inhibition of ox-LDL-induced proliferation and migration. According to the graphic summary in Figure 5, our study showed that Tan IIA inhibited TRPC3 expression by up-regulating miR-137 in ox-LDL-induced VSMC cell lines, and inhibited cell proliferation, migration, and promoted cell apoptosis, which providing a novel idea for therapy of VSMC dysfunction.
The current research disclosed the pharmacological activity of Tan IIA in VSMCs treated with ox-LDL by inhibiting the proliferation and migration, possibly through increasing miR-137 and further reducing TRPC3. This research indicated a new understanding on the anti-atherosclerotic activity of Tan IIA. It provided an experimental basis for the research and application of Tan IIA as a drug in diseases. The research ideas involved in this research also provided a strategy for the study of more disease mechanisms.
ACKNOWLEDGMENTS
The authors give their sincere gratitude to the reviewers for their constructive comments.
CONFLICT OF INTEREST STATEMENT
The authors declare that there is no conflict of interest.
