- Email: [email protected]
db mice through up-regulation of FoxO1 and TRPC6
Biomedicine & Pharmacotherapy 74 (2015) 35–41
Available online at
ScienceDirect www.sciencedirect.com
Original Article
Down-regulation of SENCR promotes smooth muscle cells proliferation and migration in db/db mice through up-regulation of FoxO1 and TRPC6 Zhi-qing Zou, Juan Xu, Li Li, Ye-shan Han * Department of Anesthesiology, Changzhou No. 2 People’s Hospital, No. 29, Xinglong Lane, Tianning District, 213003 Changzhou, Jiangsu Province, P.R. China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 June 2015 Accepted 25 June 2015
Background: The inappropriate proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in the atherosclerotic process. SENCR was reported to be associated with cell migration in human smooth muscle cells. However, the regulation role of SENCR in SMCs is still not fully understood. Methods: qRT-PCR and Western blotting were performed to detect the mRNA and protein levels of SENCR, FOXO1 and TRPC6 in SMCs of db/db mice and SMCs exposed to high glucose. The regulation of SENCR on the expression of FoxO1 and TRPC6 were examined with luciferase report assays. Furthermore, we investigated the effect of SENCR on VSMCs proliferation and migration using MTT assay and cell migration assay, respectively. Results: Here, we found that SENCR was down-regulated in db/db mice and in SMCs exposed to high glucose. According to the result of luciferase assays, it was shown that SMCs knockdown enhanced the expression of FoxO1 and FoxO1 overexpression increased the expression of TRPC6. In addition, qRT-PCR revealed that SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration. Conclusion: In this study, our data indicated that down-regulation of SENCR promoted smooth muscle cells proliferation and migration in db/db mice through up-regulation of FoxO1 and TRPC6. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: SENCR Smooth muscle cells Proliferation Migration FoxO1 TRPC6
1. Introduction The increased risk for type 2 diabetes mellitus (T2DM) is the developing of several serious complications, among which cardiovascular disease, is the most common disease and mainly involves the accelerated development of atherosclerotic vascular changes [1]. The most common pathological change in atherosclerosis is the inappropriate proliferation of vascular smooth muscle cells (VSMCs), which occurs in response to arterial injury and plays a crucial role in the atherosclerotic process [2]. It is reported that hyperproliferation and migration of VSMCs have been shown to lead to lesion formation in restenosis, atherosclerosis, and hypertension [3]. However, the molecular mechanism behind abnormal VSMC proliferation in diabetes is still not fully understood. Long noncoding RNAs (lncRNAs) have emerged as novel regulators of gene expression and played roles in diverse biological processes, such as proliferation, differentiation, and development through various modes of action [4,5]. In cardiovascular disease,
* Corresponding author. Tel.: +86 519 86633371; fax: +86 519 86633371. E-mail address: [email protected] (Y.-s. Han). http://dx.doi.org/10.1016/j.biopha.2015.06.009 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
few lncRNAs are reported to involved in cardiovascular development and pathophysiology, such as lncRNA Braveheart, CHRF, MALAT1 and LIPCAR [6]. Bell et al. used RNA sequencing of human coronary artery smooth muscle cells and identified several unannotated lncRNAs. Among these lncRNAs, they pinpointed a vascular cell-enriched lncRNA that they termed smooth muscle and endothelial cell-enriched migration/differentiation-associated long noncoding RNA, or in short SENCR, which resides within the first intron of Friend leukemia virus integration 1 (FLI1) in an antisense orientation [7]. Several studies demonstrated that SENCR is likely to be related with maintaining a normal, non-motile contractile phenotype in SMCs and its down-regulation in human coronary artery smooth muscle cells (HCASMCs) displayed increase in cell migration in scratch wound [8]. Therefore, elucidating the regulation role of SENCR in SMCs is helpful for further understanding its function. Various growth factors and cytokines are also known to be involved in SMCs proliferation and migration. FoxO1 (FKHR) proteins is a member of the transcription factor Forkhead box O (FoxO) family, which have important roles in regulating cellular differentiation, proliferation, survival in various cell lines, including cancer cells, fibroblasts, myoblasts, endothelial cells, and
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SMCs. It has been shown that contraction of VSMs mostly relies on the elevation of the intracellular free Ca2+ concentration. TRPC6 is a member of the short transient receptor potential (TRP) channel gene subfamily, which has been implicated as molecular candidates for channels mediating receptor-stimulated Ca2+ influx. In this study, we investigated the dysregulation of SENCR, FoxO1 and TRP6 in SMCs of db/db mice or SMCs exposed to high glucose. And we found that SMCs knockdown might enhance the expression of FoxO1 and FoxO1 overexpression increased the expression of TRPC6. In addition, SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration.
placed into the upper chamber per well with the non-coated membrane. Platelet-derived growth factor (PDGF) at 1 and 10 ng/ mL dissolved in DMEM medium containing 0.1% FBS was added in the bottom chamber. VSMCs (5104 cells per well) suspended in 100 L of DMEM containing 0.1% BSA was added to the upper chamber. After incubation for 5 hours at 37 8C and 5% CO2, cells on both side of the membrane were fixed and stained with Diff-Quick staining kit (Baxter Healthcare Corp). Cells on the upper side of the membrane were removed with a cotton swab. The average number of cells from 5 randomly chosen high power (200) fields on the lower side of the membrane was counted.
2. Materials and methods
2.6. Real-time RT-PCR
2.1. Experimental animals and collection of VSMCs
Quantitative real-time reverse transcription (RT-PCR) was used to determine the mRNA expression of SENCR, FoxO1 and TRP6 in SMCs. Total RNA was extracted with Trizol and treated with RNasefree DNase I. First-strand cDNA was generated from total RNA by RT with the RevertAidHMinus First Strand cDNA synthesis kit. RTPCR was performed using primers with either 50 -6-carboxyfluorescein (FAM) labeled probes (Integrated DNA Technologies, Inc.) or SybrGreen master mix according to the manufacturer’s instructions. The expression level of each candidate gene was internally normalized against that of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All mRNA levels were calculated using DD the 2 Ct method.
Animal experiments were approved by the Experimental Animal Ethics Committee of Changzhou No. 2 People’s Hospital. Male db/db mice at 8-, 12- and 16-weeks-old were used. Age matched nondiabetic db/m mice were used as the controls. Mice were housed in micro-isolator cages in a pathogen-free facility. After 1-week acclimation, mice were euthanized with CO2 and decapitated. The thoracic aorta was immediately dissected and enzymatically digested at 37 8C for 3.5 h using a 0.25% trypsin solution. Following digestion, tissue fragments were explanted in a 35-mm culture dish. Contaminated fibroblasts were separated from the VSMCs due to their differing adhesion abilities. The VSMCs used for real-time polymerase chain reaction (PCR) and western blotting experiments were frozen on dry ice and stored at 80 8C. 2.2. Cell culture, transfection, and luciferase assays Mouse vascular smooth muscle cells and human smooth muscle cell lines C-12511 were obtained from ATCC (Manassas, Virginia) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 8C. pcDNA-SENCR and were used to overexpress SENCR and FoxO1. The luciferase reporter constructs were cotransfected using Lipofectamine 2000 (Invitrogen) with pcDNA-SENCR, pcDNA-FoxO1, si-SENCR or siFoxO1 into SMCs and luciferase activity was measured using the luciferase assay kit. Relative promoter activities were expressed as luminescence relative units normalized for co-transfected bgalactosidase expression in the cell extracts.
2.7. Western blot analysis VSMCs were re-suspended in lysis buffer and 2 mg/mL protease inhibitor cocktail (Roche Diagnostics Corp). Protein concentrations were determined using the Bradford Protein assay kit (Beyotime Biotechnology, China). Equal amount of protein were analyzed by SDS-PAGE and immunoblotting. Primary antibodies used include the following: FoxO1, TRPC6 and GAPDH (BD Transduction Laboratories). Secondary antibodies were fluorescence-labeled antibodies. Bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnology). 2.8. Statistics All data are given as means SD. The statistical significance of differences between mean values was assessed with Student’s t-test. Differences were regarded as statistically significant for P < 0.05.
2.3. Chromatin-immunoprecipitation sequencing 3. Results Chromatin immunoprecipitation (ChIP) assays were carried out as described previously using the ChIP Assay Kit from Upstate Biotech following the manufacturer’s protocol [9]. Primer sequence for FoxO1 is available upon request. 2.4. VSMC proliferation assay MTT assay was performed to measure the VSMC proliferation. Briefly, VSMCs were harvested by trypsinization and plated in a 96well plate at a density of 2 103 cells/mL. Then, VSMCs were grown in 100 mL of medium at 37 8C for 24 h, followed by incubation with 20 mL MTT (3-[4,5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide) for 4 h. Then, 150 mL dimethyl sulfoxide (DMSO) was added to each well and the absorbance was measured at 490 nm using a microplate reader.
3.1. SENCR was down-regulated in db/db mice The thickness of aortic smooth muscle layer in db/db mice was significantly enhanced comparing to that in control mice. Moreover, in db/db mice, aortic smooth muscle layer appeared to be more thick in a time-dependent manner (Fig. 1A). Then, SMCs were separated from db/db mice at different weeks and corresponding control mice. RT-PCR and western blot were performed to examine the expression of SENCR, FoxO1 and TRPC6 in separated SMCs. It has been shown that SENCR was down-regulated in db/db mice in a time-dependent manner (Fig. 1B). On the other hand, the expression of FoxO1 and TRPC6 were significantly higher in db/db mice than that in control at transcription and translation level (Fig. 1C).
2.5. VSMC migration assay
3.2. SENCR was down-regulated in mouse VSMCs exposed to high glucose
Cell migration assay was performed in a 24-well plate with 8 mm pore size chamber inserts (Corning). 5 104 cells were
To further verify the dysregulation of SENCR, FoxO1 and TRPC6 in db/db mice, we detected their expression in primary mouse
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Fig. 1. SENCR was down-regulated in SMCs of db/db mice. A. The thickness of aortic smooth muscle layer in db/db mice and control mice. B. The mRNA level of SENCR in smooth muscle cells separated from db/db mice and control. C. The expression of FoxO1 and TRPC6 in smooth muscle cells separated from db/db mice and control. Mean SD, all results were repeated for three times; *VS control group, *P < 0.05; **P < 0.01.
vascular smooth muscle cells (VSMCs) and smooth muscle cell lines C-12511. Mouse VSMCs and C-12511 were exposed to high glucose (27.5 mM) for 24 h. It has been shown that the mRNA level of SENCR was decreased in cells exposed to high glucose (Fig. 2A). When C-12511 cells were transfected with b-gal and report plasmid containing transcription-binding sites of FoxO1 and exposed to high glucose treatment or low glucose treatment. As shown in Fig. 2B, the higher transcription activity of FoxO1 was observed in C-12511 cells exposed to high glucose treatment than that in cells exposed to low glucose treatment. In addition, the expression of FoxO1 and TRPC6 were also significantly higher in C-12511 cells exposed to high glucose treatment at transcription and translation level (Fig. 2C). Taken together, these data indicated that the dysregulation of SENCR, FoxO1 and TRPC6 might involve in the development of SMCs in db/db mice. 3.3. SENCR regulated the expression of FoxO1 in SMCs To determine the effect of SENCR on the expression of FoxO1, siSENCR were transfected into SMCs. Obviously, si-SENCR effectively suppressed the expression of SENCR in SMCs (Fig. 3A). Moreover, the mRNA level of FoxO1 and transcription activity of FoxO1 were
significantly enhanced in SMCs transfected with si-SENCR (Fig. 3B and C). We further found that si-SENCR increased the binding activity of FoxO1 with H3 histone through RT-PCR (Fig. 3D). Next, C-12511 cells were exposed to high glucose or low glucose or overexpressed SENCR by pcDNA-SENCR, to examine the regulation role of SENCR. As shown in Fig. 3E, overexpression of SENCR reversed the up-regulation of FoxO1 induced by high glucose. At the same time, the increasing transcription activity of FoxO1 induced by high glucose was also reversed by pcDNA-SENCR (Fig. 3F). These findings suggested that SENCR exerted the suppressive effects on the expression of FoxO1 in SMCs. 3.4. FoxO1 regulated the expression of TRPC6 in C-12511 cells In this study, the results of ChIP indicated that FoxO1 had binding sites of TRPC6 promoter (Fig. 4A). To demonstrate the regulation role of FoxO1 on the expression of TRPC6, C-12511 cells overexpressed FoxO1 through transfected with pcDNA-FoxO1. It has been shown that the mRNA and protein levels of TRPC6 were all enhanced by overexpressing FoxO1 (Fig. 4B). On the other hand, si-FoxO1 could reverse the up-regulation of TRPC6 induced by high glucose (Fig. 4C). When C-12511 cells were transfected with b-gal
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Fig. 2. SENCR was down-regulated in mouse VSMCs and C-12511 cells exposed to glucose. Mouse VSMCs and smooth muscle cell lines C-12511 were both exposed to low glucose (5.5 mM) and high glucose (27.5 mM). A. The mRNA level of SENCR in these two kinds of cells. B. The transcription activity of FoxO1 in C-12511 cells transfected with b-gal and report plasmid containing transcription binding sites of FoxO1 and exposed to high glucose. C. The mRNA and protein level of FoxO1 and TRPC6 in these two kinds of cells. Mean SD, all results were repeated for three times; **VS control group, **P < 0.01.
and report plasmid of TRPC6 promoter and exposed to high glucose or low glucose. As shown in Fig. 4D, si-FoxO1 also reversed the increasing binding activity of FoxO1 with TRPC6 promoter induced by high glucose.
reversed the effect of high glucose on mouse VSMCs proliferation and migration.
3.5. SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration
In the present study, we provide evidence for the involvement of SENCR in smooth muscle cells proliferation and migration in db/db mice. And we found that SENCR knockdown might enhance the expression of FoxO1 and FoxO1 overexpression increased the expression of TRPC6 in SMCs. In light of the molecular biological data, we suggest that SENCR is a candidate target molecular for treatment of SMCs proliferation in db/db mice. We observed that the thickness of aortic smooth muscle layer in db/db mice was significantly greater than that in control mice. Serial intravascular ultrasonographic studies have indicated that excessive intimal hyperplasia of vascular SMCs associated with type 2 diabetes might result in increased restenosis, which played
The effect of SENCR on mouse VSMCs proliferation and migration were examined. VSMCs were treated with high glucose or low glucose or transfected with pcDNA-SENCR. As shown in Fig. 5A, high glucose significantly enhanced the cell viability of VSMCs comparing to the effect of low glucose. Moreover, SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation. With respect to cell migration, high glucose promoted cell migration of VSMCs and SENCR overexpression also reversed the effect of high glucose on mouse VSMCs proliferation (Fig. 5B). These data demonstrated that SENCR overexpression
4. Discussion
Z.- Zou et al. / Biomedicine & Pharmacotherapy 74 (2015) 35–41
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Fig. 3. SENCR regulated the expression of FoxO1. A. The mRNA level of SENCR in SMCs transfected with si-SENCR. B. The mRNA level of FoxO1 in SMCs transfected with siSENCR. C. The transcription activity of FoxO1 in SMCs transfected with si-SENCR. D. The binging activity of FoxO1 with H3 histone in SMCs transfected with si-SENCR. **VS control group, **P < 0.01. E. The expression of FoxO1 in C-12511 cells transfected with pcDNA-SENCR. F. The transcription activity of FoxO1 in C-12511 cells transfected with pcDNA-SENCR. **VS 5.5 mM, P < 0.01; ##VS 27.5 mM + pcDNA, P < 0.01. Mean SD, all results were repeated for three times.
a role in the vascular complications of type 2 diabetes [10]. The proliferation of vascular SMCs is a crucial process in the pathogenesis of atherosclerosis. Generally, there is a balance between the proliferation and apoptosis of VSMCs under physiological conditions. When this balance is disturbed by
hyperglycemia and hyperinsulinemia, VSMC proliferation is increased and promotes the development of atherosclerosis [11]. Therefore, identifying the regulators of this proliferative response in VSMC may lead to therapeutic strategies aimed at blocking or inhibiting VSMC growth.
Fig. 4. FoxO1 regulated the expression of TRPC6 in C-12511 cells. A. FoxO1 could combine with TRPC6 promoter. B. The expression of TRPC6 in C-12511 cells transfected with pcDNA-FoxO1. **VS control group, **P < 0.01. C. The expression of TRPC6 in C-12511 cells transfected with si-FoxO1. D. The binging activity of FoxO1 with TRPC6 promoter in C-12511 cells transfected with si-FoxO1. **VS 5.5 mM, P < 0.01; ##VS 27.5 mM + pcDNA, P < 0.01. Mean SD, all results were repeated for three times.
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Fig. 5. SENCR overexpression reversed the effect of high glucose on SMCs proliferation and migration. A. Cell viability of SMCs exposed to glucose or pcDNA-SENCR. B. Cell migration of SMCs exposed to glucose or pcDNA-SENCR. Mean SD, all results were repeated for three times. **VS 5.5 mM, P < 0.01; ##VS 27.5 mM + pcDNA, P < 0.01.
It has been reported that several cytokines and non-coding RNAs are involved in VSMC proliferation and migration [12,13]. In particular, a novel lncRNA SENCR was described to be smooth muscle cell-and endothelial cell-enriched [7]. Bell et al. reported that SENCR silencing upregulated several genes associated with cell migration and attenuation of SENCR expression resulted in hypermotile phenotype of human smooth muscle cells [7]. In this study, we also found that the expression of SENCR was decreased in smooth muscle cells of db/db mice and in VSMCs exposed to high glucose. In addition, VSMC proliferation and migration assay demonstrated that SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration, which was consistent with previous study. To elucidate the mechanism how SENCR involved in VSMCs proliferation and migration, we examine the regulation of SENCR on the expression of FoxO1. FoxO1 is a prominent member of the Forkhead box family and is involved in regulating metabolism, cell proliferation, oxidative stress response and cell death [14]. Several reports indicated that FoxO1 played crucial role in SMCs proliferation and migration [15]. Park et al. reported that Foxo1 transcription factor is related to the recruitment of smooth muscle cells to nascent blood vessels [15]. Moreover, ubiquitin-mediated degradation of FoxO1 contributed to promote SMCs proliferation and survival by C Terminus of Hsc70-interacting protein [16]. It has been reported that forkhead signaling pathway contributed to the regulation of VSMC proliferation, cell cycle progression, and neointimal hyperplasia [17]. Our results showed that mRNA level of FoxO1 could be enhanced by SENCR knockdown. In db/db mice, the mRNA and protein level of FoxO1 was significantly upregulated comparing to control mice. These data indicated that downregulation of SENCR promoted SMCs proliferation and migration in db/db mice through upregulation of FoxO1. Except for the dysregulation of FoxO1 by SENCR in SMCs, we also observed the differential expression of another factors: TRPC6, which was upregulated in SMCs of db/db mice. TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells [18]. Yu et al. reported that upregulation of TRPC6 was involved in platelet-derived growth factor-mediated pulmonary artery smooth muscle cells proliferation [19]. In addition, deletion of TRPC4 and TRPC6 in mice was reported to impair smooth muscle contraction and intestinal motility in vivo [20]. Here, we further determined that FoxO1 regulated the expression of TRPC6 through binding to its promoter.
In summary, the in vivo and in vitro experiments in this study have shown that SENCR was down-regulated in db/db mice and VSMCs exposed to high glucose. While SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration, indicating that SENCR may be involved in the pathological process of SMCs proliferation. We further showed that SENCR promoted SMCs proliferation and migration in db/db mice through regulation of FoxO1 and TRPC6. These findings suggest that SENCR is a candidate target molecular for treatment of SMCs proliferation in db/db mice. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by grant from the Major Scientific and Technological Projects of Changzhou Health Bureau (2013.01– 2015.01, ZD201211). References [1] B. Wirostko, T.Y. Wong, R. Simo, Vascular endothelial growth factor and diabetic complications, Prog. Retin. Eye Res. 27 (2008) 608–621. [2] X. Sun, F. Han, J. Yi, N. Hou, Z. Cao, The effect of telomerase activity on vascular smooth muscle cell proliferation in type 2 diabetes in vivo and in vitro, Mol. Med. Rep. 7 (2013) 1636–1640. [3] A.R. Brasier, A. Recinos 3rd., M.S. Eledrisi, Vascular inflammation and the renin-angiotensin system, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 1257–1266. [4] A.M. Khalil, M. Guttman, M. Huarte, M. Garber, A. Raj, D. Rivea Morales, et al., Many human large intergenic noncoding RNAs associate with chromatinmodifying complexes and affect gene expression, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 11667–11672. [5] M. Guttman, J. Donaghey, B.W. Carey, M. Garber, J.K. Grenier, G. Munson, et al., lincRNAs act in the circuitry controlling pluripotency and differentiation, Nature 477 (2011) 295–300. [6] T. Thum, R. Kumarswamy, The smooth long noncoding RNA SENCR, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 1124–1125. [7] R.D. Bell, X. Long, M. Lin, J.H. Bergmann, V. Nanda, S.L. Cowan, et al., Identification and initial functional characterization of a human vascular cellenriched long noncoding RNA, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 1249–1259. [8] A. Leung, K. Stapleton, R. Natarajan, Functional long non-coding RNAs in vascular smooth muscle cells, Curr. Top. Microbiol. Immunol. (2015). [9] Z.-P. Liu, Z. Wang, H. Yanagisawa, E.N. Olson, Phenotypic modulation of smooth muscle cells through interaction of FoxO4 and myocardin, Dev. Cell 9 (2005) 261–270.
Z.- Zou et al. / Biomedicine & Pharmacotherapy 74 (2015) 35–41 [10] A. Rudijanto, The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis, Acta Med. Indones. 39 (2007) 86–93. [11] T. Jacob, N. Clouden, A. Hingorani, E. Ascher, The effect of cotinine on telomerase activity in human vascular smooth muscle cells, J. Cardiovasc. Surg. 50 (2009) 345–349. [12] A. Leung, C. Trac, W. Jin, L. Lanting, A. Akbany, P. Saetrom, et al., Novel long noncoding RNAs are regulated by angiotensin II in vascular smooth muscle cells, Circ. Res. 113 (2013) 266–278. [13] T. Thum, J. Fiedler, LINCing MALAT1 and angiogenesis, Circ. Res. 114 (2014) 1366–1368. [14] P. Puthanveetil, D. Zhang, Y. Wang, F. Wang, A. Wan, A. Abrahani, et al., Diabetes triggers a PARP1 mediated death pathway in the heart through participation of FoxO1, J. Mol. Cell. Cardiol. 53 (2012) 677–686. [15] S.-H. Park, H. Sakamoto, K. Tsuji-Tamura, T. Furuyama, M. Ogawa, FoxO1 is essential for in vitro vascular formation from embryonic stem cells, Biochem. Biophys. Res. Commun. 390 (2009) 861–866.
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[16] F. Li, P. Xie, Y. Fan, H. Zhang, L. Zheng, D. Gu, et al., C terminus of Hsc70-interacting protein promotes smooth muscle cell proliferation and survival through ubiquitinmediated degradation of FoxO1, J. Biol. Chem. 284 (2009) 20090–20098. [17] M.R. Abid, K. Yano, S. Guo, V.I. Patel, G. Shrikhande, K.C. Spokes, et al., Forkhead transcription factors inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia, J. Biol. Chem. 280 (2005) 29864–29873. [18] S. Jung, R. Strotmann, G. Schultz, T.D. Plant, TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells, Am. J. Phys. Cell Physiol. (2002). [19] Y. Yu, M. Sweeney, S. Zhang, O. Platoshyn, J. Landsberg, A. Rothman, et al., PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression, Am. J. Phys. Cell Physiol. 284 (2003) C316–C330. [20] V.V. Tsvilovskyy, A.V. Zholos, T. Aberle, S.E. Philipp, A. Dietrich, M.X. Zhu, et al., Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo, Gastroenterology 137 (2009) 1415–1424.
Available online at
ScienceDirect www.sciencedirect.com
Original Article
Down-regulation of SENCR promotes smooth muscle cells proliferation and migration in db/db mice through up-regulation of FoxO1 and TRPC6 Zhi-qing Zou, Juan Xu, Li Li, Ye-shan Han * Department of Anesthesiology, Changzhou No. 2 People’s Hospital, No. 29, Xinglong Lane, Tianning District, 213003 Changzhou, Jiangsu Province, P.R. China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 June 2015 Accepted 25 June 2015
Background: The inappropriate proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in the atherosclerotic process. SENCR was reported to be associated with cell migration in human smooth muscle cells. However, the regulation role of SENCR in SMCs is still not fully understood. Methods: qRT-PCR and Western blotting were performed to detect the mRNA and protein levels of SENCR, FOXO1 and TRPC6 in SMCs of db/db mice and SMCs exposed to high glucose. The regulation of SENCR on the expression of FoxO1 and TRPC6 were examined with luciferase report assays. Furthermore, we investigated the effect of SENCR on VSMCs proliferation and migration using MTT assay and cell migration assay, respectively. Results: Here, we found that SENCR was down-regulated in db/db mice and in SMCs exposed to high glucose. According to the result of luciferase assays, it was shown that SMCs knockdown enhanced the expression of FoxO1 and FoxO1 overexpression increased the expression of TRPC6. In addition, qRT-PCR revealed that SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration. Conclusion: In this study, our data indicated that down-regulation of SENCR promoted smooth muscle cells proliferation and migration in db/db mice through up-regulation of FoxO1 and TRPC6. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: SENCR Smooth muscle cells Proliferation Migration FoxO1 TRPC6
1. Introduction The increased risk for type 2 diabetes mellitus (T2DM) is the developing of several serious complications, among which cardiovascular disease, is the most common disease and mainly involves the accelerated development of atherosclerotic vascular changes [1]. The most common pathological change in atherosclerosis is the inappropriate proliferation of vascular smooth muscle cells (VSMCs), which occurs in response to arterial injury and plays a crucial role in the atherosclerotic process [2]. It is reported that hyperproliferation and migration of VSMCs have been shown to lead to lesion formation in restenosis, atherosclerosis, and hypertension [3]. However, the molecular mechanism behind abnormal VSMC proliferation in diabetes is still not fully understood. Long noncoding RNAs (lncRNAs) have emerged as novel regulators of gene expression and played roles in diverse biological processes, such as proliferation, differentiation, and development through various modes of action [4,5]. In cardiovascular disease,
* Corresponding author. Tel.: +86 519 86633371; fax: +86 519 86633371. E-mail address: [email protected] (Y.-s. Han). http://dx.doi.org/10.1016/j.biopha.2015.06.009 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
few lncRNAs are reported to involved in cardiovascular development and pathophysiology, such as lncRNA Braveheart, CHRF, MALAT1 and LIPCAR [6]. Bell et al. used RNA sequencing of human coronary artery smooth muscle cells and identified several unannotated lncRNAs. Among these lncRNAs, they pinpointed a vascular cell-enriched lncRNA that they termed smooth muscle and endothelial cell-enriched migration/differentiation-associated long noncoding RNA, or in short SENCR, which resides within the first intron of Friend leukemia virus integration 1 (FLI1) in an antisense orientation [7]. Several studies demonstrated that SENCR is likely to be related with maintaining a normal, non-motile contractile phenotype in SMCs and its down-regulation in human coronary artery smooth muscle cells (HCASMCs) displayed increase in cell migration in scratch wound [8]. Therefore, elucidating the regulation role of SENCR in SMCs is helpful for further understanding its function. Various growth factors and cytokines are also known to be involved in SMCs proliferation and migration. FoxO1 (FKHR) proteins is a member of the transcription factor Forkhead box O (FoxO) family, which have important roles in regulating cellular differentiation, proliferation, survival in various cell lines, including cancer cells, fibroblasts, myoblasts, endothelial cells, and
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SMCs. It has been shown that contraction of VSMs mostly relies on the elevation of the intracellular free Ca2+ concentration. TRPC6 is a member of the short transient receptor potential (TRP) channel gene subfamily, which has been implicated as molecular candidates for channels mediating receptor-stimulated Ca2+ influx. In this study, we investigated the dysregulation of SENCR, FoxO1 and TRP6 in SMCs of db/db mice or SMCs exposed to high glucose. And we found that SMCs knockdown might enhance the expression of FoxO1 and FoxO1 overexpression increased the expression of TRPC6. In addition, SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration.
placed into the upper chamber per well with the non-coated membrane. Platelet-derived growth factor (PDGF) at 1 and 10 ng/ mL dissolved in DMEM medium containing 0.1% FBS was added in the bottom chamber. VSMCs (5104 cells per well) suspended in 100 L of DMEM containing 0.1% BSA was added to the upper chamber. After incubation for 5 hours at 37 8C and 5% CO2, cells on both side of the membrane were fixed and stained with Diff-Quick staining kit (Baxter Healthcare Corp). Cells on the upper side of the membrane were removed with a cotton swab. The average number of cells from 5 randomly chosen high power (200) fields on the lower side of the membrane was counted.
2. Materials and methods
2.6. Real-time RT-PCR
2.1. Experimental animals and collection of VSMCs
Quantitative real-time reverse transcription (RT-PCR) was used to determine the mRNA expression of SENCR, FoxO1 and TRP6 in SMCs. Total RNA was extracted with Trizol and treated with RNasefree DNase I. First-strand cDNA was generated from total RNA by RT with the RevertAidHMinus First Strand cDNA synthesis kit. RTPCR was performed using primers with either 50 -6-carboxyfluorescein (FAM) labeled probes (Integrated DNA Technologies, Inc.) or SybrGreen master mix according to the manufacturer’s instructions. The expression level of each candidate gene was internally normalized against that of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All mRNA levels were calculated using DD the 2 Ct method.
Animal experiments were approved by the Experimental Animal Ethics Committee of Changzhou No. 2 People’s Hospital. Male db/db mice at 8-, 12- and 16-weeks-old were used. Age matched nondiabetic db/m mice were used as the controls. Mice were housed in micro-isolator cages in a pathogen-free facility. After 1-week acclimation, mice were euthanized with CO2 and decapitated. The thoracic aorta was immediately dissected and enzymatically digested at 37 8C for 3.5 h using a 0.25% trypsin solution. Following digestion, tissue fragments were explanted in a 35-mm culture dish. Contaminated fibroblasts were separated from the VSMCs due to their differing adhesion abilities. The VSMCs used for real-time polymerase chain reaction (PCR) and western blotting experiments were frozen on dry ice and stored at 80 8C. 2.2. Cell culture, transfection, and luciferase assays Mouse vascular smooth muscle cells and human smooth muscle cell lines C-12511 were obtained from ATCC (Manassas, Virginia) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 8C. pcDNA-SENCR and were used to overexpress SENCR and FoxO1. The luciferase reporter constructs were cotransfected using Lipofectamine 2000 (Invitrogen) with pcDNA-SENCR, pcDNA-FoxO1, si-SENCR or siFoxO1 into SMCs and luciferase activity was measured using the luciferase assay kit. Relative promoter activities were expressed as luminescence relative units normalized for co-transfected bgalactosidase expression in the cell extracts.
2.7. Western blot analysis VSMCs were re-suspended in lysis buffer and 2 mg/mL protease inhibitor cocktail (Roche Diagnostics Corp). Protein concentrations were determined using the Bradford Protein assay kit (Beyotime Biotechnology, China). Equal amount of protein were analyzed by SDS-PAGE and immunoblotting. Primary antibodies used include the following: FoxO1, TRPC6 and GAPDH (BD Transduction Laboratories). Secondary antibodies were fluorescence-labeled antibodies. Bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnology). 2.8. Statistics All data are given as means SD. The statistical significance of differences between mean values was assessed with Student’s t-test. Differences were regarded as statistically significant for P < 0.05.
2.3. Chromatin-immunoprecipitation sequencing 3. Results Chromatin immunoprecipitation (ChIP) assays were carried out as described previously using the ChIP Assay Kit from Upstate Biotech following the manufacturer’s protocol [9]. Primer sequence for FoxO1 is available upon request. 2.4. VSMC proliferation assay MTT assay was performed to measure the VSMC proliferation. Briefly, VSMCs were harvested by trypsinization and plated in a 96well plate at a density of 2 103 cells/mL. Then, VSMCs were grown in 100 mL of medium at 37 8C for 24 h, followed by incubation with 20 mL MTT (3-[4,5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide) for 4 h. Then, 150 mL dimethyl sulfoxide (DMSO) was added to each well and the absorbance was measured at 490 nm using a microplate reader.
3.1. SENCR was down-regulated in db/db mice The thickness of aortic smooth muscle layer in db/db mice was significantly enhanced comparing to that in control mice. Moreover, in db/db mice, aortic smooth muscle layer appeared to be more thick in a time-dependent manner (Fig. 1A). Then, SMCs were separated from db/db mice at different weeks and corresponding control mice. RT-PCR and western blot were performed to examine the expression of SENCR, FoxO1 and TRPC6 in separated SMCs. It has been shown that SENCR was down-regulated in db/db mice in a time-dependent manner (Fig. 1B). On the other hand, the expression of FoxO1 and TRPC6 were significantly higher in db/db mice than that in control at transcription and translation level (Fig. 1C).
2.5. VSMC migration assay
3.2. SENCR was down-regulated in mouse VSMCs exposed to high glucose
Cell migration assay was performed in a 24-well plate with 8 mm pore size chamber inserts (Corning). 5 104 cells were
To further verify the dysregulation of SENCR, FoxO1 and TRPC6 in db/db mice, we detected their expression in primary mouse
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Fig. 1. SENCR was down-regulated in SMCs of db/db mice. A. The thickness of aortic smooth muscle layer in db/db mice and control mice. B. The mRNA level of SENCR in smooth muscle cells separated from db/db mice and control. C. The expression of FoxO1 and TRPC6 in smooth muscle cells separated from db/db mice and control. Mean SD, all results were repeated for three times; *VS control group, *P < 0.05; **P < 0.01.
vascular smooth muscle cells (VSMCs) and smooth muscle cell lines C-12511. Mouse VSMCs and C-12511 were exposed to high glucose (27.5 mM) for 24 h. It has been shown that the mRNA level of SENCR was decreased in cells exposed to high glucose (Fig. 2A). When C-12511 cells were transfected with b-gal and report plasmid containing transcription-binding sites of FoxO1 and exposed to high glucose treatment or low glucose treatment. As shown in Fig. 2B, the higher transcription activity of FoxO1 was observed in C-12511 cells exposed to high glucose treatment than that in cells exposed to low glucose treatment. In addition, the expression of FoxO1 and TRPC6 were also significantly higher in C-12511 cells exposed to high glucose treatment at transcription and translation level (Fig. 2C). Taken together, these data indicated that the dysregulation of SENCR, FoxO1 and TRPC6 might involve in the development of SMCs in db/db mice. 3.3. SENCR regulated the expression of FoxO1 in SMCs To determine the effect of SENCR on the expression of FoxO1, siSENCR were transfected into SMCs. Obviously, si-SENCR effectively suppressed the expression of SENCR in SMCs (Fig. 3A). Moreover, the mRNA level of FoxO1 and transcription activity of FoxO1 were
significantly enhanced in SMCs transfected with si-SENCR (Fig. 3B and C). We further found that si-SENCR increased the binding activity of FoxO1 with H3 histone through RT-PCR (Fig. 3D). Next, C-12511 cells were exposed to high glucose or low glucose or overexpressed SENCR by pcDNA-SENCR, to examine the regulation role of SENCR. As shown in Fig. 3E, overexpression of SENCR reversed the up-regulation of FoxO1 induced by high glucose. At the same time, the increasing transcription activity of FoxO1 induced by high glucose was also reversed by pcDNA-SENCR (Fig. 3F). These findings suggested that SENCR exerted the suppressive effects on the expression of FoxO1 in SMCs. 3.4. FoxO1 regulated the expression of TRPC6 in C-12511 cells In this study, the results of ChIP indicated that FoxO1 had binding sites of TRPC6 promoter (Fig. 4A). To demonstrate the regulation role of FoxO1 on the expression of TRPC6, C-12511 cells overexpressed FoxO1 through transfected with pcDNA-FoxO1. It has been shown that the mRNA and protein levels of TRPC6 were all enhanced by overexpressing FoxO1 (Fig. 4B). On the other hand, si-FoxO1 could reverse the up-regulation of TRPC6 induced by high glucose (Fig. 4C). When C-12511 cells were transfected with b-gal
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Fig. 2. SENCR was down-regulated in mouse VSMCs and C-12511 cells exposed to glucose. Mouse VSMCs and smooth muscle cell lines C-12511 were both exposed to low glucose (5.5 mM) and high glucose (27.5 mM). A. The mRNA level of SENCR in these two kinds of cells. B. The transcription activity of FoxO1 in C-12511 cells transfected with b-gal and report plasmid containing transcription binding sites of FoxO1 and exposed to high glucose. C. The mRNA and protein level of FoxO1 and TRPC6 in these two kinds of cells. Mean SD, all results were repeated for three times; **VS control group, **P < 0.01.
and report plasmid of TRPC6 promoter and exposed to high glucose or low glucose. As shown in Fig. 4D, si-FoxO1 also reversed the increasing binding activity of FoxO1 with TRPC6 promoter induced by high glucose.
reversed the effect of high glucose on mouse VSMCs proliferation and migration.
3.5. SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration
In the present study, we provide evidence for the involvement of SENCR in smooth muscle cells proliferation and migration in db/db mice. And we found that SENCR knockdown might enhance the expression of FoxO1 and FoxO1 overexpression increased the expression of TRPC6 in SMCs. In light of the molecular biological data, we suggest that SENCR is a candidate target molecular for treatment of SMCs proliferation in db/db mice. We observed that the thickness of aortic smooth muscle layer in db/db mice was significantly greater than that in control mice. Serial intravascular ultrasonographic studies have indicated that excessive intimal hyperplasia of vascular SMCs associated with type 2 diabetes might result in increased restenosis, which played
The effect of SENCR on mouse VSMCs proliferation and migration were examined. VSMCs were treated with high glucose or low glucose or transfected with pcDNA-SENCR. As shown in Fig. 5A, high glucose significantly enhanced the cell viability of VSMCs comparing to the effect of low glucose. Moreover, SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation. With respect to cell migration, high glucose promoted cell migration of VSMCs and SENCR overexpression also reversed the effect of high glucose on mouse VSMCs proliferation (Fig. 5B). These data demonstrated that SENCR overexpression
4. Discussion
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Fig. 3. SENCR regulated the expression of FoxO1. A. The mRNA level of SENCR in SMCs transfected with si-SENCR. B. The mRNA level of FoxO1 in SMCs transfected with siSENCR. C. The transcription activity of FoxO1 in SMCs transfected with si-SENCR. D. The binging activity of FoxO1 with H3 histone in SMCs transfected with si-SENCR. **VS control group, **P < 0.01. E. The expression of FoxO1 in C-12511 cells transfected with pcDNA-SENCR. F. The transcription activity of FoxO1 in C-12511 cells transfected with pcDNA-SENCR. **VS 5.5 mM, P < 0.01; ##VS 27.5 mM + pcDNA, P < 0.01. Mean SD, all results were repeated for three times.
a role in the vascular complications of type 2 diabetes [10]. The proliferation of vascular SMCs is a crucial process in the pathogenesis of atherosclerosis. Generally, there is a balance between the proliferation and apoptosis of VSMCs under physiological conditions. When this balance is disturbed by
hyperglycemia and hyperinsulinemia, VSMC proliferation is increased and promotes the development of atherosclerosis [11]. Therefore, identifying the regulators of this proliferative response in VSMC may lead to therapeutic strategies aimed at blocking or inhibiting VSMC growth.
Fig. 4. FoxO1 regulated the expression of TRPC6 in C-12511 cells. A. FoxO1 could combine with TRPC6 promoter. B. The expression of TRPC6 in C-12511 cells transfected with pcDNA-FoxO1. **VS control group, **P < 0.01. C. The expression of TRPC6 in C-12511 cells transfected with si-FoxO1. D. The binging activity of FoxO1 with TRPC6 promoter in C-12511 cells transfected with si-FoxO1. **VS 5.5 mM, P < 0.01; ##VS 27.5 mM + pcDNA, P < 0.01. Mean SD, all results were repeated for three times.
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Fig. 5. SENCR overexpression reversed the effect of high glucose on SMCs proliferation and migration. A. Cell viability of SMCs exposed to glucose or pcDNA-SENCR. B. Cell migration of SMCs exposed to glucose or pcDNA-SENCR. Mean SD, all results were repeated for three times. **VS 5.5 mM, P < 0.01; ##VS 27.5 mM + pcDNA, P < 0.01.
It has been reported that several cytokines and non-coding RNAs are involved in VSMC proliferation and migration [12,13]. In particular, a novel lncRNA SENCR was described to be smooth muscle cell-and endothelial cell-enriched [7]. Bell et al. reported that SENCR silencing upregulated several genes associated with cell migration and attenuation of SENCR expression resulted in hypermotile phenotype of human smooth muscle cells [7]. In this study, we also found that the expression of SENCR was decreased in smooth muscle cells of db/db mice and in VSMCs exposed to high glucose. In addition, VSMC proliferation and migration assay demonstrated that SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration, which was consistent with previous study. To elucidate the mechanism how SENCR involved in VSMCs proliferation and migration, we examine the regulation of SENCR on the expression of FoxO1. FoxO1 is a prominent member of the Forkhead box family and is involved in regulating metabolism, cell proliferation, oxidative stress response and cell death [14]. Several reports indicated that FoxO1 played crucial role in SMCs proliferation and migration [15]. Park et al. reported that Foxo1 transcription factor is related to the recruitment of smooth muscle cells to nascent blood vessels [15]. Moreover, ubiquitin-mediated degradation of FoxO1 contributed to promote SMCs proliferation and survival by C Terminus of Hsc70-interacting protein [16]. It has been reported that forkhead signaling pathway contributed to the regulation of VSMC proliferation, cell cycle progression, and neointimal hyperplasia [17]. Our results showed that mRNA level of FoxO1 could be enhanced by SENCR knockdown. In db/db mice, the mRNA and protein level of FoxO1 was significantly upregulated comparing to control mice. These data indicated that downregulation of SENCR promoted SMCs proliferation and migration in db/db mice through upregulation of FoxO1. Except for the dysregulation of FoxO1 by SENCR in SMCs, we also observed the differential expression of another factors: TRPC6, which was upregulated in SMCs of db/db mice. TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells [18]. Yu et al. reported that upregulation of TRPC6 was involved in platelet-derived growth factor-mediated pulmonary artery smooth muscle cells proliferation [19]. In addition, deletion of TRPC4 and TRPC6 in mice was reported to impair smooth muscle contraction and intestinal motility in vivo [20]. Here, we further determined that FoxO1 regulated the expression of TRPC6 through binding to its promoter.
In summary, the in vivo and in vitro experiments in this study have shown that SENCR was down-regulated in db/db mice and VSMCs exposed to high glucose. While SENCR overexpression reversed the effect of high glucose on mouse VSMCs proliferation and migration, indicating that SENCR may be involved in the pathological process of SMCs proliferation. We further showed that SENCR promoted SMCs proliferation and migration in db/db mice through regulation of FoxO1 and TRPC6. These findings suggest that SENCR is a candidate target molecular for treatment of SMCs proliferation in db/db mice. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by grant from the Major Scientific and Technological Projects of Changzhou Health Bureau (2013.01– 2015.01, ZD201211). References [1] B. Wirostko, T.Y. Wong, R. Simo, Vascular endothelial growth factor and diabetic complications, Prog. Retin. Eye Res. 27 (2008) 608–621. [2] X. Sun, F. Han, J. Yi, N. Hou, Z. Cao, The effect of telomerase activity on vascular smooth muscle cell proliferation in type 2 diabetes in vivo and in vitro, Mol. Med. Rep. 7 (2013) 1636–1640. [3] A.R. Brasier, A. Recinos 3rd., M.S. Eledrisi, Vascular inflammation and the renin-angiotensin system, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 1257–1266. [4] A.M. Khalil, M. Guttman, M. Huarte, M. Garber, A. Raj, D. Rivea Morales, et al., Many human large intergenic noncoding RNAs associate with chromatinmodifying complexes and affect gene expression, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 11667–11672. [5] M. Guttman, J. Donaghey, B.W. Carey, M. Garber, J.K. Grenier, G. Munson, et al., lincRNAs act in the circuitry controlling pluripotency and differentiation, Nature 477 (2011) 295–300. [6] T. Thum, R. Kumarswamy, The smooth long noncoding RNA SENCR, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 1124–1125. [7] R.D. Bell, X. Long, M. Lin, J.H. Bergmann, V. Nanda, S.L. Cowan, et al., Identification and initial functional characterization of a human vascular cellenriched long noncoding RNA, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 1249–1259. [8] A. Leung, K. Stapleton, R. Natarajan, Functional long non-coding RNAs in vascular smooth muscle cells, Curr. Top. Microbiol. Immunol. (2015). [9] Z.-P. Liu, Z. Wang, H. Yanagisawa, E.N. Olson, Phenotypic modulation of smooth muscle cells through interaction of FoxO4 and myocardin, Dev. Cell 9 (2005) 261–270.
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