- Email: [email protected]
db mice through down-regulation of SIRT1
Biochemical and Biophysical Research Communications 463 (2015) 1159e1164
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1 Juan Xu a, Li Li b, Hui-fang Yun b, Ye-shan Han b, * a b
Department of Gynecology, Changzhou Maternity and Children Health Hospital, Changzhou, Jiangsu 213003, PR China Department of Anesthesiology, Changzhou No. 2 People's Hospital, Changzhou, Jiangsu 213003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2015 Accepted 10 June 2015 Available online 14 June 2015
Background: Diabetic vascular smooth muscle cells (VSMCs) exhibit significantly increased rates of proliferation and migration, which was the most common pathological change in atherosclerosis. In addition, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified. Methods: The expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and in SMC lines C-12511 exposed to high glucose with qRT-PCR and western blot. The regulation of miR-138 on the expression of SMCs was detected with luciferase report assay. VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. Results: We discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 30 -UTR was highly increased by miR-138 inhibitor and reduced by miR138 mimic. In addition, we examined that the up-regulation of NF-kB induced by high glucose in SMCs was reversed by resveratrol and miR-138 inhibitor. MTT and migration assays showed that miR-138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Conclusion: In this study, we revealed that miR-138 might promote proliferation and migration of SMC in db/db mice through suppressing the expression of SIRT1. © 2015 Elsevier Inc. All rights reserved.
Keywords: miR-138 Smooth muscle cells Proliferation Migration Diabetes mellitus SIRT1
1. Introduction Nowadays, cardiovascular complications, such as atherosclerosis, have been confirmed as the leading cause of morbidity and mortality in patients with diabetes mellitus (DM) [1]. The most common pathological change in atherosclerosis is the proliferation of vascular smooth muscle cells (VSMCs), which occurs in response to arterial injury and plays a crucial role in the atherosclerotic process [2]. Diabetic VSMCs exhibit significantly increased rates of proliferation, adhesion, and migration as well as abnormal cell culture morphology suggestive of abnormal contact inhibition [3]. However, the molecular mechanism of abnormal VSMC proliferation in diabetes mellitus patients is poorly understand. Recently, Sebastian Albinsson et al. has reviewed the microRNAs (miRNAs) that played a role in VSMC differentiation and
* Corresponding author. No. 29, Xinglong Lane, Tianning District, Changzhou, Jiangsu Province 213003, PR China. E-mail address: [email protected] (Y.-s. Han). http://dx.doi.org/10.1016/j.bbrc.2015.06.076 0006-291X/© 2015 Elsevier Inc. All rights reserved.
proliferation by modulating the expression of several transcription factors [4]. These small non-coding RNAs regulate target genes by inducing mRNA degradation or translational repression. In 2007, the role of miRNAs in VSMCs was first reported by microarray analysis and Northern blot [5]. Later, a breakthrough was achieved with the finding that miR-143/145 exerted potent actions in VSMCs [6]. To date, several other miRNAs were found to be involved in VSMC differentiation and proliferation, such as miR-21 and miR221 [4]. However, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified and clarified their interactions in vascular disease. Sirtuin 1 (SIRT1), the best characterized member of class III HDACs, has been implicated in aging, metabolism, and tolerance to oxidative stress by deacetylating and fine-tuning the activities of protein factors including LXR, p53, and NF-kB [7]. SIRT1 was also reported to serve as a key regulator in vascular endothelial homeostasis by controlling angiogenesis, decreasing atherosclerosis and endothelial dysfunction [8]. Hui-Na Zhang et al. reported that
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the up-regulation of SIRT1 by TNF-a may be an ubiquitous autoprotective response of VSMCs to inflammatory events [9]. Investigating the regulation of SIRT1 expression will help provide a more thorough understanding of its role in inflammatory reactions. In the present research, we discovered that SMC separated from T2DM rats expressed increased levels of miR-138 and decreased level of SIRT1. We further detected the regulation of miR-138 on SIRT1 expression and elucidated its role in driving VSMC proliferation. 2. Materials and methods 2.1. Animals 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 C 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 C. 2.2. Western blot analysis For Western Blotting, proteins of cells were resolved by SDSPAGE and transferred onto PVDF membrane (Roche). The transferred membranes were probed with primary antibodies of acetylated p65 and SIRT1 at 4 C for 24 h. HRP-conjugated IgG (Amersham Pharmacia Biotech, Buckinghamshire, UK) were used as secondary antibodies and detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA). Tubulin was used as a protein loading control.
2.3. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA was reversely transcribed using TaqMan MicroRNA Reverse Transcription Kit for analysis of miR-138 expression. For the detection of SIRT1 mRNA, reverse transcription and qRT-PCR were performed using SYBR Green PCR master mix. U6 snRNA was used as an endogenous control for miR-138 mRNA expression and b-Actin mRNA was used as the standard for SIRT1 mRNA expression. The relative expression level was computed using the 2DDCt analysis method. Experiments were performed in triplicate. 2.4. Vectors, transfection and luciferase reporter assay The SIRT1 gene was PCR-amplified and cloned into a pcDNA vector to generate pcDNA-sirt1. The 30 -untranslated regions (30 UTR) of NF-kB and SIRT1 generated by PCR amplification and were cloned into the pGL3-luciferase reporter plasmid (Promega). Transfection of siRNA, plasmids, miR-138 inhibitor or miR-138 mimic were conducted using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. For luciferase reporter assay, cells (1 104) were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of luciferase reporter plasmids or miR-138 inhibitor, miR-138 mimic, pcDNA-sirt1 (Promega), were transfected into cells using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Luciferase signals were measured using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. 2.5. Proliferation analysis Methylthiazoletetrazolium (MTT) assay was performed to assess the effect of miR-138 inhibitor on VSMC proliferation. VSMC were plated sparsely in 96-well flat-bottomed microplates in 100 mL of DMEM þ2.5% FBS and were incubated in a humidified 5% CO2
Fig. 1. The expression of miR-138 and SIRT1 in SMCs. (A) The thickness of aortic smooth muscle layer in db/db mice and control mice. (B) The mRNA level of miR-138 in SMCs separated from db/db mice and control. (C) The expression of SIRT1 in SMCs separated from db/db mice and control. (D) The mRNA level of miR-138 in mouse VSMCs and smooth muscle cell lines C-12511 which were both exposed to low glucose (5.5 mM) and high glucose (30 mM). (E) The mRNA level of SIRT1 in mouse VSMCs and smooth muscle cell lines C-12511 which were both exposed to low glucose (5.5 mM) and high glucose (30 mM). (F) The protein level of SIRT1 and acetylized p65 in mouse VSMCs and smooth muscle cell lines C-12511 which were both exposed to low glucose (5.5 mM) and high glucose (30 mM). Mean ± SD, all results were repeated for three times; *VS control group, *P < 0.05; **P < 0.01.
J. Xu et al. / Biochemical and Biophysical Research Communications 463 (2015) 1159e1164
atmosphere at 37 C for 24 h. Then 25 mL 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide solution (5 mg/mL) was added to each well, followed by a 4-h incubation at 37 C. Then, the medium carefully was removed, and 150 mL dimethylsulfoxide (DMSO) was added to dissolve the produced formazan crystals. The optical density immediately was measured at 490 nm using a microplate reader (Bio-Tek Instrument, Carpinteria, CA, USA). 2.6. VSMC migration assay Cell migration was analyzed using a modified two-chamber transwell system (BD Biosciences) following the manufacturer's instructions. Cells were detached by trypsin-EDTA and washed once with serum-free medium. Cells were then resuspended in serum-free medium, and 0.5 ml of either complete culture media or serum-free media containing 50 ng/ml of miRNA-138 inhibitor was added to each bottom well. 1 105 cells were added in each transwell insert and allowed to migrate for or 24 h in a 37 C cell incubator. Cells in the upper surface of the transwell were removed using cotton swabs. Migrated cells attached on the undersurface were fixed with 4% paraformaldehyde for 10 min and stained with crystal violet solution (0.5% in water) for 10 min. Cells were counted under microscope at 100 magnification.
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3. Results 3.1. The expression of miR-138 and SIRT1 in SMCs When comparing the thickness of aortic smooth muscle layer from db/db mice with that from control mice, we found that the thickness of aortic smooth muscle layer from db/db mice was significantly enhanced and became more thick with the increase of age (Fig. 1A). Furthermore, the expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and control mice. As shown in Fig. 1B, higher mRNA level of miR-138 was observed in SMCs from db/db mice. While, the mRNA and protein level of SIRT1 in SMCs from db/db mice were greatly reduced in a time-dependent manner (Fig. 1C). We further detected the expression of miR-138 and SIRT1 in VSMCs and SMC lines C-12511 in vitro, which were exposed to low glucose (5.5 mM) or high glucose (30 mM). Consistent with the results in vivo experiment, the mRNA level of miR-138 was SIRT1 increased in VSMCs and C12511 exposed to high glucose (Fig. 1D) and the expression of SIRT1 were down-regulated (Fig. 1E). Moreover, in VSMCs and C-12511 exposed to high glucose, increased expression of acetylized p65 was detected (Fig. 1F). These data suggested that the dysregulation of miR-138 and SIRT1 might be involved in SMC proliferation in db/ db mice.
2.7. Statistical analysis 3.2. MiR-138 regulated the expression of SIRT1 in SMCs Statistical analyses were performed using SPSS 16.0 software (SPSS Inc.). The values were expressed as the mean ± standard deviation (SD) of three independent experiments, and the relationship between SIRT1 and miR-138 expressions was tested with two-tailed Pearson's correlation. The significance of the differences between two groups was calculated using a two-tailed Student's ttest. P-values < 0.05 were considered to be statistically significant.
To elucidate the mechanism of miR-138 contributing to SMC proliferation in db/db mice, we investigated the regulation of miR138 on the expression of SIRT1 in C-12511 cells. When C-12511 transfected with miR-138 inhibitor to suppress the expression of miR-138, luciferase report assay showed that the activity of SIRT1 30 -UTR was highly increased. In addition, the mRNA and protein
Fig. 2. MiR-138 regulated the expression of SIRT1 in SMCs. (A) The effect of miR-138 inhibitor on the expression of SIRT1. (B) The effect of miR-138 mimic on the expression of SIRT1. Mean ± SD, all results were repeated for three times; **VS control group, **P < 0.01.
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level of SIRT1 were also enhanced (Fig. 2A). On the other hand, miR138 mimic significantly decreased the expression of SIRT1 in C12511 cells, as shown in Fig. 2B. Taken together, these findings indicated that miR-138 could regulate the expression of SIRT1 in SMCs. 3.3. The effect of SIRT1 on the expression of acetylized p65 and NFkB in C-12511 cells Acetylized p65 and NF-kB were reported to regulate by SIRT1. In C-12511 cells, we found that high glucose induced the upregulation of acetylized p65 and NF-kB. When C-12511 cells overexpressed SIRT1 and exposed to high glucose, the expression of acetylized p65 and NF-kB were significantly reduced comparing to that in C-12511 cells just exposed to high glucose. We verified this result through transfecting resveratrol (RSV) into C-12511 cells, which was an agonist of SIRT1. As shown in Fig. 3, consistent with above results, resveratrol reversed the up-regulation of acetylized p65 and NF-kB induced by high glucose in C-12511 cells. 3.4. The effect of miR-138 inhibitor or si-sirt1 on the expression of NF-kB in C-12511 exposed to glucose and SMCs proliferation and migration induced by high glucose To investigate the effect of miR-138 and SIRT1 on the expression of NF-kB in C-12511 exposed to glucose, C-12511 cells were
transfected with miR-138 inhibitor or si-sirt1. As demonstrated in Fig. 4A, miR-138 inhibitor reversed the effect of high glucose on the expression of NF-kB. While, higher activity of NF-kB was observed in C-12511 cells transfected with miR-138 inhibitor and si-sirt1 than that in C-12511 cells transfected with miR-138 inhibitor and si-control. This result indicated that si-sirt1 reversed the effect of miR-138 inhibitor on the activity of NF-kB. Next, VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. It has been shown that high glucose significantly stimulated the proliferation and migration of VSMCs comparing to that with low glucose (Fig. 4B and C). Moreover, miR-138 inhibitor reduced the cell viability and migration in VSMCs exposed to high glucose, which indicated that miR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose. 4. Discussion In this study, we discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 30 -UTR was highly increased by miR-138 inhibitor and reduced by miR-138 mimic, which indicated that miR-138 could regulate the expression of SIRT1 in SMCs. In addition, we examined that the up-regulation of NF-kB induced by high glucose in SMCs was reversed by resveratrol
Fig. 3. The effect of SIRT1 on the expression of acetylized p65 and NF-kB in C-12511 cells. (A) SIRT1 overexpression reversed the up-regulation of acetylized p65 and NF-kB induced by high glucose. (B) Resveratrol reversed the up-regulation of acetylized p65 and NF-kB induced by high glucose. Mean ± SD, all results were repeated for three times; **VS 5.5 mM, P < 0.01; ##VS 30 mM þ pcDNA, P < 0.01.
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Fig. 4. The effect of miR-138 inhibitor or si-sirt1 on the expression of NF-kB in C-12511 exposed to glucose and SMCs proliferation and migration induced by high glucose. (A) The effect of miR-138 inhibitor or si-sirt1 on the expression of NF-kB in C-12511 exposed to glucose. (B) Cell viability of SMCs exposed to glucose or miR-138 inhibitor. (C) Cell migration of SMCs exposed to glucose or miR-138 inhibitor. Mean ± SD, all results were repeated for three times. **VS 5.5 mM, P < 0.01; ##VS 30 mM þ NC, P < 0.01; &VS 30 mM þ miR-138 inhibitor þ si-control, P < 0.01.
and miR-138 inhibitor. MTT and migration assays showed that miR138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Taken together, our results suggest that miR-138 promotes smooth muscle cells proliferation and migration in db/ db mice through down-regulation of SIRT1. As a novel class of gene regulators, miRNAs play important roles not only in normal development and physiological conditions, but also in disease status. Although multiple identified miRNAs have been characterized as important regulators for cell growth, differentiation, and apoptosis in human diseases, the evidence about dysregulation of miRNAs in vascular diseases was first reported in 2007 [5]. They confirmed the cellular mechanisms of miR-21 mediated effect on neointimal lesion formation in vivo in the vascular wall after angioplasty. Recently, several independent groups demonstrated the importance of miR-145 in VSMC differentiation [10,11]. In addition, miR-221 was also found to be implicated in the regulation of VSMC phenotype [12]. MiR-138 is previously proven to be an important regulator in heart development [13]. Several reports indicated that the downregulation of miR-138 contributes to cancer cell proliferation and invasion and inhibits apoptosis in multiple human cancers [14e16]. On the other hand, the up-regulation of miR-138 has also been shown to be associated with tumor recurrence and survival in tumor-initiating glioma stem cells (GSC) [17]. These findings indicate that miR-138 functions as both an oncomir and tumorsuppressive miRNA depending on the tumor type. In pulmonary artery smooth muscle cells (PASMCs), miR-138 expression was found to be up-regulated induced by hypoxia and accelerating the proliferation of PASMCs. In this study, we identified the upregulation of miR-138 in SMC separated from db/db mice. And we found that miR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose. To further elucidate the mechanism by which miR-138 action leads to VSMCs proliferation and migration, we detected the expression of SIRT1 in SMC transfected with miR-138 mimic or miR-138 inhibitor. It has been suggested that SIRT1 is a downstream target of miR-138 in adult sensory neurons in vitro and in vivo [18]. Pia Rivetti di Val Cervo et al. also identified SIRT1 as a direct target of miR-138 [19]. We also found that miR-138 could regulate the expression of SIRT1 in SMCs. Furthermore, when C12511 cells overexpressed SIRT1 and exposed to high glucose, the expression of acetylized p65 and NF-kB were significantly reduced which was verified by resveratrol. RelA/p65-NF-kB deacetylation by SIRT1 prevented the macrophage foam cell formation and reduced atherosclerosis [20]. SIRT1 also reported to retard calcification in
VSMCs and reduce neointima formation after injury by reducing cell proliferation and migration [21]. Isabelle Gorenne et al. showed that reduced SIRT1 activity in VSMCs was associated with defective DNA repair, persistent DNA damage, DDR activation, reduced cell proliferation, and apoptosis [22]. Markedly induced expression of pro-inflammatory cytokines are always accompanied with responses of VSMCs to inflammatory lesions such as proliferation and migration [23]. SIRT1 inhibition of NF-kB activity through deacetylation of p65/RelA at lysine 310 has been well documented in previous reports [24]. In this study, miR-138 inhibitor was found to reverse the effect of high glucose on the expression of NF-kB and sisirt1 was found to reverse the effect of miR-138 inhibitor on the activity of NF-kB. These results indicated that miR-138 might involve in smooth muscle cells proliferation and migration through down-regulation of SIRT1. In conclusion, we revealed that miR-138 was up-regulated in SMC separated from db/db mice or exposed to high glucose, and its contritution to SMC proliferation and migration, further suggesting that it might promote proliferation and migration of SMC in db/db mice, which is at least through suppressing the expression of SIRT1 as shown in this study. The identification of miR-138 and its regulation to SIRT1 might provide insights into the developing efficient therapeutic strategy to suppress atherosclerosis because of diabetes mellitus. Conflict of interest The authors have no actual or potential conflicts of interest to declare. Acknowledgments This work was supported by grant from the Major Scientific and Technological Projects of Changzhou Health Bureau (2013.01 e2015.01, ZD201211). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.06.076. References , Vascular endothelial growth factor and dia[1] B. Wirostko, T.Y. Wong, R. Simo betic complications, Prog. Retin. Eye Res. 27 (2008) 608e621.
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[2] D. Bruemmer, R.E. Law, Thiazolidinedione regulation of smooth muscle cell proliferation, Am. J. Med. 115 (2003) 87e92. [3] P.L. Faries, D.I. Rohan, H. Takahara, M.C. Wyers, M.A. Contreras, W.C. Quist, et al., Human vascular smooth muscle cells of diabetic origin exhibit increased proliferation, adhesion, and migration, J. Vasc. Surg. 33 (2001) 601e607. [4] S. Albinsson, Can microRNAs control vascular smooth muscle phenotypic modulation and the response to injury? Physiol. Genomics 43 (2011) 529e533. [5] R. Ji, Y. Cheng, J. Yue, J. Yang, X. Liu, H. Chen, et al., MicroRNA expression signature and antisense-mediated depletion reveal an essential role of MicroRNA in vascular neointimal lesion formation, Circulation Res. 100 (2007) 1579e1588. [6] N. Xu, T. Papagiannakopoulos, G. Pan, J.A. Thomson, K.S. Kosik, MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells, Cell 137 (2009) 647e658. [7] J. Xia, SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells, Biochem. Biophys. Res. Commun. 428 (2012) 264e270. [8] Y.H. Kim, J.U. Bae, S.J. Lee, S.Y. Park, C.D. Kim, SIRT1 attenuates PAF-induced MMP-2 production via down-regulation of PAF receptor expression in vascular smooth muscle cells, Vasc. Pharmacol. [9] H.N. Zhang, L. Li, P. Gao, H.Z. Chen, R. Zhang, Y.S. Wei, et al., Involvement of the p65/RelA subunit of NF-kB in TNF-a-induced SIRT1 expression in vascular smooth muscle cells, Biochem. Biophys. Res. Commun. 397 (2010) 569e575. [10] T. Boettger, Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster, J. Clin. Investigation 119 (2009) 2634e2647. [11] Y. Cheng, MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neointimal lesion formation, Circulation Res. 105 (2009) 158e166. [12] B.N. Davis, A.C. Hilyard, P.H. Nguyen, G. Lagna, A. Hata, Induction of microRNA-221 by platelet-derived growth factor signaling is critical for modulation of vascular smooth muscle phenotype, J. Biol. Chem. 284 (2009) 3728e3738.
[13] S. He, miR-138 protects cardiomyocytes from hypoxia-induced apoptosis via MLK3/JNK/c-jun pathway, Biochem. Biophys. Res. Commun. 441 (2013) 763e769. [14] X. Liu, MicroRNA-138 suppresses invasion and promotes apoptosis in head and neck squamous cell carcinoma cell lines, Cancer Lett. 286 (2009) 217e222. [15] X. Liu, MiR-138 suppressed nasopharyngeal carcinoma growth and tumorigenesis by targeting the CCND1 oncogene, Cell. Cycle 11 (2012) 2495e2506. [16] H. Gong, Downregulation of miR-138 sustains NF-kB activation and promotes lipid raft formation in esophageal squamous cell carcinoma, Clin. Cancer Res. 19 (2013). [17] X.H. Chan, S. Nama, F. Gopal, P. Rizk, S. Ramasamy, G. Sundaram, et al., Targeting glioma stem cells by functional inhibition of a prosurvival oncomiR138 in malignant gliomas, Cell. Reports 2 (2012) 591e602. [18] C.-M. Liu, R.-Y. Wang, Z.-X. Jiao, B.-Y. Zhang, F.-Q. Zhou, MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration, Genes Dev. 27 (2013) 1473e1483. [19] P.R. di Val Cervo, A.M. Lena, M. Nicoloso, S. Rossi, M. Mancini, H. Zhou, et al., p63emicroRNA feedback in keratinocyte senescence, Proc. Natl. Acad. Sci. 109 (2012) 1133e1138. €fer, J. Hofmann, L. Rohrer, C. Besler, et al., SIRT1 [20] S. Stein, C. Lohmann, N. Scha decreases Lox-1-mediated foam cell formation in atherogenesis, Eur. Heart J. (2010) ehq107. [21] L. Li, H.-N. Zhang, H.-Z. Chen, P. Gao, L.-H. Zhu, H.-L. Li, et al., SIRT1 acts as a modulator of neointima formation following vascular injury in mice, Circulation Res. 108 (2011) 1180e1189. [22] I. Gorenne, S. Kumar, K. Gray, N. Figg, H. Yu, J. Mercer, et al., Vascular smooth muscle cell sirtuin 1 protects against DNA damage and inhibits atherosclerosis, Circulation 127 (2013) 386e396. [23] H.-N. Zhang, L. Li, P. Gao, H.-Z. Chen, R. Zhang, Y.-S. Wei, et al., Involvement of the p65/RelA subunit of NF-kB in TNF-a-induced SIRT1 expression in vascular smooth muscle cells, Biochem. Biophys. Res. Commun. 397 (2010) 569e575. [24] F. Yeung, J.E. Hoberg, C.S. Ramsey, M.D. Keller, D.R. Jones, R.A. Frye, et al., Modulation of NF-kB-dependent transcription and cell survival by the SIRT1 deacetylase, EMBO J. 23 (2004) 2369e2380.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1 Juan Xu a, Li Li b, Hui-fang Yun b, Ye-shan Han b, * a b
Department of Gynecology, Changzhou Maternity and Children Health Hospital, Changzhou, Jiangsu 213003, PR China Department of Anesthesiology, Changzhou No. 2 People's Hospital, Changzhou, Jiangsu 213003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2015 Accepted 10 June 2015 Available online 14 June 2015
Background: Diabetic vascular smooth muscle cells (VSMCs) exhibit significantly increased rates of proliferation and migration, which was the most common pathological change in atherosclerosis. In addition, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified. Methods: The expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and in SMC lines C-12511 exposed to high glucose with qRT-PCR and western blot. The regulation of miR-138 on the expression of SMCs was detected with luciferase report assay. VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. Results: We discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 30 -UTR was highly increased by miR-138 inhibitor and reduced by miR138 mimic. In addition, we examined that the up-regulation of NF-kB induced by high glucose in SMCs was reversed by resveratrol and miR-138 inhibitor. MTT and migration assays showed that miR-138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Conclusion: In this study, we revealed that miR-138 might promote proliferation and migration of SMC in db/db mice through suppressing the expression of SIRT1. © 2015 Elsevier Inc. All rights reserved.
Keywords: miR-138 Smooth muscle cells Proliferation Migration Diabetes mellitus SIRT1
1. Introduction Nowadays, cardiovascular complications, such as atherosclerosis, have been confirmed as the leading cause of morbidity and mortality in patients with diabetes mellitus (DM) [1]. The most common pathological change in atherosclerosis is the proliferation of vascular smooth muscle cells (VSMCs), which occurs in response to arterial injury and plays a crucial role in the atherosclerotic process [2]. Diabetic VSMCs exhibit significantly increased rates of proliferation, adhesion, and migration as well as abnormal cell culture morphology suggestive of abnormal contact inhibition [3]. However, the molecular mechanism of abnormal VSMC proliferation in diabetes mellitus patients is poorly understand. Recently, Sebastian Albinsson et al. has reviewed the microRNAs (miRNAs) that played a role in VSMC differentiation and
* Corresponding author. No. 29, Xinglong Lane, Tianning District, Changzhou, Jiangsu Province 213003, PR China. E-mail address: [email protected] (Y.-s. Han). http://dx.doi.org/10.1016/j.bbrc.2015.06.076 0006-291X/© 2015 Elsevier Inc. All rights reserved.
proliferation by modulating the expression of several transcription factors [4]. These small non-coding RNAs regulate target genes by inducing mRNA degradation or translational repression. In 2007, the role of miRNAs in VSMCs was first reported by microarray analysis and Northern blot [5]. Later, a breakthrough was achieved with the finding that miR-143/145 exerted potent actions in VSMCs [6]. To date, several other miRNAs were found to be involved in VSMC differentiation and proliferation, such as miR-21 and miR221 [4]. However, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified and clarified their interactions in vascular disease. Sirtuin 1 (SIRT1), the best characterized member of class III HDACs, has been implicated in aging, metabolism, and tolerance to oxidative stress by deacetylating and fine-tuning the activities of protein factors including LXR, p53, and NF-kB [7]. SIRT1 was also reported to serve as a key regulator in vascular endothelial homeostasis by controlling angiogenesis, decreasing atherosclerosis and endothelial dysfunction [8]. Hui-Na Zhang et al. reported that
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the up-regulation of SIRT1 by TNF-a may be an ubiquitous autoprotective response of VSMCs to inflammatory events [9]. Investigating the regulation of SIRT1 expression will help provide a more thorough understanding of its role in inflammatory reactions. In the present research, we discovered that SMC separated from T2DM rats expressed increased levels of miR-138 and decreased level of SIRT1. We further detected the regulation of miR-138 on SIRT1 expression and elucidated its role in driving VSMC proliferation. 2. Materials and methods 2.1. Animals 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 C 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 C. 2.2. Western blot analysis For Western Blotting, proteins of cells were resolved by SDSPAGE and transferred onto PVDF membrane (Roche). The transferred membranes were probed with primary antibodies of acetylated p65 and SIRT1 at 4 C for 24 h. HRP-conjugated IgG (Amersham Pharmacia Biotech, Buckinghamshire, UK) were used as secondary antibodies and detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA). Tubulin was used as a protein loading control.
2.3. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA was reversely transcribed using TaqMan MicroRNA Reverse Transcription Kit for analysis of miR-138 expression. For the detection of SIRT1 mRNA, reverse transcription and qRT-PCR were performed using SYBR Green PCR master mix. U6 snRNA was used as an endogenous control for miR-138 mRNA expression and b-Actin mRNA was used as the standard for SIRT1 mRNA expression. The relative expression level was computed using the 2DDCt analysis method. Experiments were performed in triplicate. 2.4. Vectors, transfection and luciferase reporter assay The SIRT1 gene was PCR-amplified and cloned into a pcDNA vector to generate pcDNA-sirt1. The 30 -untranslated regions (30 UTR) of NF-kB and SIRT1 generated by PCR amplification and were cloned into the pGL3-luciferase reporter plasmid (Promega). Transfection of siRNA, plasmids, miR-138 inhibitor or miR-138 mimic were conducted using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. For luciferase reporter assay, cells (1 104) were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of luciferase reporter plasmids or miR-138 inhibitor, miR-138 mimic, pcDNA-sirt1 (Promega), were transfected into cells using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Luciferase signals were measured using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. 2.5. Proliferation analysis Methylthiazoletetrazolium (MTT) assay was performed to assess the effect of miR-138 inhibitor on VSMC proliferation. VSMC were plated sparsely in 96-well flat-bottomed microplates in 100 mL of DMEM þ2.5% FBS and were incubated in a humidified 5% CO2
Fig. 1. The expression of miR-138 and SIRT1 in SMCs. (A) The thickness of aortic smooth muscle layer in db/db mice and control mice. (B) The mRNA level of miR-138 in SMCs separated from db/db mice and control. (C) The expression of SIRT1 in SMCs separated from db/db mice and control. (D) The mRNA level of miR-138 in mouse VSMCs and smooth muscle cell lines C-12511 which were both exposed to low glucose (5.5 mM) and high glucose (30 mM). (E) The mRNA level of SIRT1 in mouse VSMCs and smooth muscle cell lines C-12511 which were both exposed to low glucose (5.5 mM) and high glucose (30 mM). (F) The protein level of SIRT1 and acetylized p65 in mouse VSMCs and smooth muscle cell lines C-12511 which were both exposed to low glucose (5.5 mM) and high glucose (30 mM). Mean ± SD, all results were repeated for three times; *VS control group, *P < 0.05; **P < 0.01.
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atmosphere at 37 C for 24 h. Then 25 mL 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide solution (5 mg/mL) was added to each well, followed by a 4-h incubation at 37 C. Then, the medium carefully was removed, and 150 mL dimethylsulfoxide (DMSO) was added to dissolve the produced formazan crystals. The optical density immediately was measured at 490 nm using a microplate reader (Bio-Tek Instrument, Carpinteria, CA, USA). 2.6. VSMC migration assay Cell migration was analyzed using a modified two-chamber transwell system (BD Biosciences) following the manufacturer's instructions. Cells were detached by trypsin-EDTA and washed once with serum-free medium. Cells were then resuspended in serum-free medium, and 0.5 ml of either complete culture media or serum-free media containing 50 ng/ml of miRNA-138 inhibitor was added to each bottom well. 1 105 cells were added in each transwell insert and allowed to migrate for or 24 h in a 37 C cell incubator. Cells in the upper surface of the transwell were removed using cotton swabs. Migrated cells attached on the undersurface were fixed with 4% paraformaldehyde for 10 min and stained with crystal violet solution (0.5% in water) for 10 min. Cells were counted under microscope at 100 magnification.
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3. Results 3.1. The expression of miR-138 and SIRT1 in SMCs When comparing the thickness of aortic smooth muscle layer from db/db mice with that from control mice, we found that the thickness of aortic smooth muscle layer from db/db mice was significantly enhanced and became more thick with the increase of age (Fig. 1A). Furthermore, the expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and control mice. As shown in Fig. 1B, higher mRNA level of miR-138 was observed in SMCs from db/db mice. While, the mRNA and protein level of SIRT1 in SMCs from db/db mice were greatly reduced in a time-dependent manner (Fig. 1C). We further detected the expression of miR-138 and SIRT1 in VSMCs and SMC lines C-12511 in vitro, which were exposed to low glucose (5.5 mM) or high glucose (30 mM). Consistent with the results in vivo experiment, the mRNA level of miR-138 was SIRT1 increased in VSMCs and C12511 exposed to high glucose (Fig. 1D) and the expression of SIRT1 were down-regulated (Fig. 1E). Moreover, in VSMCs and C-12511 exposed to high glucose, increased expression of acetylized p65 was detected (Fig. 1F). These data suggested that the dysregulation of miR-138 and SIRT1 might be involved in SMC proliferation in db/ db mice.
2.7. Statistical analysis 3.2. MiR-138 regulated the expression of SIRT1 in SMCs Statistical analyses were performed using SPSS 16.0 software (SPSS Inc.). The values were expressed as the mean ± standard deviation (SD) of three independent experiments, and the relationship between SIRT1 and miR-138 expressions was tested with two-tailed Pearson's correlation. The significance of the differences between two groups was calculated using a two-tailed Student's ttest. P-values < 0.05 were considered to be statistically significant.
To elucidate the mechanism of miR-138 contributing to SMC proliferation in db/db mice, we investigated the regulation of miR138 on the expression of SIRT1 in C-12511 cells. When C-12511 transfected with miR-138 inhibitor to suppress the expression of miR-138, luciferase report assay showed that the activity of SIRT1 30 -UTR was highly increased. In addition, the mRNA and protein
Fig. 2. MiR-138 regulated the expression of SIRT1 in SMCs. (A) The effect of miR-138 inhibitor on the expression of SIRT1. (B) The effect of miR-138 mimic on the expression of SIRT1. Mean ± SD, all results were repeated for three times; **VS control group, **P < 0.01.
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level of SIRT1 were also enhanced (Fig. 2A). On the other hand, miR138 mimic significantly decreased the expression of SIRT1 in C12511 cells, as shown in Fig. 2B. Taken together, these findings indicated that miR-138 could regulate the expression of SIRT1 in SMCs. 3.3. The effect of SIRT1 on the expression of acetylized p65 and NFkB in C-12511 cells Acetylized p65 and NF-kB were reported to regulate by SIRT1. In C-12511 cells, we found that high glucose induced the upregulation of acetylized p65 and NF-kB. When C-12511 cells overexpressed SIRT1 and exposed to high glucose, the expression of acetylized p65 and NF-kB were significantly reduced comparing to that in C-12511 cells just exposed to high glucose. We verified this result through transfecting resveratrol (RSV) into C-12511 cells, which was an agonist of SIRT1. As shown in Fig. 3, consistent with above results, resveratrol reversed the up-regulation of acetylized p65 and NF-kB induced by high glucose in C-12511 cells. 3.4. The effect of miR-138 inhibitor or si-sirt1 on the expression of NF-kB in C-12511 exposed to glucose and SMCs proliferation and migration induced by high glucose To investigate the effect of miR-138 and SIRT1 on the expression of NF-kB in C-12511 exposed to glucose, C-12511 cells were
transfected with miR-138 inhibitor or si-sirt1. As demonstrated in Fig. 4A, miR-138 inhibitor reversed the effect of high glucose on the expression of NF-kB. While, higher activity of NF-kB was observed in C-12511 cells transfected with miR-138 inhibitor and si-sirt1 than that in C-12511 cells transfected with miR-138 inhibitor and si-control. This result indicated that si-sirt1 reversed the effect of miR-138 inhibitor on the activity of NF-kB. Next, VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. It has been shown that high glucose significantly stimulated the proliferation and migration of VSMCs comparing to that with low glucose (Fig. 4B and C). Moreover, miR-138 inhibitor reduced the cell viability and migration in VSMCs exposed to high glucose, which indicated that miR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose. 4. Discussion In this study, we discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 30 -UTR was highly increased by miR-138 inhibitor and reduced by miR-138 mimic, which indicated that miR-138 could regulate the expression of SIRT1 in SMCs. In addition, we examined that the up-regulation of NF-kB induced by high glucose in SMCs was reversed by resveratrol
Fig. 3. The effect of SIRT1 on the expression of acetylized p65 and NF-kB in C-12511 cells. (A) SIRT1 overexpression reversed the up-regulation of acetylized p65 and NF-kB induced by high glucose. (B) Resveratrol reversed the up-regulation of acetylized p65 and NF-kB induced by high glucose. Mean ± SD, all results were repeated for three times; **VS 5.5 mM, P < 0.01; ##VS 30 mM þ pcDNA, P < 0.01.
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Fig. 4. The effect of miR-138 inhibitor or si-sirt1 on the expression of NF-kB in C-12511 exposed to glucose and SMCs proliferation and migration induced by high glucose. (A) The effect of miR-138 inhibitor or si-sirt1 on the expression of NF-kB in C-12511 exposed to glucose. (B) Cell viability of SMCs exposed to glucose or miR-138 inhibitor. (C) Cell migration of SMCs exposed to glucose or miR-138 inhibitor. Mean ± SD, all results were repeated for three times. **VS 5.5 mM, P < 0.01; ##VS 30 mM þ NC, P < 0.01; &VS 30 mM þ miR-138 inhibitor þ si-control, P < 0.01.
and miR-138 inhibitor. MTT and migration assays showed that miR138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Taken together, our results suggest that miR-138 promotes smooth muscle cells proliferation and migration in db/ db mice through down-regulation of SIRT1. As a novel class of gene regulators, miRNAs play important roles not only in normal development and physiological conditions, but also in disease status. Although multiple identified miRNAs have been characterized as important regulators for cell growth, differentiation, and apoptosis in human diseases, the evidence about dysregulation of miRNAs in vascular diseases was first reported in 2007 [5]. They confirmed the cellular mechanisms of miR-21 mediated effect on neointimal lesion formation in vivo in the vascular wall after angioplasty. Recently, several independent groups demonstrated the importance of miR-145 in VSMC differentiation [10,11]. In addition, miR-221 was also found to be implicated in the regulation of VSMC phenotype [12]. MiR-138 is previously proven to be an important regulator in heart development [13]. Several reports indicated that the downregulation of miR-138 contributes to cancer cell proliferation and invasion and inhibits apoptosis in multiple human cancers [14e16]. On the other hand, the up-regulation of miR-138 has also been shown to be associated with tumor recurrence and survival in tumor-initiating glioma stem cells (GSC) [17]. These findings indicate that miR-138 functions as both an oncomir and tumorsuppressive miRNA depending on the tumor type. In pulmonary artery smooth muscle cells (PASMCs), miR-138 expression was found to be up-regulated induced by hypoxia and accelerating the proliferation of PASMCs. In this study, we identified the upregulation of miR-138 in SMC separated from db/db mice. And we found that miR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose. To further elucidate the mechanism by which miR-138 action leads to VSMCs proliferation and migration, we detected the expression of SIRT1 in SMC transfected with miR-138 mimic or miR-138 inhibitor. It has been suggested that SIRT1 is a downstream target of miR-138 in adult sensory neurons in vitro and in vivo [18]. Pia Rivetti di Val Cervo et al. also identified SIRT1 as a direct target of miR-138 [19]. We also found that miR-138 could regulate the expression of SIRT1 in SMCs. Furthermore, when C12511 cells overexpressed SIRT1 and exposed to high glucose, the expression of acetylized p65 and NF-kB were significantly reduced which was verified by resveratrol. RelA/p65-NF-kB deacetylation by SIRT1 prevented the macrophage foam cell formation and reduced atherosclerosis [20]. SIRT1 also reported to retard calcification in
VSMCs and reduce neointima formation after injury by reducing cell proliferation and migration [21]. Isabelle Gorenne et al. showed that reduced SIRT1 activity in VSMCs was associated with defective DNA repair, persistent DNA damage, DDR activation, reduced cell proliferation, and apoptosis [22]. Markedly induced expression of pro-inflammatory cytokines are always accompanied with responses of VSMCs to inflammatory lesions such as proliferation and migration [23]. SIRT1 inhibition of NF-kB activity through deacetylation of p65/RelA at lysine 310 has been well documented in previous reports [24]. In this study, miR-138 inhibitor was found to reverse the effect of high glucose on the expression of NF-kB and sisirt1 was found to reverse the effect of miR-138 inhibitor on the activity of NF-kB. These results indicated that miR-138 might involve in smooth muscle cells proliferation and migration through down-regulation of SIRT1. In conclusion, we revealed that miR-138 was up-regulated in SMC separated from db/db mice or exposed to high glucose, and its contritution to SMC proliferation and migration, further suggesting that it might promote proliferation and migration of SMC in db/db mice, which is at least through suppressing the expression of SIRT1 as shown in this study. The identification of miR-138 and its regulation to SIRT1 might provide insights into the developing efficient therapeutic strategy to suppress atherosclerosis because of diabetes mellitus. Conflict of interest The authors have no actual or potential conflicts of interest to declare. Acknowledgments This work was supported by grant from the Major Scientific and Technological Projects of Changzhou Health Bureau (2013.01 e2015.01, ZD201211). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.06.076. References , Vascular endothelial growth factor and dia[1] B. Wirostko, T.Y. Wong, R. Simo betic complications, Prog. Retin. Eye Res. 27 (2008) 608e621.
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