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Study on the mechanism of HIF1a-SOX9 in glucose-induced cardiomyocyte hypertrophy
Biomedicine & Pharmacotherapy 74 (2015) 57–62
Available online at
ScienceDirect www.sciencedirect.com
Original Article
Study on the mechanism of HIF1a-SOX9 in glucose-induced cardiomyocyte hypertrophy Qianqian Gao a, Lina Guan b, Shanshan Hu c, Yanwei Yao a, Xiaolin Ren a, Zhenwei Zhang a, Canling Cheng a, Yi Liu d, Chun Zhang d, Jinpeng Huang d, Dongmei Su b,*, Xu Ma b,* a
Linyi Academy of Technology Cooperation and Application, Linyi, Shandong Province, China Department of Genetics, National Research Institute for Family Planning, Beijing, China Department of Ophthalmology, Hongqi Hospital of Mudanjiang Medical College, Mudanjiang, Heilongjiang Province, China d Department of Biotechnology, College of Bioengineering, Beijing Polytechnic, Beijing, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1st June 2015 Accepted 9 July 2015
A major cause of morbidity and mortality in cardiovascular disease is pathological cardiac hypertrophy. With an increase in the cellular surface area and upregulation of the atrial natriuretic peptide (ANP) gene, cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. ANP is a hypertrophic marker. Many works have been done to explore how the glucose induces the cardiac hypertrophy. However, it is not enough for us to figure it out. In this study, the influences of different glucose concentrations on cardiomyocytes were examined in vitro. The results showed that cardiomyocytes cultured with 25 mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35 mM glucose tended to undergo apoptosis. An increased expression of SOX9 was observed when cardiomyocytes were cultured with 25 mM glucose, but when the concentration of glucose was increased to 35 mM, the expression of SOX9 decreased. We used the RNAi approach to knockdown SOX9 expression, to assess its effects on cardiomyocyte hypertrophy. The results showed that knockdown of the SOX9 gene suppressed the 25 mM glucose-induced cardiomyocyte hypertrophy. The upregulation of the ANP gene was associated with overexpression of SOX9. Additionally, the results showed that high glucose (HG, 25 mM) treatment increased the expression of hypoxia-inducible factor (HIF)1a. Further study showed that HIF1a participated in regulating SOX9 expression in response to HG. This study revealed a novel regulatory mechanism of HIF1a-SOX9 in high glucose-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Cardiomyocyte hypertrophy SOX9 HIF1a
1. Introduction Myocyte usually increases cell size and protein synthesis to compensate the increase in the workload, which is the character of cardiac hypertrophy. That is associated with an overtly increased risk of ventricular dysfunction, heart failure, and malignant arrhythmias in obese individuals [1,2]. Abnormal glucose homeostasis affects cardiac structure and function [3,4]. In response to high glucose induction, the ventricular myocardium will undergo hypertrophic growth [5,6]. Abbreviations: ANP, atrial natriuretic peptide; RT-PCR, reverse transcription; Sox9, SRY (sex determining region Y)-box 9; HIF1a, hypoxia inducible factor 1 alpha. * Corresponding authors at: Department of Genetics, National Research Institute for Family Planning, 12, Dahuisi Road, Haidian, Beijing 100081, China. E-mail addresses: [email protected] (D. Su), [email protected], [email protected] (X. Ma). http://dx.doi.org/10.1016/j.biopha.2015.07.009 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
Sox9 is a member of Sry-related high mobility group box family of transcription factors [7]. By binding to the Pik3ca promoter to induce Akt phosphorylation, Sox9 expression is essential for hypertrophy and sustains chondrocyte-specific survival mechanisms in differentiated chondrocytes in mouse [8]. However, the expression and role of SOX9 in human cardiomyocyte hypertrophy was not elucidated. HIF1a plays a significant role in adaptive cardiopulmonary responses and the transcriptional regulation in the heart is considerably influenced by dysfunction of HIF1 pathways [9,10]. Glycolytic genes activated by HIF-1a are critical for metabolic adaptation to hypoxia by increasing conversion of glucose to pyruvate and subsequently to lactate [11]. As a key mediator of glycolysis and lipid anabolism, HIF1a jointly upregulates in hypertrophic cardiomyopathy and is cooperated with PPARg to mediate pivotal changes in cardiac hypertrophy in vivo [12]. Combining with the evidence about HIF1a, we speculated that
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there is change of HIF1a expression in response to high glucose, and HIF-1a may play an important role in high glucose-induced cardiomyocyte hypertrophy. This study identifies the molecular mechanisms leading to cardiomyocyte hypertrophy, in response to high concentrations of extracellular glucose, which may be important in the treatment of diabetic heart disease. Specifically, we focus on the expression and regulatory mechanism of Sox9 and HIF1a in cardiomyocyte hypertrophy induced by high glucose. Firstly, we examined the influence of different glucose concentrations on cardiomyocytes in vitro. Our results showed that cardiomyocytes cultured with 25 mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35 mM glucose tended to undergo apoptosis. The upregulation of Sox9 is associated with high glucose-induced cardiomyocytes hypertrophy. Further study showed that SOX9 is required for high glucose-induced cardio[(Fig._1)TD$IG]myocyte hypertrophy by knockdown SOX9 expression using the
RNAi approach. Meanwhile, SOX9 overexpression also partly induced cardiomyocyte hypertrophy. In addition, our results showed that the upregulation of HIF1a and cardiomyocytes hypertrophy is positively correlated. Moreover, we found that HIF1a participated in regulating SOX9 expression in response to high glucose. Above all, this study is highly relevant to the understanding of the effects of the HIF1a-SOX9 upregulation on HG-induced cardiomyocyte hypertrophy, as well as the related molecular mechanism. 2. Materials and methods 2.1. Cell culture, transfection and treatments Human cardiac cardiomyocytes (HCM, ScienCell Research Laboratories) were cultured and transfected as described before in previous work [13]. Human cardiac myocytes (HCM, ScienCell
Fig. 1. Different glucose concentrations sent human cardiomyocytes to different fates. (A) Comparison of the cell size in different glucose contents under Optical Microscope. HCM cells were treated with different concentration of glucose for 48 h, and were visualized under Optical Microscope. (B) The analysis of the cell surface was done with Image-Pro plus Data Analysis Software. Quantification of the cell surface area was measured with 300 random cells from three experiments after different concentration of glucose treatment for 48 h, and the average value was used for the analysis. *P < 0.05; **P < 0.01 versus the untreated group. (C) The change in endogenous of ANP mRNA levels in response to different concentration of glucose stimulus. The cells were treated with different concentration of glucose for 2 days, and ANP mRNA was measured using quantitative RT-PCR. b-Actin was used as an internal control. (D) The apoptosis detection of cardiomyocytes under different glucose contents treatment by Hoechst 33342 staining assay. HCM cells were treated with different concentration of glucose for 2 days and then were stained with Hoechst 33342 and to assess the apoptosis. The panel showed the nuclear morphology stained with blue by using Hoechst 33342. (E) The apoptosis rates were analyzed after different concentration of glucose treatment. The apoptosis rates which were calculated based on at least 100 cells from three experiments and are shown as means SD. *P < 0.05; **P < 0.01 versus the untreated cells.
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Research Laboratories) were maintained in cardiac myocyte medium supplemented with 10% fetal bovine serum, cardiac myocyte growth supplement, 100 mg/mL penicillin, and 100 mg/ mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 8C. The Sox9, Hif1a siRNA vectors and DNA plasmids were transfected into cardiomyocytes using lipofectamine 2000 (Invitrogen) procedures. Cardiomyocytes were treated with 25 mM and 35 mM glucose, respectively. Cardiomyocytes treated with 5 mM glucose were used as control sample.
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anti-sense, 50 -CTCACTGAGCACTTGT-30 [15]. The primer pairs for the SOX9 gene were: sense, 50 -AAGACATTTAAGCTAAAGGCAACTCGTAC-30 and antisense, 50 -TGATCACAC GATTCTCCATCATCCTC-30 [16]. The b-actin primer pairs were: sense, 50 -TCGTGCGTGACATTAAGGAG-30 and antisense, 50 -ATGCCAGGGT ACATGGTGGT-30 [15]. 2.5. Western blotting
The apoptosis of cells was assessed through observation of morphological changes of nuclei using Hoechst 33342 (Sigma). Briefly, cells were cultured in 6-well plates and stained with Hoechst 33342 and examined with fluorescence microscopy. In 50 randomly selected fields, the numbers of apoptotic nuclei were counted. The apoptotic characteristics of the cells were examined.
The protocol for Western blotting was described previously [17]. After treatments cells were harvested and lysed in the lysis buffer (Invitrogen, 880125A) for 30 min at 4 8C. Cell extracts were separated in 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. Then the membrane was incubated with rabbit polyclonal anti-HIF1a (ABclonal, A0009), rabbit polyclonal anti-SOX9 (Abcam, ab26414), mouse monoclonal anti-b-actin (Sigma-Aldrich, A1978). The signals were visualized by using the Chemiluminescent Substrate method with the SuperSignal West Pico kit provided by Pierce Co.
2.3. Morphometric analysis
2.6. RNA interference (RNAi)
Cellular hypertrophy was assessed by cell surface area. Cells were visualized with a fluorescence microscope (Nikon80i). Cell surface was measured using Image-ProPlus6.1 Data Analysis Software [14]. By measuring 50 random cells from three experiments, quantification of cell surface area and the average value was used for analysis.
The siRNA targeting sequences of Sox9 and HIF1a was 50 -AACTCCAGCTCCTACTACAGC-30 [18] and 50 -CAAGCAACTGTCATATATA-30 [19], respectively. The control siRNA sequence was 50 -CGTCAACATGGCTTTCACC-30 . Oligo nucleotides that represent small hairpin RNAs targeting these sequences were designed, synthesized and cloned into the pSilencer4.1-CMV neo vector (Ambion) between BamHI and HindIII sites according to the manufacturer’s instructions.
2.2. Hoechst 33342 stain
2.4. RNA extraction, reverse transcription, and quantitative real-time PCR
2.7. Statistical analysis According to the Promega Total RNA Isolation System manual, cellular RNA (1 mg per sample) was extracted from cells. Quantitative RT (reverse transcription)-PCR (RT-PCR) was performed with the Access RT-PCR System supplied by Promega. The primer pairs for the ANP gene were: sense, 50 -CAGACCAGAGCTAATCC-30 and
[(Fig._2)TD$IG]
The Student t test was used to calculate the statistical significance of the experimental data. The significance level was set as *P < 0.05; #P < 0.05; **P < 0.01; ##P < 0.01. Error bars denote standard deviations.
Fig. 2. The expression Sox9 was analyzed in cardiomyocytes in response to different glucose treatment. (A) and (B) The mRNA expression of SOX9 in cardiomyocytes in different conditions was detected with RT-PCR and real-time PCR, respectively. (C) The protein expression of SOX9 in cardiomyocytes in different conditions was detected with Western blot.
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3. Results 3.1. Different glucose concentrations sent human cardiomyocytes to different fates First, we investigated the fate of human cardiomyocytes after treatment with different concentrations of glucose. The cells were visualized with a microscope and measured with ImageProPlus6.1 after different concentrations of glucose added. The results showed that the cell size after 25 mM glucose treatment was obviously larger than that of the control, while there was no obvious difference in cell size between the control and the cells
[(Fig._3)TD$IG]
treated with 35 mM glucose (Fig. 1A and B). The expression of the hypertrophic marker, ANP, obviously increased in the cardiomyocytes after treatment with 25 mM glucose (Fig. 1C). Thus, it appears that the cells treated with 25 mM glucose tended to undergo hypertrophy compared with the control. The apoptosis analysis with Hoechst 33342 staining showed that the cells treated with 35 mM glucose tended to undergo apoptosis compared with the control and the 25 mM glucose-treated cells (Fig. 1D). The amount of apoptotic cells after 35 mM glucose treatment was about 8-fold that of the control (Fig. 1E). These results showed that different glucose concentrations resulted in different cell fates.
Fig. 3. SOX9 affects high glucose-induce cardiomyocyte hypertrophy. (A) Western blotting confirmation of the siRNA-mediated SOX9 knockdown. The negative control was an irrelevant siRNA. (B) Knockdown of SOX9 restrained cell hypertrophy induced by high glucose by morphometric analysis, cells transfected with SOX9 siRNA vectors were treated with glucose for 2 days, and cell hypertrophy was visualized and analyzed using morphometric analysis. *P < 0.05; **P < 0.01 versus the untreated group; #P < 0.05; ## P < 0.01 versus the HG-treated group. (C) Knockdown of SOX9 restrained upregulation of ANP induced by high glucose. Cells transfected with SOX9 siRNA vectors were treated with glucose for 2 days and then lysed for quantitative RT-PCR analysis. b-Actin was used as the internal reference. (D) Verification of SOX9 overexpression with Western blot. HCM cells transfected with SOX9 expression plasmids were lysed for the Western blotting assay. The negative control was the empty vector. (E) SOX9 overexpression induced cell hypertrophy as determined by morphometric analysis. The cell hypertrophy analyzed by morphometric analysis after transfection with the SOX9 expression plasmid. (F) The upregulation of ANP after transfection with the SOX9 expression plasmid.
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3.2. Expression of SOX9 gene after different glucose concentrations treatment The mRNA level of Sox9 in cells at different glucose concentrations was detected using RT-PCR and real-time PCR (Fig. 2A and B). The mRNA level of Sox9 in 25 mM glucose induced cell is about 3-fold that of the control, while there is no big difference between the control and cells treated with 35 mM glucose. The protein expression detection result verified the same phenomenon (Fig. 2C). These results indicate that Sox9 may participate in the cardiomyocyte hypertrophy. 3.3. SOX9 plays an important role in mediating glucose-induced hypertrophy in cardiomyocytes To further characterize the function of SOX9 in HG-induced hypertrophy, we used the RNAi approach to knockdown SOX9 expression to assess its effects. The endogenous SOX9 protein expression was markedly reduced by transfection of the specific SOX9 siRNA, as confirmed by Western blotting (Fig. 3A). Suppression of endogenous SOX9 expression by transfection with SOX9 siRNA obviously restrained the 25 mM glucose-induced hypertrophy by morphometric analysis (Fig. 3B). The increased expression of ANP after 25 mM glucose treatment was also decreased after SOX9 siRNA transfection (Fig. 3C). Furthermore, our results showed that the overexpression of SOX9 led to hypertrophy, with the characteristics of increased cell size and ANP gene upregulation (Fig. 3D–F). These results indicate that SOX9 was required for glucose-induced hypertrophy. 3.4. HIF1a participates in the regulation of Sox9 expression Then we intended to investigate whether any upstream genes of SOX9 are involved in this process. Western blotting analysis [(Fig._4)TD$IG] showed that expression of HIF1a was increased after 25 mM
Fig. 4. HIF1a participates in the upregulation of SOX9 in cardiomyocyte. The control means without glucose. The vector means flank vector. Actin was internal control. (A) The expressions of HIF1-a in cardiomyocytes after 25 mM glucose treatment were detected with Western blot. (B) Verification of HIF1-a overexpression with Western blot. (C) The expression of SOX9 increases when the HIF1a was over-expressed. Western blot detected the expression of SOX9 after transfection with the HIF1a expression plasmid. (D) Western blotting confirmation of the siRNA-mediated HIF1a knockdown. The negative control was an irrelevant siRNA. (E) Knockdown of HIF1a restrained the increases of SOX9 expression in response to high glucose. Cells transfected with HIF1a siRNA vectors were treated with 25 mM glucose for 2 days and then lysed for Western blot analysis. b-Actin was used as the internal reference.
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glucose treatment (Fig. 4A). When the HIF1a was over-expressed (Fig. 4B), the SOX9 expression also increases after transfection with HIF1a expression plasmid (Fig. 4C). In addition, our results showed that the increases in SOX9 expression after 25 mM treatment were remarkably attenuated when the HIF1a expression was knocked down using the RNA interference strategy (Fig. 4D and E). Together, upregulation of HIF1a gene was associated with glucose-induced hypertrophy, and increased expression of HIF1a was required for upregulation of SOX9 gene. 4. Discussion Cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. Pathologically increased in the circulating blood of diabetic patients, glucose may play a significant role in diabetic cardiomyopathy [20]. Recent studies showed that pregnant women with hyperglycemia can affect fetal heart development in its structure and function [21,22]. Understanding the molecular mechanism of glucose-induced myocardial hypertrophy may supply new therapeutic strategies for reducing neonatal heart malformations. The aim of this study was to investigate the mechanism of high glucose-induced myocardial hypertrophy in more detail in vitro. Our data in this report has validated that glucose is an effective inducer of myocardial hypertrophy, with the concentrations of glucose (25 mM). When the concentration of glucose increases, the cardiomyocytes went to apoptosis after treatment with 35 mM glucose (Fig. 1D and E). Thus, the appropriate concentration of glucose to induce hypertrophy human cardiomyocytes was 25 mM in vitro. Previous work showed many genes and protein kinase cascades pathway play important roles in hypertrophy, such as JAK2, PKC, MAPK, and ERK1/2 [23–26]. However, there are many blind nodes in glucose-induced cardiac hypertrophy. Our study revealed that the upregulation of the SOX9 gene was associated with high glucose-induced myocardial hypertrophy (Fig. 1D); moreover, the suppression of the endogenous SOX9 expression restrained the glucose-induced hypertrophy in cardiomyocytes (Fig. 2A–C). These data have shown that SOX9 plays a critical role in mediating glucose-induced myocardial hypertrophy. In addition, our results showed that the expression of SOX9 decreased when cardiomyocytes underwent apoptosis after 35 mM glucose treatment, indicating SOX9 may also participate in suppressing high glucose induced apoptosis in cardiomyocytes. This report focused on the role and mechanism of SOX9 in glucoseinduced myocardial hypertrophy. Further study will be done in mechanism of SOX9 in myocardial apoptosis. Above all, SOX9 not only induces cardiomyocyte hypertrophy, but also may participate in mediating cardiomyocyte apoptosis, indicating that SOX9 may be a new molecular target for diabetic cardiomyopathy. Data presented in this report demonstrated that HG-induced hypertrophy was associated with upregulation of HIF1a gene (Fig. 3A), which is consistent with the results from Krishnan et al. [12], reporting that HIF1a is upregulated in myocardial hypertrophy. But the effects and molecular mechanism of HIF1a upregulation in contributing to cardiac hypertrophy are not fully elucidated. Our data in this report showed that SOX9 expression was increased when the HIF1a was over-expressed (Fig. 4C). In addition, our results showed that the increases in SOX9 expression after 25 mM treatment were remarkably attenuated when the HIF1a expression was knocked down using the RNA interference strategy (Fig. 4D and E). Together, increased expression of HIF1a was required for upregulation of SOX9 gene in response to cardiac hypertrophy. However, HIF1a plays key role in cardiac hypertrophy, and there may be other down-stream genes that were regulated by HIF1a besides SOX9 in response to cardiac hypertrophy. Further study will focus on it.
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5. Conclusions Cardiac hypertrophy is associated with a dramatic change in the gene expression profile of cardiomyocytes. In our study, we show that upregulation of the SOX9 gene is associated with 25 mM glucose treatment, and is required for 25 mM glucose-induced hypertrophy. We also showed that 25 mM glucose treatments led to the increase of the HIF1a gene expression. Further study showed that the increase in the HIF1a expression, in response to 25 mM glucose treatment, was contributed to the upregulation of SOX9 gene. This study is highly relevant to the understanding of the effects of the HIF1a-SOX9 upregulation on HG-induced cardiomyocyte apoptosis, as well as the related molecular mechanisms. Funding This research was supported by grants from the Central Publicinterest Scientific Institution Basal Research Fund (Grant No. 2012GJSSJKA02) and the National Natural Science Foundation of China (Grant No. 31301145). Conflict of interest The authors declare that there are no conflicts of interests. Acknowledgements We are grateful to everyone who helped us to successfully complete this research. References [1] D. Levy, R.J. Garrison, D.D. Savage, W.B. Kannel, W.P. Castelli, Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study, N. Engl. J. Med. (1990) 1561–1566. [2] A. Rababa’h, S. Singh, S.V. Suryavanshi, S.E. Altarabsheh, S.V. Deo, B.K. McConnell, Compartmentalization role of A-kinase anchoring proteins (AKAPs) in mediating protein kinase A (PKA) signaling and cardiomyocyte hypertrophy, Int. J. Mol. Sci. 16 (2015) 218–229. [3] I.G. Poornima, P. Parikh, R.P. Shannon, Diabetic cardiomyopathy: the search for a unifying hypothesis, Circ. Res. 98 (2006) 596–605. [4] S. Umadevi, V. Gopi, S.P. Simna, A. Parthasarathy, S.M. Yousuf, V. Elangovan, Studies on the cardioprotective role of gallic acid against AGE-induced cell proliferation and oxidative stress in H9C2 (2–1) cells, Cardiovasc. Toxicol. 12 (2012) 304–311. [5] A. Malhotra, B.P. Kang, S. Cheung, D. Opawumi, L.G. Meggs, Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I, Diabetes 50 (2001) 1918–1926. [6] M. Wang, J. Wang, R. Tan, Q. Wu, H. Qiu, J. Yang, et al., Effect of berberine on PPARa/NO activation in high glucose- and insulin-induced cardiomyocyte hypertrophy, Evid. Based Complement. Altern. Med. 2013 (2013) 285489. [7] M. Sun, H. Uozaki, R. Hino, A. Kunita, A. Shinozaki, T. Ushiku, et al., SOX9 expression and its methylation status in gastric cancer, Virchows Archiv. Int. J. Pathol. 460 (2012) 271–279.
[8] D. Ikegami, H. Akiyama, A. Suzuki, T. Nakamura, T. Nakano, H. Yoshikawa, et al., Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways, Development 138 (2011) 1507–1519. [9] M.R. Shin, H.J. Lee, S.K. Kang, Q.S. Auh, Y.M. Lee, Y.C. Kim, et al., Isocudraxanthone K induces growth inhibition and apoptosis in oral cancer cells via hypoxia inducible factor-1alpha, BioMed Res. Int. 2014 (2014) 934691. [10] R. Bohuslavova, F. Kolar, L. Kuthanova, J. Neckar, A. Tichopad, G. Pavlinkova, Gene expression profiling of sex differences in HIF1-dependent adaptive cardiac responses to chronic hypoxia, J. Appl. Physiol. 109 (2010) 1195–1202. [11] J.W. Kim, I. Tchernyshyov, G.L. Semenza, C.V. Dang, HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia, Cell Metab. 3 (2006) 177–185. [12] J. Krishnan, M. Suter, R. Windak, T. Krebs, A. Felley, C. Montessuit, et al., Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy, Cell Metab. 9 (2009) 512–524. [13] D. Su, S. Jing, L. Guan, Q. Li, H. Zhang, X. Gao, et al., Role of Nodal-PITX2C signaling pathway in glucose-induced cardiomyocyte hypertrophy, Biochim. Biol. Cell. (Biochem. Cell Biol.), 92 (2014) 183–190. [14] K. Jeong, H. Kwon, C. Min, Y. Pak, Modulation of the caveolin-3 localization to caveolae and STAT3 to mitochondria by catecholamine-induced cardiac hypertrophy in H9c2 cardiomyoblasts, Exp. Mol. Med. 41 (2009) 226–235. [15] B. Baur, C. Solbach, E. Andreolli, G. Winter, H.J. Machulla, S.N. Reske, Synthesis, radiolabelling and in vitro characterization of the gallium-68-, Yttrium-90and lutetium-177-labelled PSMA ligand, CHX-A’’-DTPA-DUPA-Pep, Pharmaceuticals 7 (2014) 517–529. [16] A. Orlandi, A. Ciucci, A. Ferlosio, R. Genta, L.G. Spagnoli, G. Gabbiani, Cardiac myxoma cells exhibit embryonic endocardial stem cell features, J. Pathol. 209 (2006) 231–239. [17] G. Ren, G. Zhang, Z. Dong, Z. Liu, L. Li, Y. Feng, et al., Recruitment of HDAC4 by transcription factor YY1 represses HOXB13 to affect cell growth in ARnegative prostate cancers, Int. J. Biochem. Cell Biol. 41 (2009) 1094–1101. [18] I. Kou, S. Ikegawa, SOX9-dependent and -independent transcriptional regulation of human cartilage link protein, J. Biol. Chem. 279 (2004) 50942–50948. [19] W.B. Cai, Y. Zhang, R. Cheng, Z. Wang, S.H. Fang, Z.M. Xu, et al., Dual inhibition of plasminogen kringle 5 on angiogenesis and chemotaxis suppresses tumor metastasis by targeting HIF-1alpha pathway, PLOS ONE 7 (2012) e53152. [20] D. Dyntar, P. Sergeev, J. Klisic, P. Ambuhl, M.C. Schaub, M.Y. Donath, High glucose alters cardiomyocyte contacts and inhibits myofibrillar formation, J. Clin. Endocrinol. Metab. 91 (2006) 1961–1967. [21] H.S. Menezes, M. Barra, A.R. Bello, C.B. Martins, P. Zielinsky, Fetal myocardial hypertrophy in an experimental model of gestational diabetes, Cardiol. Young 11 (2001) 609–613. [22] G. Passarella, G. Trifiro, M. Gasparetto, G.S. Moreolo, O. Milanesi, Disorders in glucidic metabolism and congenital heart diseases: detection and prevention, Pediatr. Cardiol. 34 (2013) 931–937. [23] S. Chen, Z.A. Khan, M. Karmazyn, S. Chakrabarti, Role of endothelin-1, sodium hydrogen exchanger-1 and mitogen activated protein kinase (MAPK) activation in glucose-induced cardiomyocyte hypertrophy, Diabetes Metab. Res. Rev. 23 (2007) 356–367. [24] F. Ding, L. Yu, M. Wang, S. Xu, Q. Xia, G. Fu, O-GlcNAcylation involvement in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2, Amino Acids 45 (2013) 339–349. [25] A. Modesti, I. Bertolozzi, T. Gamberi, M. Marchetta, C. Lumachi, M. Coppo, et al., Hyperglycemia activates JAK2 signaling pathway in human failing myocytes via angiotensin II-mediated oxidative stress, Diabetes 54 (2005) 394–401. [26] M. Wang, W.B. Zhang, J.H. Zhu, G.S. Fu, B.Q. Zhou, Breviscapine ameliorates hypertrophy of cardiomyocytes induced by high glucose in diabetic rats via the PKC signaling pathway, Acta Pharmacol. Sin. 30 (2009) 1081–1091.
Available online at
ScienceDirect www.sciencedirect.com
Original Article
Study on the mechanism of HIF1a-SOX9 in glucose-induced cardiomyocyte hypertrophy Qianqian Gao a, Lina Guan b, Shanshan Hu c, Yanwei Yao a, Xiaolin Ren a, Zhenwei Zhang a, Canling Cheng a, Yi Liu d, Chun Zhang d, Jinpeng Huang d, Dongmei Su b,*, Xu Ma b,* a
Linyi Academy of Technology Cooperation and Application, Linyi, Shandong Province, China Department of Genetics, National Research Institute for Family Planning, Beijing, China Department of Ophthalmology, Hongqi Hospital of Mudanjiang Medical College, Mudanjiang, Heilongjiang Province, China d Department of Biotechnology, College of Bioengineering, Beijing Polytechnic, Beijing, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1st June 2015 Accepted 9 July 2015
A major cause of morbidity and mortality in cardiovascular disease is pathological cardiac hypertrophy. With an increase in the cellular surface area and upregulation of the atrial natriuretic peptide (ANP) gene, cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. ANP is a hypertrophic marker. Many works have been done to explore how the glucose induces the cardiac hypertrophy. However, it is not enough for us to figure it out. In this study, the influences of different glucose concentrations on cardiomyocytes were examined in vitro. The results showed that cardiomyocytes cultured with 25 mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35 mM glucose tended to undergo apoptosis. An increased expression of SOX9 was observed when cardiomyocytes were cultured with 25 mM glucose, but when the concentration of glucose was increased to 35 mM, the expression of SOX9 decreased. We used the RNAi approach to knockdown SOX9 expression, to assess its effects on cardiomyocyte hypertrophy. The results showed that knockdown of the SOX9 gene suppressed the 25 mM glucose-induced cardiomyocyte hypertrophy. The upregulation of the ANP gene was associated with overexpression of SOX9. Additionally, the results showed that high glucose (HG, 25 mM) treatment increased the expression of hypoxia-inducible factor (HIF)1a. Further study showed that HIF1a participated in regulating SOX9 expression in response to HG. This study revealed a novel regulatory mechanism of HIF1a-SOX9 in high glucose-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Cardiomyocyte hypertrophy SOX9 HIF1a
1. Introduction Myocyte usually increases cell size and protein synthesis to compensate the increase in the workload, which is the character of cardiac hypertrophy. That is associated with an overtly increased risk of ventricular dysfunction, heart failure, and malignant arrhythmias in obese individuals [1,2]. Abnormal glucose homeostasis affects cardiac structure and function [3,4]. In response to high glucose induction, the ventricular myocardium will undergo hypertrophic growth [5,6]. Abbreviations: ANP, atrial natriuretic peptide; RT-PCR, reverse transcription; Sox9, SRY (sex determining region Y)-box 9; HIF1a, hypoxia inducible factor 1 alpha. * Corresponding authors at: Department of Genetics, National Research Institute for Family Planning, 12, Dahuisi Road, Haidian, Beijing 100081, China. E-mail addresses: [email protected] (D. Su), [email protected], [email protected] (X. Ma). http://dx.doi.org/10.1016/j.biopha.2015.07.009 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
Sox9 is a member of Sry-related high mobility group box family of transcription factors [7]. By binding to the Pik3ca promoter to induce Akt phosphorylation, Sox9 expression is essential for hypertrophy and sustains chondrocyte-specific survival mechanisms in differentiated chondrocytes in mouse [8]. However, the expression and role of SOX9 in human cardiomyocyte hypertrophy was not elucidated. HIF1a plays a significant role in adaptive cardiopulmonary responses and the transcriptional regulation in the heart is considerably influenced by dysfunction of HIF1 pathways [9,10]. Glycolytic genes activated by HIF-1a are critical for metabolic adaptation to hypoxia by increasing conversion of glucose to pyruvate and subsequently to lactate [11]. As a key mediator of glycolysis and lipid anabolism, HIF1a jointly upregulates in hypertrophic cardiomyopathy and is cooperated with PPARg to mediate pivotal changes in cardiac hypertrophy in vivo [12]. Combining with the evidence about HIF1a, we speculated that
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there is change of HIF1a expression in response to high glucose, and HIF-1a may play an important role in high glucose-induced cardiomyocyte hypertrophy. This study identifies the molecular mechanisms leading to cardiomyocyte hypertrophy, in response to high concentrations of extracellular glucose, which may be important in the treatment of diabetic heart disease. Specifically, we focus on the expression and regulatory mechanism of Sox9 and HIF1a in cardiomyocyte hypertrophy induced by high glucose. Firstly, we examined the influence of different glucose concentrations on cardiomyocytes in vitro. Our results showed that cardiomyocytes cultured with 25 mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35 mM glucose tended to undergo apoptosis. The upregulation of Sox9 is associated with high glucose-induced cardiomyocytes hypertrophy. Further study showed that SOX9 is required for high glucose-induced cardio[(Fig._1)TD$IG]myocyte hypertrophy by knockdown SOX9 expression using the
RNAi approach. Meanwhile, SOX9 overexpression also partly induced cardiomyocyte hypertrophy. In addition, our results showed that the upregulation of HIF1a and cardiomyocytes hypertrophy is positively correlated. Moreover, we found that HIF1a participated in regulating SOX9 expression in response to high glucose. Above all, this study is highly relevant to the understanding of the effects of the HIF1a-SOX9 upregulation on HG-induced cardiomyocyte hypertrophy, as well as the related molecular mechanism. 2. Materials and methods 2.1. Cell culture, transfection and treatments Human cardiac cardiomyocytes (HCM, ScienCell Research Laboratories) were cultured and transfected as described before in previous work [13]. Human cardiac myocytes (HCM, ScienCell
Fig. 1. Different glucose concentrations sent human cardiomyocytes to different fates. (A) Comparison of the cell size in different glucose contents under Optical Microscope. HCM cells were treated with different concentration of glucose for 48 h, and were visualized under Optical Microscope. (B) The analysis of the cell surface was done with Image-Pro plus Data Analysis Software. Quantification of the cell surface area was measured with 300 random cells from three experiments after different concentration of glucose treatment for 48 h, and the average value was used for the analysis. *P < 0.05; **P < 0.01 versus the untreated group. (C) The change in endogenous of ANP mRNA levels in response to different concentration of glucose stimulus. The cells were treated with different concentration of glucose for 2 days, and ANP mRNA was measured using quantitative RT-PCR. b-Actin was used as an internal control. (D) The apoptosis detection of cardiomyocytes under different glucose contents treatment by Hoechst 33342 staining assay. HCM cells were treated with different concentration of glucose for 2 days and then were stained with Hoechst 33342 and to assess the apoptosis. The panel showed the nuclear morphology stained with blue by using Hoechst 33342. (E) The apoptosis rates were analyzed after different concentration of glucose treatment. The apoptosis rates which were calculated based on at least 100 cells from three experiments and are shown as means SD. *P < 0.05; **P < 0.01 versus the untreated cells.
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Research Laboratories) were maintained in cardiac myocyte medium supplemented with 10% fetal bovine serum, cardiac myocyte growth supplement, 100 mg/mL penicillin, and 100 mg/ mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 8C. The Sox9, Hif1a siRNA vectors and DNA plasmids were transfected into cardiomyocytes using lipofectamine 2000 (Invitrogen) procedures. Cardiomyocytes were treated with 25 mM and 35 mM glucose, respectively. Cardiomyocytes treated with 5 mM glucose were used as control sample.
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anti-sense, 50 -CTCACTGAGCACTTGT-30 [15]. The primer pairs for the SOX9 gene were: sense, 50 -AAGACATTTAAGCTAAAGGCAACTCGTAC-30 and antisense, 50 -TGATCACAC GATTCTCCATCATCCTC-30 [16]. The b-actin primer pairs were: sense, 50 -TCGTGCGTGACATTAAGGAG-30 and antisense, 50 -ATGCCAGGGT ACATGGTGGT-30 [15]. 2.5. Western blotting
The apoptosis of cells was assessed through observation of morphological changes of nuclei using Hoechst 33342 (Sigma). Briefly, cells were cultured in 6-well plates and stained with Hoechst 33342 and examined with fluorescence microscopy. In 50 randomly selected fields, the numbers of apoptotic nuclei were counted. The apoptotic characteristics of the cells were examined.
The protocol for Western blotting was described previously [17]. After treatments cells were harvested and lysed in the lysis buffer (Invitrogen, 880125A) for 30 min at 4 8C. Cell extracts were separated in 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. Then the membrane was incubated with rabbit polyclonal anti-HIF1a (ABclonal, A0009), rabbit polyclonal anti-SOX9 (Abcam, ab26414), mouse monoclonal anti-b-actin (Sigma-Aldrich, A1978). The signals were visualized by using the Chemiluminescent Substrate method with the SuperSignal West Pico kit provided by Pierce Co.
2.3. Morphometric analysis
2.6. RNA interference (RNAi)
Cellular hypertrophy was assessed by cell surface area. Cells were visualized with a fluorescence microscope (Nikon80i). Cell surface was measured using Image-ProPlus6.1 Data Analysis Software [14]. By measuring 50 random cells from three experiments, quantification of cell surface area and the average value was used for analysis.
The siRNA targeting sequences of Sox9 and HIF1a was 50 -AACTCCAGCTCCTACTACAGC-30 [18] and 50 -CAAGCAACTGTCATATATA-30 [19], respectively. The control siRNA sequence was 50 -CGTCAACATGGCTTTCACC-30 . Oligo nucleotides that represent small hairpin RNAs targeting these sequences were designed, synthesized and cloned into the pSilencer4.1-CMV neo vector (Ambion) between BamHI and HindIII sites according to the manufacturer’s instructions.
2.2. Hoechst 33342 stain
2.4. RNA extraction, reverse transcription, and quantitative real-time PCR
2.7. Statistical analysis According to the Promega Total RNA Isolation System manual, cellular RNA (1 mg per sample) was extracted from cells. Quantitative RT (reverse transcription)-PCR (RT-PCR) was performed with the Access RT-PCR System supplied by Promega. The primer pairs for the ANP gene were: sense, 50 -CAGACCAGAGCTAATCC-30 and
[(Fig._2)TD$IG]
The Student t test was used to calculate the statistical significance of the experimental data. The significance level was set as *P < 0.05; #P < 0.05; **P < 0.01; ##P < 0.01. Error bars denote standard deviations.
Fig. 2. The expression Sox9 was analyzed in cardiomyocytes in response to different glucose treatment. (A) and (B) The mRNA expression of SOX9 in cardiomyocytes in different conditions was detected with RT-PCR and real-time PCR, respectively. (C) The protein expression of SOX9 in cardiomyocytes in different conditions was detected with Western blot.
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3. Results 3.1. Different glucose concentrations sent human cardiomyocytes to different fates First, we investigated the fate of human cardiomyocytes after treatment with different concentrations of glucose. The cells were visualized with a microscope and measured with ImageProPlus6.1 after different concentrations of glucose added. The results showed that the cell size after 25 mM glucose treatment was obviously larger than that of the control, while there was no obvious difference in cell size between the control and the cells
[(Fig._3)TD$IG]
treated with 35 mM glucose (Fig. 1A and B). The expression of the hypertrophic marker, ANP, obviously increased in the cardiomyocytes after treatment with 25 mM glucose (Fig. 1C). Thus, it appears that the cells treated with 25 mM glucose tended to undergo hypertrophy compared with the control. The apoptosis analysis with Hoechst 33342 staining showed that the cells treated with 35 mM glucose tended to undergo apoptosis compared with the control and the 25 mM glucose-treated cells (Fig. 1D). The amount of apoptotic cells after 35 mM glucose treatment was about 8-fold that of the control (Fig. 1E). These results showed that different glucose concentrations resulted in different cell fates.
Fig. 3. SOX9 affects high glucose-induce cardiomyocyte hypertrophy. (A) Western blotting confirmation of the siRNA-mediated SOX9 knockdown. The negative control was an irrelevant siRNA. (B) Knockdown of SOX9 restrained cell hypertrophy induced by high glucose by morphometric analysis, cells transfected with SOX9 siRNA vectors were treated with glucose for 2 days, and cell hypertrophy was visualized and analyzed using morphometric analysis. *P < 0.05; **P < 0.01 versus the untreated group; #P < 0.05; ## P < 0.01 versus the HG-treated group. (C) Knockdown of SOX9 restrained upregulation of ANP induced by high glucose. Cells transfected with SOX9 siRNA vectors were treated with glucose for 2 days and then lysed for quantitative RT-PCR analysis. b-Actin was used as the internal reference. (D) Verification of SOX9 overexpression with Western blot. HCM cells transfected with SOX9 expression plasmids were lysed for the Western blotting assay. The negative control was the empty vector. (E) SOX9 overexpression induced cell hypertrophy as determined by morphometric analysis. The cell hypertrophy analyzed by morphometric analysis after transfection with the SOX9 expression plasmid. (F) The upregulation of ANP after transfection with the SOX9 expression plasmid.
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3.2. Expression of SOX9 gene after different glucose concentrations treatment The mRNA level of Sox9 in cells at different glucose concentrations was detected using RT-PCR and real-time PCR (Fig. 2A and B). The mRNA level of Sox9 in 25 mM glucose induced cell is about 3-fold that of the control, while there is no big difference between the control and cells treated with 35 mM glucose. The protein expression detection result verified the same phenomenon (Fig. 2C). These results indicate that Sox9 may participate in the cardiomyocyte hypertrophy. 3.3. SOX9 plays an important role in mediating glucose-induced hypertrophy in cardiomyocytes To further characterize the function of SOX9 in HG-induced hypertrophy, we used the RNAi approach to knockdown SOX9 expression to assess its effects. The endogenous SOX9 protein expression was markedly reduced by transfection of the specific SOX9 siRNA, as confirmed by Western blotting (Fig. 3A). Suppression of endogenous SOX9 expression by transfection with SOX9 siRNA obviously restrained the 25 mM glucose-induced hypertrophy by morphometric analysis (Fig. 3B). The increased expression of ANP after 25 mM glucose treatment was also decreased after SOX9 siRNA transfection (Fig. 3C). Furthermore, our results showed that the overexpression of SOX9 led to hypertrophy, with the characteristics of increased cell size and ANP gene upregulation (Fig. 3D–F). These results indicate that SOX9 was required for glucose-induced hypertrophy. 3.4. HIF1a participates in the regulation of Sox9 expression Then we intended to investigate whether any upstream genes of SOX9 are involved in this process. Western blotting analysis [(Fig._4)TD$IG] showed that expression of HIF1a was increased after 25 mM
Fig. 4. HIF1a participates in the upregulation of SOX9 in cardiomyocyte. The control means without glucose. The vector means flank vector. Actin was internal control. (A) The expressions of HIF1-a in cardiomyocytes after 25 mM glucose treatment were detected with Western blot. (B) Verification of HIF1-a overexpression with Western blot. (C) The expression of SOX9 increases when the HIF1a was over-expressed. Western blot detected the expression of SOX9 after transfection with the HIF1a expression plasmid. (D) Western blotting confirmation of the siRNA-mediated HIF1a knockdown. The negative control was an irrelevant siRNA. (E) Knockdown of HIF1a restrained the increases of SOX9 expression in response to high glucose. Cells transfected with HIF1a siRNA vectors were treated with 25 mM glucose for 2 days and then lysed for Western blot analysis. b-Actin was used as the internal reference.
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glucose treatment (Fig. 4A). When the HIF1a was over-expressed (Fig. 4B), the SOX9 expression also increases after transfection with HIF1a expression plasmid (Fig. 4C). In addition, our results showed that the increases in SOX9 expression after 25 mM treatment were remarkably attenuated when the HIF1a expression was knocked down using the RNA interference strategy (Fig. 4D and E). Together, upregulation of HIF1a gene was associated with glucose-induced hypertrophy, and increased expression of HIF1a was required for upregulation of SOX9 gene. 4. Discussion Cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. Pathologically increased in the circulating blood of diabetic patients, glucose may play a significant role in diabetic cardiomyopathy [20]. Recent studies showed that pregnant women with hyperglycemia can affect fetal heart development in its structure and function [21,22]. Understanding the molecular mechanism of glucose-induced myocardial hypertrophy may supply new therapeutic strategies for reducing neonatal heart malformations. The aim of this study was to investigate the mechanism of high glucose-induced myocardial hypertrophy in more detail in vitro. Our data in this report has validated that glucose is an effective inducer of myocardial hypertrophy, with the concentrations of glucose (25 mM). When the concentration of glucose increases, the cardiomyocytes went to apoptosis after treatment with 35 mM glucose (Fig. 1D and E). Thus, the appropriate concentration of glucose to induce hypertrophy human cardiomyocytes was 25 mM in vitro. Previous work showed many genes and protein kinase cascades pathway play important roles in hypertrophy, such as JAK2, PKC, MAPK, and ERK1/2 [23–26]. However, there are many blind nodes in glucose-induced cardiac hypertrophy. Our study revealed that the upregulation of the SOX9 gene was associated with high glucose-induced myocardial hypertrophy (Fig. 1D); moreover, the suppression of the endogenous SOX9 expression restrained the glucose-induced hypertrophy in cardiomyocytes (Fig. 2A–C). These data have shown that SOX9 plays a critical role in mediating glucose-induced myocardial hypertrophy. In addition, our results showed that the expression of SOX9 decreased when cardiomyocytes underwent apoptosis after 35 mM glucose treatment, indicating SOX9 may also participate in suppressing high glucose induced apoptosis in cardiomyocytes. This report focused on the role and mechanism of SOX9 in glucoseinduced myocardial hypertrophy. Further study will be done in mechanism of SOX9 in myocardial apoptosis. Above all, SOX9 not only induces cardiomyocyte hypertrophy, but also may participate in mediating cardiomyocyte apoptosis, indicating that SOX9 may be a new molecular target for diabetic cardiomyopathy. Data presented in this report demonstrated that HG-induced hypertrophy was associated with upregulation of HIF1a gene (Fig. 3A), which is consistent with the results from Krishnan et al. [12], reporting that HIF1a is upregulated in myocardial hypertrophy. But the effects and molecular mechanism of HIF1a upregulation in contributing to cardiac hypertrophy are not fully elucidated. Our data in this report showed that SOX9 expression was increased when the HIF1a was over-expressed (Fig. 4C). In addition, our results showed that the increases in SOX9 expression after 25 mM treatment were remarkably attenuated when the HIF1a expression was knocked down using the RNA interference strategy (Fig. 4D and E). Together, increased expression of HIF1a was required for upregulation of SOX9 gene in response to cardiac hypertrophy. However, HIF1a plays key role in cardiac hypertrophy, and there may be other down-stream genes that were regulated by HIF1a besides SOX9 in response to cardiac hypertrophy. Further study will focus on it.
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5. Conclusions Cardiac hypertrophy is associated with a dramatic change in the gene expression profile of cardiomyocytes. In our study, we show that upregulation of the SOX9 gene is associated with 25 mM glucose treatment, and is required for 25 mM glucose-induced hypertrophy. We also showed that 25 mM glucose treatments led to the increase of the HIF1a gene expression. Further study showed that the increase in the HIF1a expression, in response to 25 mM glucose treatment, was contributed to the upregulation of SOX9 gene. This study is highly relevant to the understanding of the effects of the HIF1a-SOX9 upregulation on HG-induced cardiomyocyte apoptosis, as well as the related molecular mechanisms. Funding This research was supported by grants from the Central Publicinterest Scientific Institution Basal Research Fund (Grant No. 2012GJSSJKA02) and the National Natural Science Foundation of China (Grant No. 31301145). Conflict of interest The authors declare that there are no conflicts of interests. Acknowledgements We are grateful to everyone who helped us to successfully complete this research. References [1] D. Levy, R.J. Garrison, D.D. Savage, W.B. Kannel, W.P. Castelli, Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study, N. Engl. J. Med. (1990) 1561–1566. [2] A. Rababa’h, S. Singh, S.V. Suryavanshi, S.E. Altarabsheh, S.V. Deo, B.K. McConnell, Compartmentalization role of A-kinase anchoring proteins (AKAPs) in mediating protein kinase A (PKA) signaling and cardiomyocyte hypertrophy, Int. J. Mol. Sci. 16 (2015) 218–229. [3] I.G. Poornima, P. Parikh, R.P. Shannon, Diabetic cardiomyopathy: the search for a unifying hypothesis, Circ. Res. 98 (2006) 596–605. [4] S. Umadevi, V. Gopi, S.P. Simna, A. Parthasarathy, S.M. Yousuf, V. Elangovan, Studies on the cardioprotective role of gallic acid against AGE-induced cell proliferation and oxidative stress in H9C2 (2–1) cells, Cardiovasc. Toxicol. 12 (2012) 304–311. [5] A. Malhotra, B.P. Kang, S. Cheung, D. Opawumi, L.G. Meggs, Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I, Diabetes 50 (2001) 1918–1926. [6] M. Wang, J. Wang, R. Tan, Q. Wu, H. Qiu, J. Yang, et al., Effect of berberine on PPARa/NO activation in high glucose- and insulin-induced cardiomyocyte hypertrophy, Evid. Based Complement. Altern. Med. 2013 (2013) 285489. [7] M. Sun, H. Uozaki, R. Hino, A. Kunita, A. Shinozaki, T. Ushiku, et al., SOX9 expression and its methylation status in gastric cancer, Virchows Archiv. Int. J. Pathol. 460 (2012) 271–279.
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