Radiotherapy and Oncology xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
MiR-210: A potential therapeutic target against radiation-induced enteropathy Saad Hamama a,b,c, Muhammad Zaeem Noman d, Pascal Gervaz e, Sylvie Delanian f, Marie-Catherine Vozenin a,b,c,g,⇑ a
INSERM U-1030 ‘‘Molecular Radiotherapy’’, Institut Gustave Roussy, Villejuif, France; b LabEX LERMIT Villejuif, France; c Université Paris Sud Paris XI, Le Kremlin Bicêtre, France; INSERM, U753, Laboratoire d’immunologie des Tumeurs Humaines: Interaction Effecteurs Cytotoxiques-Système Tumoral, Institut Gustave-Roussy, Villejuif, France; e Geneva University Hospital and Medical School, Geneva, Switzerland; f Unité de Radiopathologie, Service Oncologie-Radiothérapie, Hôpital Saint-Louis, APHP, Paris, France; g Laboratoire de RadioOncologie, Swiss Cancer Center of Lausanne, CHUV, Lausanne, Switzerland
d
a r t i c l e
i n f o
Article history: Received 13 July 2013 Received in revised form 1 October 2013 Accepted 16 October 2013 Available online xxxx
a b s t r a c t A previously undescribed and robust miR210 overexpression is shown in intestinal samples obtained from patients with radiation enteropathy and fibrotic cultured cells. In addition, miR-210 overexpression is repressed by antifibrotic treatment combining pentoxifylline and a-tocopherol. Ó 2013 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2013) xxx–xxx
Keywords: Anti-fibrotic treatment Radiation-induced fibrosis Radiation enteropathy microRNA miR-210 Trolox Pentoxifylline
Pelvic radiotherapy for treatment of urogenital or anorectal malignancies may induce radiation-induced enteropathy characterized by transmural fibrosis that may affect small intestine, colon, rectum and the anal sphincter wall. Fibrosis impairs contractility, leading to dysfunction including diarrhea and constipation, fecal incontinence and ultimately occlusion [1]. Today, therapeutic options against radiation-induced fibrosis such as radiation-induced enteropathy remain very limited, therefore identifying new therapeutic targets is an important clinical issue. Among the possible therapeutic candidates, microRNAs were recently studied. Liu et al. demonstrated the pathogenic inter-relation between miR-21 and the fibrogenic growth factor TGF-b1 [2]. TGF-b1 is also known to modulate the transcription of microRNAs [3] and their maturation via activation of Smad proteins [4]. Due to their critical biological roles in fine tuning cellular and tissue homeostasis, we investigated the possible involvement of microRNAs in radiation enteropathy by molecular profiling and
⇑ Corresponding author. Address: Laboratoire de RadioOncologie, Swiss Cancer Center of Lausanne, CHUV, 1010 Lausanne, Switzerland. E-mail addresses: [email protected] (S. Hamama), muhammed-zaeem. [email protected] (M.Z. Noman), [email protected] (P. Gervaz), sylvie.delanian @sls.aphp.fr (S. Delanian), [email protected] (M.-C. Vozenin).
showed robust induction of miR-210 in a validated cellular model of radiation-induced fibrosis [5,6] comprised of primary smooth muscle cells derived from human radiation enteropathy compared with their healthy counterparts (Fig 1A). Secondly, we confirmed miR-210 induction in rectal samples surgically removed 12 weeks after radiotherapy in a cohort of 16 patients irradiated for rectal cancer with a total dose of 50.4 Gray versus 4 non-irradiated patients’ samples (Fig 1B). Human samples were histologically and molecularly characterized as fibrotic with consistent induction of fibrogenic growth factors such as TGF-b1 and CTGF (Fig 1C). Thirdly, we explored possible modulation of miR-210 after anti-fibrotic therapy using the reference treatment combining pentoxifylline–trolox [7] and found that miR-210 was repressed by 40% when cells were incubated with trolox (p < 0.05) (Fig 1D). Interestingly, addition of pentoxyfilline–trolox did not enhance the inhibitory effect on miR-210, suggesting that miR-210 inhibition was trolox-mediated and suggesting the involvement of antioxidant pathways in miR-210 modulation. Since miR-210 is a well known hypoxia microRNA [8], we investigated the role of hypoxia and more specifically, HIF-1a in miR-210 up-regulation. Consistently, when primary smooth muscle cells were cultured under hypoxic conditions (1% O2) significant induction of miR-210 was observed both in normal and fibrotic cells with higher level in fibrotic cells
0167-8140/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radonc.2013.10.030
Please cite this article in press as: Hamama S et al. MiR-210: A potential therapeutic target against radiation-induced enteropathy. Radiother Oncol (2013), http://dx.doi.org/10.1016/j.radonc.2013.10.030
2
Mir-210 in radiation-induced fibrosis
(A)
(B)
MiR-210 expression in normal and fibrotic cells
MiR-210 relative expression
3
*
2.5
*
2 1.5
Hybridization qRT-PCR
1 0.5 0
Normal
Fibrosis
(C)
(D)
Fig. 1. (A) MiR-210 overexpression in fibrotic versus normal cells in normoxic conditions (21% O2). (⁄p 6 0.05). MiR-210 levels were detected using 8 15 K Agilent Human v3 (G4471A) microRNA array and results were confirmed by qRT-PCR using RNU6B as an endogenous control. (B) Levels of miR-210 are significantly increased in anal sphincter surgical specimens isolated from patients (n = 16) suffering from radiation enteropathy versus normal biopsies (n = 4) (⁄p 6 0.05). MiR-210 levels were detected by qRT-PCR using RNU6B as an endogenous control. (C) Levels of TGF-b1 and CTGF mRNA were much higher in anal sphincter surgical specimen isolated from patients suffering from radiation enteropathy versus normal biopsies: (⁄⁄p 6 0.01). mRNA levels were detected by qRT-PCR using 18S as an endogenous control. (D) 24 h treatment with trolox (50 lg/mL) or pentoxifylline–trolox (50 lg/mL of each molecule) reverses miR-210 overexpression detected by qRT-PCR and normalized to RNU6B. Results are always expressed as mean ± SEM. Significance was established with GraphPad Prism (GraphPad Software) using the non-parametric Kruskal–Wallis or Student’s t-test, each when appropriate, for the analysis of at least three independent in vitro experiments. Differences were considered significant at a value of p 6 0.05. (P: pentoxifylline, T: trolox; PT: pentoxifylline–trolox treatment 10 and 50 lg/mL, continuous and dashed lines: miR-210 levels in normal and fibrotic cells, respectively).
(Fig 2A). Trolox and pentoxifylline–trolox combination again reduced miR-210 expression in fibrotic cells (Fig 2A) when cultured in hypoxic conditions. Studying HIF-1a expression, we surprisingly found that it was not modified by any treatment (Fig 2B), neither was its target VEGF (Fig 2C) suggesting that the down-regulation of miR-210 expression induced by trolox and pentoxifylline-trolox combination (PT) was HIF-1a-independent. Lastly, we investigated whether direct targeted inhibition of miR-210 displayed anti-fibrotic action per se. Cells were then transfected using an antagomiR210 and the mRNA expression of TGF-b1, CTGF, PAI-1, FN-1, and Col-Ia1 was investigated. We found a reduction of Col-Ia1 mRNA expression (p < 0.05) 48 h after transfection (Fig 2D) suggesting a role for miR-210 in the regulation of extracellular matrix homeostasis in fibrotic tissues. However, the mRNA expression of TGFb1, CTGF, PAI-1, and FN-1 was not modified at the studied time points, suggesting a specific action of miR-210 inhibition of matrix genes. MiR-210 has been shown to be implicated in various pathophysiological functions such as apoptosis angiogenesis, cell cycle regulation, DNA damage repair, mitochondrial metabolism, stem cell survival, [9], and increased radioresistance [10] but to our knowledge this is the first publication to report its association with fibrosis. Interestingly, miR-210 action in the regulation of hypoxiainduced pathogenesis is also well documented and mediated by HIF-1a through the hypoxia responsive element (HRE) located on the proximal promoter of miR-210 [9]. However, our results suggest that an additional yet never reported regulation of miR-210 occurs in radiation-induced fibrosis since we found no modulation of the HIF1-a cascade in our system. Still, Vitamin
E-based anti-fibrotic treatments (i.e., trolox alone and pentoxifylline–trolox combination) reduced miR-210 expression in hypoxic conditions. More interestingly the antagomiR-210 decreased Col Ia1 mRNA expression in fibrotic cells. The intrinsic function of microRNAs implies that their inhibition induces increased expression of their targets. Here the downregulation of Col Ia1 could suggest a novel indirect up-regulation of a co-repressor of Col Ia1 exists and remains to be identified. To explore this putative indirect mechanism, in silico analysis of miR-210 targets using various software including TargetScan, MiRanda, PicTar, and MicroCosm were performed but revealed no obvious candidates and more experiments are needed to identify this mediator. In conclusion, our study is the first to show the overexpression of miR-210 in radiation-induced intestinal fibrosis. Furthermore, we showed that miR-210 overexpression was repressed by anti-fibrotic treatment and direct miR-210 inhibition exerted an anti-fibrotic action through the reduction of Col Ia1 mRNA expression in fibrotic cells. Further studies are needed to clearly define the molecular mechanisms involved but the specific inhibition of fibrillar collagen triggered by the antagomiR-210 will probably be highly clinically valuable as abnormal and excessive collagen deposition [11] is responsible for the loss of intestinal compliance leading to stenosis and poor function in radiation enteropathy.
Conflict of interest None.
Please cite this article in press as: Hamama S et al. MiR-210: A potential therapeutic target against radiation-induced enteropathy. Radiother Oncol (2013), http://dx.doi.org/10.1016/j.radonc.2013.10.030
3
S. Hamama et al. / Radiotherapy and Oncology xxx (2013) xxx–xxx
(A)
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1 hour 3 hours HIF-1α 120 KDa 6 hours 24 hours Representative
(C)
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F-n
F-h
P50
T50
(D)
PT50 42 KDa 46 KDa
Col-Iα1 mRNA expression at 48 hours post-transfection with miR-210 inhibitor 1.2
MiR-210/Col-Iα1 relative expression
VEGF GAPDH
GAPDH 38 KDa
1
0.8
*
miR-210
0.6
Col-Iα1 *
0.4
0.2
0 Negative control
miR-210 inhibitor
Fig. 2. (A) Modulation of miR-210 expression by pentoxifylline, trolox, and pentoxifylline–trolox treatments when primary intestinal smooth muscle cells are cultured under hypoxia (1% O2) compared to normoxia (21% O2). Trolox and pentoxifylline–trolox combination significantly reduce miR-210 expression both under hypoxic and normoxic conditions. (⁄p 6 0.05 versus fibrotic cells in normoxic conditions, #p 6 0.05 versus fibrotic cells in hypoxic conditions). MiR-210 levels were detected by qRT-PCR using RNU6B as an endogenous control. (B) Detection of HIF-1a by Western blotting in normal cells and fibrotic cells under hypoxic conditions (1% O2) at different time points. The abundance of the stabilized form of HIF-1a is not modulated by any treatment at any time point from 1 to 24 h post-treatment. (C) HIF-1a-independence is confirmed by monitoring of VEGF, a well-known target of HIF-1a, by Western blot in normal cells, fibrotic cells, and treated fibrotic cells in hypoxic conditions (1% O2) at 6 h posttreatment. None of the treatments modulate the expression of VEGF indicating absence of modulation of HIF-1 activity. (D) Transfection of primary smooth muscle cells with miR-210 mirVana™ miRNA inhibitor (Ambion) versus mirVana™ miRNA inhibitor Negative Control (Ambion) at 5nM was performed using A-033 ready-to-use AmaxaÒ optimized protocol on the Nucleofector™ II Device (Amaxa). Results show that the miR-210 inhibitor reduced Col-Ia1 mRNA expression 48 h post-transfection (⁄p 6 0.05 versus negative control). The decrease of miR-210 expression is an internal control. (⁄p 6 0.05 versus negative control). Results are always expressed as mean ± SEM. Significance was established with GraphPad Prism (GraphPad Software) using the non-parametric Kruskal–Wallis or Student’s t-test, each when appropriate, for the analysis of at least three independent in vitro experiments. Differences were considered significant at a value of p 6 0.05. (P: pentoxifylline; T: trolox; PT: pentoxifylline–trolox treatment 10 and 50 lg/mL; N-n: normal cells-normoxia; N-h: normal cells-hypoxia; F-n: fibrotic cells-normoxia; F-h: fibrotic cells-hypoxia).
Acknowledgements MicroRNA array analysis was a project selected by the scientific committee of the apprenticeship tax of Institut Gustave Roussy (IGR). It was then realized with appreciated scientific and technical support of the genomics and bioinformatics platform of the Institut Gustave Roussy. We would like to thank Pr M. McKay for his help in the preparation of the paper. References [1] Gervaz P, Morel P, Vozenin-Brotons MC. Molecular aspects of intestinal radiation-induced fibrosis. Curr Mol Med 2009;9:273–80. [2] Liu G, Friggeri A, Yang Y, Milosevic J, Ding Q, Thannickal VJ, et al. MiR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp Med 2010;207:1589–97. [3] Kato M, Putta S, Wang M, Yuan H, Lanting L, Nair I, et al. TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN. Nat Cell Biol 2009;11:881–9. [4] Davis BN, Hilyard AC, Lagna G, Hata A. SMAD proteins control DROSHAmediated microRNA maturation. Nature 2008;454:56–61.
[5] Bourgier C, Haydont V, Milliat F, Francois A, Holler V, Lasser P, et al. Inhibition of Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration of the cytoskeleton and connective tissue growth factor expression. Gut 2005;54:336–43. [6] Hamama S, Gilbert-Sirieix M, Vozenin MC, Delanian S. Radiation-induced enteropathy: molecular basis of pentoxifylline-vitamin E anti-fibrotic effect involved TGF-beta1 cascade inhibition. Radiother Oncol 2012;105:305–12. [7] Delanian S, Chatel C, Porcher R, Depondt J, Lefaix JL. Complete restoration of refractory mandibular osteoradionecrosis by prolonged treatment with a pentoxifylline-tocopherol-clodronate combination (PENTOCLO): a phase II trial. Int J Radiat Oncol Biol Phys 2011;80:832–9. [8] Kulshreshtha R, Ferracin M, Negrini M, Calin GA, Davuluri RV, Ivan M. Regulation of microRNA expression: the hypoxic component. Cell Cycle 2007;6:1426–31. [9] Chan YC, Banerjee J, Choi SY, Sen CK. MiR-210: the master hypoxamir. Microcirculation 2012;19:215–23. [10] Grosso S, Doyen J, Parks SK, Bertero T, Paye A, Cardinaud B, et al. MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines. Cell Death Dis 2013;4:e544. [11] Strup-Perrot C, Mathe D, Linard C, Violot D, Milliat F, Francois A, et al. Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis. Am J Physiol Gastrointest Liver Physiol 2004;287:G875–85.
Please cite this article in press as: Hamama S et al. MiR-210: A potential therapeutic target against radiation-induced enteropathy. Radiother Oncol (2013), http://dx.doi.org/10.1016/j.radonc.2013.10.030
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
MiR-210: A potential therapeutic target against radiation-induced enteropathy Saad Hamama a,b,c, Muhammad Zaeem Noman d, Pascal Gervaz e, Sylvie Delanian f, Marie-Catherine Vozenin a,b,c,g,⇑ a
INSERM U-1030 ‘‘Molecular Radiotherapy’’, Institut Gustave Roussy, Villejuif, France; b LabEX LERMIT Villejuif, France; c Université Paris Sud Paris XI, Le Kremlin Bicêtre, France; INSERM, U753, Laboratoire d’immunologie des Tumeurs Humaines: Interaction Effecteurs Cytotoxiques-Système Tumoral, Institut Gustave-Roussy, Villejuif, France; e Geneva University Hospital and Medical School, Geneva, Switzerland; f Unité de Radiopathologie, Service Oncologie-Radiothérapie, Hôpital Saint-Louis, APHP, Paris, France; g Laboratoire de RadioOncologie, Swiss Cancer Center of Lausanne, CHUV, Lausanne, Switzerland
d
a r t i c l e
i n f o
Article history: Received 13 July 2013 Received in revised form 1 October 2013 Accepted 16 October 2013 Available online xxxx
a b s t r a c t A previously undescribed and robust miR210 overexpression is shown in intestinal samples obtained from patients with radiation enteropathy and fibrotic cultured cells. In addition, miR-210 overexpression is repressed by antifibrotic treatment combining pentoxifylline and a-tocopherol. Ó 2013 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2013) xxx–xxx
Keywords: Anti-fibrotic treatment Radiation-induced fibrosis Radiation enteropathy microRNA miR-210 Trolox Pentoxifylline
Pelvic radiotherapy for treatment of urogenital or anorectal malignancies may induce radiation-induced enteropathy characterized by transmural fibrosis that may affect small intestine, colon, rectum and the anal sphincter wall. Fibrosis impairs contractility, leading to dysfunction including diarrhea and constipation, fecal incontinence and ultimately occlusion [1]. Today, therapeutic options against radiation-induced fibrosis such as radiation-induced enteropathy remain very limited, therefore identifying new therapeutic targets is an important clinical issue. Among the possible therapeutic candidates, microRNAs were recently studied. Liu et al. demonstrated the pathogenic inter-relation between miR-21 and the fibrogenic growth factor TGF-b1 [2]. TGF-b1 is also known to modulate the transcription of microRNAs [3] and their maturation via activation of Smad proteins [4]. Due to their critical biological roles in fine tuning cellular and tissue homeostasis, we investigated the possible involvement of microRNAs in radiation enteropathy by molecular profiling and
⇑ Corresponding author. Address: Laboratoire de RadioOncologie, Swiss Cancer Center of Lausanne, CHUV, 1010 Lausanne, Switzerland. E-mail addresses: [email protected] (S. Hamama), muhammed-zaeem. [email protected] (M.Z. Noman), [email protected] (P. Gervaz), sylvie.delanian @sls.aphp.fr (S. Delanian), [email protected] (M.-C. Vozenin).
showed robust induction of miR-210 in a validated cellular model of radiation-induced fibrosis [5,6] comprised of primary smooth muscle cells derived from human radiation enteropathy compared with their healthy counterparts (Fig 1A). Secondly, we confirmed miR-210 induction in rectal samples surgically removed 12 weeks after radiotherapy in a cohort of 16 patients irradiated for rectal cancer with a total dose of 50.4 Gray versus 4 non-irradiated patients’ samples (Fig 1B). Human samples were histologically and molecularly characterized as fibrotic with consistent induction of fibrogenic growth factors such as TGF-b1 and CTGF (Fig 1C). Thirdly, we explored possible modulation of miR-210 after anti-fibrotic therapy using the reference treatment combining pentoxifylline–trolox [7] and found that miR-210 was repressed by 40% when cells were incubated with trolox (p < 0.05) (Fig 1D). Interestingly, addition of pentoxyfilline–trolox did not enhance the inhibitory effect on miR-210, suggesting that miR-210 inhibition was trolox-mediated and suggesting the involvement of antioxidant pathways in miR-210 modulation. Since miR-210 is a well known hypoxia microRNA [8], we investigated the role of hypoxia and more specifically, HIF-1a in miR-210 up-regulation. Consistently, when primary smooth muscle cells were cultured under hypoxic conditions (1% O2) significant induction of miR-210 was observed both in normal and fibrotic cells with higher level in fibrotic cells
0167-8140/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radonc.2013.10.030
Please cite this article in press as: Hamama S et al. MiR-210: A potential therapeutic target against radiation-induced enteropathy. Radiother Oncol (2013), http://dx.doi.org/10.1016/j.radonc.2013.10.030
2
Mir-210 in radiation-induced fibrosis
(A)
(B)
MiR-210 expression in normal and fibrotic cells
MiR-210 relative expression
3
*
2.5
*
2 1.5
Hybridization qRT-PCR
1 0.5 0
Normal
Fibrosis
(C)
(D)
Fig. 1. (A) MiR-210 overexpression in fibrotic versus normal cells in normoxic conditions (21% O2). (⁄p 6 0.05). MiR-210 levels were detected using 8 15 K Agilent Human v3 (G4471A) microRNA array and results were confirmed by qRT-PCR using RNU6B as an endogenous control. (B) Levels of miR-210 are significantly increased in anal sphincter surgical specimens isolated from patients (n = 16) suffering from radiation enteropathy versus normal biopsies (n = 4) (⁄p 6 0.05). MiR-210 levels were detected by qRT-PCR using RNU6B as an endogenous control. (C) Levels of TGF-b1 and CTGF mRNA were much higher in anal sphincter surgical specimen isolated from patients suffering from radiation enteropathy versus normal biopsies: (⁄⁄p 6 0.01). mRNA levels were detected by qRT-PCR using 18S as an endogenous control. (D) 24 h treatment with trolox (50 lg/mL) or pentoxifylline–trolox (50 lg/mL of each molecule) reverses miR-210 overexpression detected by qRT-PCR and normalized to RNU6B. Results are always expressed as mean ± SEM. Significance was established with GraphPad Prism (GraphPad Software) using the non-parametric Kruskal–Wallis or Student’s t-test, each when appropriate, for the analysis of at least three independent in vitro experiments. Differences were considered significant at a value of p 6 0.05. (P: pentoxifylline, T: trolox; PT: pentoxifylline–trolox treatment 10 and 50 lg/mL, continuous and dashed lines: miR-210 levels in normal and fibrotic cells, respectively).
(Fig 2A). Trolox and pentoxifylline–trolox combination again reduced miR-210 expression in fibrotic cells (Fig 2A) when cultured in hypoxic conditions. Studying HIF-1a expression, we surprisingly found that it was not modified by any treatment (Fig 2B), neither was its target VEGF (Fig 2C) suggesting that the down-regulation of miR-210 expression induced by trolox and pentoxifylline-trolox combination (PT) was HIF-1a-independent. Lastly, we investigated whether direct targeted inhibition of miR-210 displayed anti-fibrotic action per se. Cells were then transfected using an antagomiR210 and the mRNA expression of TGF-b1, CTGF, PAI-1, FN-1, and Col-Ia1 was investigated. We found a reduction of Col-Ia1 mRNA expression (p < 0.05) 48 h after transfection (Fig 2D) suggesting a role for miR-210 in the regulation of extracellular matrix homeostasis in fibrotic tissues. However, the mRNA expression of TGFb1, CTGF, PAI-1, and FN-1 was not modified at the studied time points, suggesting a specific action of miR-210 inhibition of matrix genes. MiR-210 has been shown to be implicated in various pathophysiological functions such as apoptosis angiogenesis, cell cycle regulation, DNA damage repair, mitochondrial metabolism, stem cell survival, [9], and increased radioresistance [10] but to our knowledge this is the first publication to report its association with fibrosis. Interestingly, miR-210 action in the regulation of hypoxiainduced pathogenesis is also well documented and mediated by HIF-1a through the hypoxia responsive element (HRE) located on the proximal promoter of miR-210 [9]. However, our results suggest that an additional yet never reported regulation of miR-210 occurs in radiation-induced fibrosis since we found no modulation of the HIF1-a cascade in our system. Still, Vitamin
E-based anti-fibrotic treatments (i.e., trolox alone and pentoxifylline–trolox combination) reduced miR-210 expression in hypoxic conditions. More interestingly the antagomiR-210 decreased Col Ia1 mRNA expression in fibrotic cells. The intrinsic function of microRNAs implies that their inhibition induces increased expression of their targets. Here the downregulation of Col Ia1 could suggest a novel indirect up-regulation of a co-repressor of Col Ia1 exists and remains to be identified. To explore this putative indirect mechanism, in silico analysis of miR-210 targets using various software including TargetScan, MiRanda, PicTar, and MicroCosm were performed but revealed no obvious candidates and more experiments are needed to identify this mediator. In conclusion, our study is the first to show the overexpression of miR-210 in radiation-induced intestinal fibrosis. Furthermore, we showed that miR-210 overexpression was repressed by anti-fibrotic treatment and direct miR-210 inhibition exerted an anti-fibrotic action through the reduction of Col Ia1 mRNA expression in fibrotic cells. Further studies are needed to clearly define the molecular mechanisms involved but the specific inhibition of fibrillar collagen triggered by the antagomiR-210 will probably be highly clinically valuable as abnormal and excessive collagen deposition [11] is responsible for the loss of intestinal compliance leading to stenosis and poor function in radiation enteropathy.
Conflict of interest None.
Please cite this article in press as: Hamama S et al. MiR-210: A potential therapeutic target against radiation-induced enteropathy. Radiother Oncol (2013), http://dx.doi.org/10.1016/j.radonc.2013.10.030
3
S. Hamama et al. / Radiotherapy and Oncology xxx (2013) xxx–xxx
(A)
(B)
N-n
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PT50
1 hour 3 hours HIF-1α 120 KDa 6 hours 24 hours Representative
(C)
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F-h
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(D)
PT50 42 KDa 46 KDa
Col-Iα1 mRNA expression at 48 hours post-transfection with miR-210 inhibitor 1.2
MiR-210/Col-Iα1 relative expression
VEGF GAPDH
GAPDH 38 KDa
1
0.8
*
miR-210
0.6
Col-Iα1 *
0.4
0.2
0 Negative control
miR-210 inhibitor
Fig. 2. (A) Modulation of miR-210 expression by pentoxifylline, trolox, and pentoxifylline–trolox treatments when primary intestinal smooth muscle cells are cultured under hypoxia (1% O2) compared to normoxia (21% O2). Trolox and pentoxifylline–trolox combination significantly reduce miR-210 expression both under hypoxic and normoxic conditions. (⁄p 6 0.05 versus fibrotic cells in normoxic conditions, #p 6 0.05 versus fibrotic cells in hypoxic conditions). MiR-210 levels were detected by qRT-PCR using RNU6B as an endogenous control. (B) Detection of HIF-1a by Western blotting in normal cells and fibrotic cells under hypoxic conditions (1% O2) at different time points. The abundance of the stabilized form of HIF-1a is not modulated by any treatment at any time point from 1 to 24 h post-treatment. (C) HIF-1a-independence is confirmed by monitoring of VEGF, a well-known target of HIF-1a, by Western blot in normal cells, fibrotic cells, and treated fibrotic cells in hypoxic conditions (1% O2) at 6 h posttreatment. None of the treatments modulate the expression of VEGF indicating absence of modulation of HIF-1 activity. (D) Transfection of primary smooth muscle cells with miR-210 mirVana™ miRNA inhibitor (Ambion) versus mirVana™ miRNA inhibitor Negative Control (Ambion) at 5nM was performed using A-033 ready-to-use AmaxaÒ optimized protocol on the Nucleofector™ II Device (Amaxa). Results show that the miR-210 inhibitor reduced Col-Ia1 mRNA expression 48 h post-transfection (⁄p 6 0.05 versus negative control). The decrease of miR-210 expression is an internal control. (⁄p 6 0.05 versus negative control). Results are always expressed as mean ± SEM. Significance was established with GraphPad Prism (GraphPad Software) using the non-parametric Kruskal–Wallis or Student’s t-test, each when appropriate, for the analysis of at least three independent in vitro experiments. Differences were considered significant at a value of p 6 0.05. (P: pentoxifylline; T: trolox; PT: pentoxifylline–trolox treatment 10 and 50 lg/mL; N-n: normal cells-normoxia; N-h: normal cells-hypoxia; F-n: fibrotic cells-normoxia; F-h: fibrotic cells-hypoxia).
Acknowledgements MicroRNA array analysis was a project selected by the scientific committee of the apprenticeship tax of Institut Gustave Roussy (IGR). It was then realized with appreciated scientific and technical support of the genomics and bioinformatics platform of the Institut Gustave Roussy. We would like to thank Pr M. McKay for his help in the preparation of the paper. References [1] Gervaz P, Morel P, Vozenin-Brotons MC. Molecular aspects of intestinal radiation-induced fibrosis. Curr Mol Med 2009;9:273–80. [2] Liu G, Friggeri A, Yang Y, Milosevic J, Ding Q, Thannickal VJ, et al. MiR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp Med 2010;207:1589–97. [3] Kato M, Putta S, Wang M, Yuan H, Lanting L, Nair I, et al. TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN. Nat Cell Biol 2009;11:881–9. [4] Davis BN, Hilyard AC, Lagna G, Hata A. SMAD proteins control DROSHAmediated microRNA maturation. Nature 2008;454:56–61.
[5] Bourgier C, Haydont V, Milliat F, Francois A, Holler V, Lasser P, et al. Inhibition of Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration of the cytoskeleton and connective tissue growth factor expression. Gut 2005;54:336–43. [6] Hamama S, Gilbert-Sirieix M, Vozenin MC, Delanian S. Radiation-induced enteropathy: molecular basis of pentoxifylline-vitamin E anti-fibrotic effect involved TGF-beta1 cascade inhibition. Radiother Oncol 2012;105:305–12. [7] Delanian S, Chatel C, Porcher R, Depondt J, Lefaix JL. Complete restoration of refractory mandibular osteoradionecrosis by prolonged treatment with a pentoxifylline-tocopherol-clodronate combination (PENTOCLO): a phase II trial. Int J Radiat Oncol Biol Phys 2011;80:832–9. [8] Kulshreshtha R, Ferracin M, Negrini M, Calin GA, Davuluri RV, Ivan M. Regulation of microRNA expression: the hypoxic component. Cell Cycle 2007;6:1426–31. [9] Chan YC, Banerjee J, Choi SY, Sen CK. MiR-210: the master hypoxamir. Microcirculation 2012;19:215–23. [10] Grosso S, Doyen J, Parks SK, Bertero T, Paye A, Cardinaud B, et al. MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines. Cell Death Dis 2013;4:e544. [11] Strup-Perrot C, Mathe D, Linard C, Violot D, Milliat F, Francois A, et al. Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis. Am J Physiol Gastrointest Liver Physiol 2004;287:G875–85.
Please cite this article in press as: Hamama S et al. MiR-210: A potential therapeutic target against radiation-induced enteropathy. Radiother Oncol (2013), http://dx.doi.org/10.1016/j.radonc.2013.10.030
