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Aberrant high expression of NRG1 gene in Hirschsprung disease
Journal of Pediatric Surgery (2012) 47, 1694–1698
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Aberrant high expression of NRG1 gene in Hirschsprung disease☆ Weibing Tang a,c,1 , Bo Li a,c,1 , Xiaoqun Xu a,c , Zhigang Zhou a,c , Wei Wu a,b , Junwei Tang a,c , Jingjing Qin a,c , Qiming Geng c , Weiwei Jiang c , Jie Zhang c , Jiahao Sha a , Yankai Xia a,b,⁎, Xinru Wang a,b a
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing 210029, China b Key Laboratory of Modern Toxicology (Nanjing Medical University), Ministry of Education, Nanjing 210029, China c Department of Pediatric Surgery, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China Received 21 December 2011; revised 21 February 2012; accepted 20 March 2012
Key words: Hirschsprung disease; NRG1; DNA methylation
Abstract Background/Purpose: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells along with variable lengths of the gastrointestinal tract. Recent studies have indicated the potential function of neuregulin-1 (NRG1) in HSCR, which encodes the heregulins and other mitogenic ligands for the ErbB family. The purpose of this study was to further clarify the role of NRG1 in the pathogenesis of HSCR. Methods: We examined the NRG1 messenger RNA (messenger RNA) and protein expression levels in gut tissues of 63 patients with sporadic HSCR (both stenotic and dilated gut tissues) and 35 controls. Moreover, using the methylation-specific polymerase chain reaction, we examined the methylation pattern of exon 1 of the NRG1 gene. Results: The mRNA expression levels of NRG1 were significantly higher in tissues of HSCR than those in controls, and the increased NRG1 protein levels in HSCR were consistent with the mRNA levels. However, no methylation pattern change was observed in exon 1 of the gene among different groups. Conclusions: Our study demonstrates that the aberrant expression of NRG1 may play an important role in the pathology of HSCR. DNA methylation of the gene seems not to be involved in the mechanism of such aberrant expression, and other factors should be explored. © 2012 Elsevier Inc. All rights reserved.
☆
Conflict of interest: None declared. ⁎ Corresponding author. State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China. Tel.: +86 25 86862939; fax: +86 25 86662863. E-mail address: [email protected] (Y. Xia). 1 These authors contributed equally. 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2012.03.061
Hirschsprung disease (HSCR), also called aganglionosis, is one of the most difficult diagnoses in pediatric surgery [1], characterized by the absence of ganglion cells in the lower digestive tract. It is the most frequent cause of a functional intestinal obstruction among newborns. The incidence of this disease is about 1:2000 to 1:5000. Hirschsprung disease usually occurs sporadically (up to70% of the cases), although
Aberrant high expression of NRG1 gene in HSCR it can be familial, and is frequently associated with recognized syndromes, chromosome, or congenital anomalies [2,3]. Although many studies have been performed to elucidate the pathological process of this disease, the exact mechanism still remains unknown. To date, more than 10 genes have been found to be involved in the pathogenesis of HSCR [2]. Among these genes, the RET proto-oncogene accounts for the highest proportion of both familial and sporadic cases [4]. It is now known that neural crest neuroblasts can colonize aganglionic and ganglionic gut in vivo, which brought a new prospect for the treatment of HSCR [5]. A previous genome-wide association study on Chinese patients with sporadic HSCR identified neuregulin-1 (NRG1) as a susceptibility locus for HSCR [6]. This study also found that there was a significant difference in NRG1 expression level between the diseased and control individuals bearing a same-risk genotype [7]. In addition, not only common variants but also rare ones of the NRG1 gene can contribute to HSCR [8]. Epigenetic alterations in DNA without concomitant changes in underlying genetic codes are now known to occur frequently in several kinds of human diseases [9-11]. Epigenetic silencing is achieved not only through DNA methylation but also by many other DNA, chromatin, and RNA modification mechanisms [12,13]. Changes in DNA methylation patterns impact several critical cellular processes including gene expression, Xinactivation, carcinogenesis, aging, and development [14-17]. Recently, it was shown that NRG1 was unmethylated in normal breast tissues and methylated in breast tumor samples; hypermethylation of the gene is correlated with lower a NRG1 gene expression level [18]. In the present study, we set out to investigate the role of NRG1 in HSCR, initially by measuring expression of NRG1 (both messenger RNA [mRNA] and protein) in human gut tissues (63 patients and 35 controls). We found that there were significant higher NRG1 expression levels in patients with HSCR than those in controls. To find out whether aberrant expression of NRG1 in HSCR is caused by abnormal DNA methylation and if there were any changes in the methylation pattern among different groups, we further examined the methylation pattern of the exon 1 of the NRG1 gene. In this work, we report that there were no significant changes in the methylation pattern of NRG1 among different groups tested. Future studies with a larger sample size are needed to confirm these results. The functional relevance of aberrant expression of NRG1 to HSCR found in this work warrants further investigation.
1. Materials and methods 1.1. Patients and samples This study was approved by the institutional review board of Nanjing Medical University (Nanjing, China), and
1695 all human subjects provided written informed consent. A total of 63 patients with HSCR were collected in Nanjing Children's Hospital affiliated to Nanjing Medical University from October 2009 to May 2011 (Nanjing Medical University Birth Cohort). All patients were confirmed with barium enema, anorectal manometry, and postoperative pathological examination. We took full-thickness tissues in the dilated segment (ganglionic bowel) and stenotic segment (aganglionic bowel) of the colon, and the tissues were immediately stored in liquid nitrogen. Tissue specimens in the normal colon of 35 patients without HSCR (intussusception, incarcerated hernia) were obtained as negative controls over the same period as that for the diseased samples.
1.2. Quantitative real-time polymerase chain reaction Total RNA was obtained from tissues using TRIzol reagent as described by the manufacturer (Invitrogen Life Technologies Co, CA, USA). For NRG1 mRNA detection, RNA (500 ng) was reverse transcribed using a reverse transcription kit (Takala, Tokyo, Japan) under 37°C for 15 minutes and 85°C for 30 seconds. The expression level of the NRG1 gene in each sample was measured by quantitative real-time polymerase chain reaction (PCR) in a volume of 10 μL with 384-well plates using ABI Prism 7900HT (Applied Biosystems, Foster City, CA). β-Actin was used as an endogenous control. Forward (F) and reverse (R) primer sequences were as follows: NRG1, (F) 5′-atgtgtcttcagagtctcccat-3′ and (R) 5′-tggacgtactgtagaagctgg-3′; β-actin, (F) 5′ccaaccgcgagaagatga-3′ and (R) 5′-ccagaggcgtacagggatag-3′. The thermocycler program included a step of denaturation at 95°C (10 minutes) and 40 cycles of 95°C (15 seconds) and 60°C (1 minute).
1.3. Western blotting Proteins were extracted from gut tissues using radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors (cOmplete, ULTRA, 132 Mini, EDTA-free, EASYpack; Roche, Basel, Switzerland). Protein concentrations were determined using the BCA (Bicinchoninic Acid) method. Equal amount of proteins (80-100 μg) were separated by 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Roche). Membranes were blocked using 5% skimmed milk and incubated with respective antibodies. Primary rabbit polyclonal rabbit antiNRG1 antibody was purchased from Santa Cruz Biotechnology (SC-348; Santa Cruz, CA). The secondary antibody used was antirabbit from Santa Cruz Biotechnology. Blots were developed using electrochemiluminescence (ECL) (Millipore, Billerica, MA). Equal loading of protein amounts was confirmed using a GAPDH antibody. Integrated density values were calculated using AlphaImager 3400 (Alpha
1696 InnoTech, San Leandro, CA). These values were then normalized to the GAPDH internal control. All experiments were repeated at least 3 times; representative results are presented in this study.
1.4. DNA isolation For DNA preparation, we collected a small piece from the full-thickness tissue in a 2-mL Eppendorf tube, incubated it with 800 μL lysate (20 mmol/L Tris-HCl [pH 8.0], 5 mmol/L EDTA [pH 8.0], 1% sodium dodecyl sulphate, 400 mmol/L NaCl) and an additional 10 μL of 10 mg/mL proteinase K at 56°C for 24 hours. After digestion, DNA was extracted with phenol-chloroform (1:1) and precipitated in ethanol and was then dissolved in 50 μL TE (Tris-EDTA) buffer (1 mmol/L Tris-HCl, 0.5 mmol/L EDTA, pH 8.0). DNA concentration was determined by spectrophotometry.
1.5. Bisulfite modification and methylation-specific PCR Genomic DNA (500 ng) was treated with sodium bisulfite using the EpiTect Bisulfite Kit (QIAGEN, Valencia, CA), according to the protocol recommended by the manufacturer. Two microliters of the final eluent was used for subsequent PCR amplification. We analyzed the methylation pattern of NRG1 exon 1, which has a region located between −136 to +79 (containing +1, the translational initiation site) rich in CpG sites. The primers used were as follows: methylated forward: 5′-GTTTTAGCGCGGTCGTTC-3′, methylated reverse: 5′-CGAACTCCGACTTCTTACCG-3′; unmethylated forward: 5′-GTAGTGTGAGTGTTTTAGTGTGGTTG-3′, unmethylated reverse: 5′-CAAACTCCAACTTCTTACCA3′ [19]. For both PCR reactions, a total of 100 ng of modified DNA was amplified in a 25-μL reaction containing 0.5 μM each of forward and reverse primer, 200 μM dNTPs, 1 × PCR buffer, and 1.25 U of Taq Hot Start DNA Polymerase (Takara Bio, Tokyo, Japan) under the following conditions: 5 minutes of denaturation at 94°C, followed by 40 cycles of 40 seconds at 94°C, 40 seconds at 61.3°C for methylated primers (58.8°C for unmethylated primers), 1 minute at 72°, and a final extension for 10 minutes at 72°C. The amplification products were separated on 2% agarose gels and stained with ethidium bromide. Most samples were subjected to 2 independent bisulfite treatments and analyzed from 2 independent PCR products. One control DNA sample methylated using SssI methyltransferase (New England Biolabs, MA, USA), according to the manufacturer's protocol, was used as a methylated positive control for those methylation-specific PCR (MSP) reactions.
W. Tang et al. using Stata 9.2 (StataCorp LP, College Station, Texas) and presented with GraphPad prism software (GraphPad Software, Inc, California, USA). For statistical analysis, 2sample Wilcoxon rank sum (Mann-Whitney) test was used to evaluate the differences between groups. Results were considered statistically significant when P b .05.
2. Results 2.1. Results of human sample data analysis The clinical data of 63 patients with HSCR and 35 normal controls were collected, including age, sex (male/female), and body weight. The ages of patients with HSCR and the controls were 3.4 ± 0.18 and 2.8 ± 0.24 months, the sex ratio was 51:12 and 26:9, and the body weight was 5.8 ± 0.28 and 5.1 ± 0.34 kg, respectively There were no significant differences between patients with HSCR and the control subjects regarding age, sex, and body weight.
2.2. Up-regulated mRNA and protein expression levels of NRG1 in HSCR To detect NRG1 expression levels in HSCR and control samples, we used TaqMan quantitative real-time PCR methods. The relative expression levels of NRG1 mRNA were normalized to β-actin expression levels. The medians of each group were 8.97 × 10−4 (HSCR-S), 3.71 × 10−4 (HSCR-D), and 4.89 × 10−5 (control). These results indicate that NRG1 mRNA levels in HSCR stenotic segment samples (P = 4.5 × 10−5) and dilated segment samples (P = 7.5 × 10−5) were both significantly higher than those in the controls. There was no significant difference between
1.6. Statistics and analysis For analysis, the results of reverse transcriptase PCR, we used the method of 2−ΔCt. Statistical analysis was performed
Fig. 1 Relative expression of NRG1 mRNA in HSCR and control samples. Data were presented as median values for NRG1 relative expression of tissues of HSCR and controls (*P b .01).
Aberrant high expression of NRG1 gene in HSCR
Fig. 2 The protein expression of NRG1 between patients with HSCR and the controls. NRG1 protein expression was determined by Western blot, and GAPDH was used as a loading control. HSCR, the stenotic segment of HSCR.
stenotic segment and dilated segment samples (P = .22) (Fig. 1). Such finding indicates that up-regulated NRG1 expression level may be associated with the development of HSCR. Moreover, Western blot analysis showed increased NRG1 protein levels in HSCR pathological segment than in the controls, which was consistent with the results obtained at the mRNA levels (Fig. 2).
2.3. Methylation pattern of the samples To investigate the possible cause of abnormal expression of NRG1, we analyzed the methylation pattern of the NRG1 exon 1 using MSP. The region targeted is located between −136 to +79 (containing +1, the translational initiation site) that is rich in CpG sites. Nevertheless, all patients with HSCR and the controls examined in this study exhibited a similar pattern, that is, the unmethylated allele (Fig. 3).
3. Discussion Hirschsprung disease is considered a disorder of the enteric nervous system (ENS), whereby the enteric ganglion precursors fail to migrate along the developing gut. Therefore, the pathogenesis of HSCR can only be conceptually understood in light of the molecular and cellular events occurring during the ENS development [20]. To date, there is strong evidence indicating that NRG1
1697 signaling is associated with the development and maintenance of the ENS [21-23]. It was previously reported that the overall NRG1 expression in the gut did not differ between patients with HSCR and the controls; they, however, only took full-thickness tissues from ganglionic portions of the bowel of those patients [7]. In the present study, we examined both ganglionic and aganglionic portions of a larger amount of samples. The results indicate that NRG1 mRNA levels in both HSCR stenotic segment and dilated segment samples were significantly higher than those in the controls. In addition, Western blotting analysis showed that increased NRG1 protein levels in patients with HSCR than in the controls, consistent with the changes observed at mRNA levels. Current studies are focused on discovering the role of NRG1 in HSCR from the point of genetic aspects [6-8], with few concerning about epigenetic factors that might be related to the regulation of NRG1 expression and the mechanism underlying this disease. DNA methylation is one of the most studied regulatory mechanisms in epigenetics. It is well established that hypomethylation is associated with transcriptional activation, whereas hypermethylation is associated with repression of transcription [14]. In this study, we hypothesized that the regulation of NRG1 expression in HSCR might be caused by aberrant DNA methylation of the gene. Although the alignment of NRG1 UniGene clusters to the assembled genomic sequence revealed 21 exons [24], the exon 1 region is the closest to the promoter and has the greatest impact on gene function. Moreover, the region located between −136 to +79 (containing +1, the translational initiation site) is rich in CpG sites. However, our results demonstrated complete unmethylation of the gene (exon 1) in all patients with HSCR and controls. Then, as it is known that microRNAs (miRNAs) might play a role in regulating gene expression by transcriptionally repressing mRNAs [25,26], we also used a bioinformatics method to predict miRNAs, which may regulate the expression of NRG1 gene. We applied miRanda (http://www.cbio.mskcc.org/ mirnaviewer), PicTar (http://pictar.mdc-berlin.de/), and TargetScan (http://www.targetscan.org) databases to predict related miRNAs of the NRG1 gene. The results of these 3 databases were inconsistent: miR-18a, miR-139,miR-130, miR-103, miR-107, miR-142, and miR-195 (miRanda); miR-18a, miR-18b, miR-34a, miR-34b, miR-139, and miR-
Fig. 3 Representative results of MSP analyses of DNA samples. M, methylated primers; U, ummethylated primers; P, positive control; S, stenotic segment samples; D, dilated segment samples; C, control samples.
1698 342 (PicTar); and miR-499, miR-876-5p, miR-494, and miR-376 (TargetScan). The results of the prediction were not coincident with each other in the bioinformatics software used above; therefore, further study of the correlated miRNAs of NRG1 is needed. Because the sample size of our study was still relatively small and we only chose one specific region of the gene to examine the methylation pattern in HSCR, it may not sufficient to conclude that the regulation of NRG1 expression is not caused by the methylation of the gene. Further studies with larger sample sizes and different ethnical populations, together with the analysis of the CpG sites at other NRG1 portions, are needed to gain more understanding of the role and mechanism of the NRG1 gene in the pathogenesis of HSCR.
Acknowledgments We thank Dr Bo Hang (Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA) for language editing and Dr Huan Chen and Changgui Lu (Nanjing Children's Hospital Affiliated to Nanjing Medical University) for sample collection. This study was supported by the grants SKLRM-KF-1104, Nanjing Medical Science and Technique Development Foundation (201108010), and Priority Academic Program Development of Jiangsu Higher Education Institutions.
References [1] Martucciello G. Hirschsprung's disease, one of the most difficult diagnoses in pediatric surgery: a review of the problems from clinical practice to the bench. Eur J Pediatr Surg 2008;18:140-9. [2] Amiel J, Sproat-Emison E, Garcia-Barcelo M, et al. Hirschsprung disease, associated syndromes and genetics: a review. J Med Genet 2008;45:1-14. [3] Moore SW. The contribution of associated congenital anomalies in understanding hirschsprung's disease. Pediatr Surg Int 2006;22: 305-15. [4] Martucciello G, Ceccherini I, Lerone M, et al. Pathogenesis of hirschsprung's disease. J Pediatr Surg 2000;35:1017-25. [5] Martucciello G, Brizzolara A, Favre A, et al. Neural crest neuroblasts can colonise aganglionic and ganglionic gut in vivo. Eur J Pediatr Surg 2007;17:34-40.
W. Tang et al. [6] Garcia-Barcelo MM, Tang CSM, Ngan ESW, et al. Genome-wide association study identifies nrg1 as a susceptibility locus for hirschsprung's disease. Proc Natl Acad Sci U S A 2009;106:2694-9. [7] Tang CS-M, Tang W-K, So M-T, et al. Fine mapping of the nrg1 Hirschsprung's disease locus. Plos One 2011:6. [8] Tang CS, Ngan ES, Tang WK, et al. Mutations in the nrg1 gene are associated with hirschsprung disease. Hum Genet 2012;131(1):67-76. [9] Handel AE, Ebers GC, Ramagopalan SV. Epigenetics: molecular mechanisms and implications for disease. Trends Mol Med 2010;16: 7-16. [10] Dallosso AR, Hancock AL, Szemes M, et al. Frequent long-range epigenetic silencing of protocadherin gene clusters on chromosome 5q31 in wilms' tumor. PLoS Genet 2009;5:e1000745. [11] Schuebel KE, Chen W, Cope L, et al. Comparing the dna hypermethylome with gene mutations in human colorectal cancer. PLoS Genet 2007;3:1709-23. [12] Shilatifard A. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annu Rev Biochem 2006;75:243-69. [13] Chuang JC, Jones PA. Epigenetics and microRNAs. Pediatr Res 2007;61:24R-9R. [14] Biermann K, Steger K. Epigenetics in male germ cells. J Androl 2007;28:466-80. [15] Heard E, Clerc P, Avner P. X-chromosome inactivation in mammals. Annu Rev Genet 1997;31:571-610. [16] Gopalakrishnan S, Van Emburgh BO, Robertson KD. Dna methylation in development and human disease. Mutat Res 2008;647:30-8. [17] Calvanese V, Lara E, Kahn A, et al. The role of epigenetics in aging and age-related diseases. Ageing Res Rev 2009;8:268-76. [18] Chua YL, Ito Y, Pole JCM, et al. The nrg1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene. Oncogene 2009;28:4041-52. [19] Fernandez SV, Snider KE, Wu YZ, et al. Dna methylation changes in a human cell model of breast cancer progression. Mutat Res-Fundam Mol Mech Mutagen 2010;688:28-35. [20] Tam PKH, Garcia-Barcelo M. Genetic basis of Hirschsprung's disease. Pediatr Surg Int 2009;25:543-58. [21] Britsch S, Goerich DE, Riethmacher D, et al. The transcription factor sox10 is a key regulator of peripheral glial development. Genes Dev 2001;15:66-78. [22] Britsch S. The neuregulin-i/erbb signaling system in development and disease. Adv Anat Embryol Cell Biol 2007;190:1-65. [23] Paratore C, Eichenberger C, Suter U, et al. Sox10 haploinsufficiency affects maintenance of progenitor cells in a mouse model of hirschsprung disease. Hum Mol Genet 2002;11:3075-85. [24] Falls DL. Neuregulins: functions, forms, and signaling strategies. Exp Cell Res 2003;284:14-30. [25] Lee ST, Chu K, Oh HJ, et al. Let-7 microRNA inhibits the proliferation of human glioblastoma cells. J Neurooncol 2011;102:19-24. [26] Farrell BC, Power EM, Mc Dermott KW. Developmentally regulated expression of sox9 and microRNAs 124, 128 and 23 in neuroepithelial stem cells in the developing spinal cord. Int J Dev Neurosci 2011;29: 31-6.
www.elsevier.com/locate/jpedsurg
Aberrant high expression of NRG1 gene in Hirschsprung disease☆ Weibing Tang a,c,1 , Bo Li a,c,1 , Xiaoqun Xu a,c , Zhigang Zhou a,c , Wei Wu a,b , Junwei Tang a,c , Jingjing Qin a,c , Qiming Geng c , Weiwei Jiang c , Jie Zhang c , Jiahao Sha a , Yankai Xia a,b,⁎, Xinru Wang a,b a
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing 210029, China b Key Laboratory of Modern Toxicology (Nanjing Medical University), Ministry of Education, Nanjing 210029, China c Department of Pediatric Surgery, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China Received 21 December 2011; revised 21 February 2012; accepted 20 March 2012
Key words: Hirschsprung disease; NRG1; DNA methylation
Abstract Background/Purpose: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells along with variable lengths of the gastrointestinal tract. Recent studies have indicated the potential function of neuregulin-1 (NRG1) in HSCR, which encodes the heregulins and other mitogenic ligands for the ErbB family. The purpose of this study was to further clarify the role of NRG1 in the pathogenesis of HSCR. Methods: We examined the NRG1 messenger RNA (messenger RNA) and protein expression levels in gut tissues of 63 patients with sporadic HSCR (both stenotic and dilated gut tissues) and 35 controls. Moreover, using the methylation-specific polymerase chain reaction, we examined the methylation pattern of exon 1 of the NRG1 gene. Results: The mRNA expression levels of NRG1 were significantly higher in tissues of HSCR than those in controls, and the increased NRG1 protein levels in HSCR were consistent with the mRNA levels. However, no methylation pattern change was observed in exon 1 of the gene among different groups. Conclusions: Our study demonstrates that the aberrant expression of NRG1 may play an important role in the pathology of HSCR. DNA methylation of the gene seems not to be involved in the mechanism of such aberrant expression, and other factors should be explored. © 2012 Elsevier Inc. All rights reserved.
☆
Conflict of interest: None declared. ⁎ Corresponding author. State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China. Tel.: +86 25 86862939; fax: +86 25 86662863. E-mail address: [email protected] (Y. Xia). 1 These authors contributed equally. 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2012.03.061
Hirschsprung disease (HSCR), also called aganglionosis, is one of the most difficult diagnoses in pediatric surgery [1], characterized by the absence of ganglion cells in the lower digestive tract. It is the most frequent cause of a functional intestinal obstruction among newborns. The incidence of this disease is about 1:2000 to 1:5000. Hirschsprung disease usually occurs sporadically (up to70% of the cases), although
Aberrant high expression of NRG1 gene in HSCR it can be familial, and is frequently associated with recognized syndromes, chromosome, or congenital anomalies [2,3]. Although many studies have been performed to elucidate the pathological process of this disease, the exact mechanism still remains unknown. To date, more than 10 genes have been found to be involved in the pathogenesis of HSCR [2]. Among these genes, the RET proto-oncogene accounts for the highest proportion of both familial and sporadic cases [4]. It is now known that neural crest neuroblasts can colonize aganglionic and ganglionic gut in vivo, which brought a new prospect for the treatment of HSCR [5]. A previous genome-wide association study on Chinese patients with sporadic HSCR identified neuregulin-1 (NRG1) as a susceptibility locus for HSCR [6]. This study also found that there was a significant difference in NRG1 expression level between the diseased and control individuals bearing a same-risk genotype [7]. In addition, not only common variants but also rare ones of the NRG1 gene can contribute to HSCR [8]. Epigenetic alterations in DNA without concomitant changes in underlying genetic codes are now known to occur frequently in several kinds of human diseases [9-11]. Epigenetic silencing is achieved not only through DNA methylation but also by many other DNA, chromatin, and RNA modification mechanisms [12,13]. Changes in DNA methylation patterns impact several critical cellular processes including gene expression, Xinactivation, carcinogenesis, aging, and development [14-17]. Recently, it was shown that NRG1 was unmethylated in normal breast tissues and methylated in breast tumor samples; hypermethylation of the gene is correlated with lower a NRG1 gene expression level [18]. In the present study, we set out to investigate the role of NRG1 in HSCR, initially by measuring expression of NRG1 (both messenger RNA [mRNA] and protein) in human gut tissues (63 patients and 35 controls). We found that there were significant higher NRG1 expression levels in patients with HSCR than those in controls. To find out whether aberrant expression of NRG1 in HSCR is caused by abnormal DNA methylation and if there were any changes in the methylation pattern among different groups, we further examined the methylation pattern of the exon 1 of the NRG1 gene. In this work, we report that there were no significant changes in the methylation pattern of NRG1 among different groups tested. Future studies with a larger sample size are needed to confirm these results. The functional relevance of aberrant expression of NRG1 to HSCR found in this work warrants further investigation.
1. Materials and methods 1.1. Patients and samples This study was approved by the institutional review board of Nanjing Medical University (Nanjing, China), and
1695 all human subjects provided written informed consent. A total of 63 patients with HSCR were collected in Nanjing Children's Hospital affiliated to Nanjing Medical University from October 2009 to May 2011 (Nanjing Medical University Birth Cohort). All patients were confirmed with barium enema, anorectal manometry, and postoperative pathological examination. We took full-thickness tissues in the dilated segment (ganglionic bowel) and stenotic segment (aganglionic bowel) of the colon, and the tissues were immediately stored in liquid nitrogen. Tissue specimens in the normal colon of 35 patients without HSCR (intussusception, incarcerated hernia) were obtained as negative controls over the same period as that for the diseased samples.
1.2. Quantitative real-time polymerase chain reaction Total RNA was obtained from tissues using TRIzol reagent as described by the manufacturer (Invitrogen Life Technologies Co, CA, USA). For NRG1 mRNA detection, RNA (500 ng) was reverse transcribed using a reverse transcription kit (Takala, Tokyo, Japan) under 37°C for 15 minutes and 85°C for 30 seconds. The expression level of the NRG1 gene in each sample was measured by quantitative real-time polymerase chain reaction (PCR) in a volume of 10 μL with 384-well plates using ABI Prism 7900HT (Applied Biosystems, Foster City, CA). β-Actin was used as an endogenous control. Forward (F) and reverse (R) primer sequences were as follows: NRG1, (F) 5′-atgtgtcttcagagtctcccat-3′ and (R) 5′-tggacgtactgtagaagctgg-3′; β-actin, (F) 5′ccaaccgcgagaagatga-3′ and (R) 5′-ccagaggcgtacagggatag-3′. The thermocycler program included a step of denaturation at 95°C (10 minutes) and 40 cycles of 95°C (15 seconds) and 60°C (1 minute).
1.3. Western blotting Proteins were extracted from gut tissues using radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors (cOmplete, ULTRA, 132 Mini, EDTA-free, EASYpack; Roche, Basel, Switzerland). Protein concentrations were determined using the BCA (Bicinchoninic Acid) method. Equal amount of proteins (80-100 μg) were separated by 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Roche). Membranes were blocked using 5% skimmed milk and incubated with respective antibodies. Primary rabbit polyclonal rabbit antiNRG1 antibody was purchased from Santa Cruz Biotechnology (SC-348; Santa Cruz, CA). The secondary antibody used was antirabbit from Santa Cruz Biotechnology. Blots were developed using electrochemiluminescence (ECL) (Millipore, Billerica, MA). Equal loading of protein amounts was confirmed using a GAPDH antibody. Integrated density values were calculated using AlphaImager 3400 (Alpha
1696 InnoTech, San Leandro, CA). These values were then normalized to the GAPDH internal control. All experiments were repeated at least 3 times; representative results are presented in this study.
1.4. DNA isolation For DNA preparation, we collected a small piece from the full-thickness tissue in a 2-mL Eppendorf tube, incubated it with 800 μL lysate (20 mmol/L Tris-HCl [pH 8.0], 5 mmol/L EDTA [pH 8.0], 1% sodium dodecyl sulphate, 400 mmol/L NaCl) and an additional 10 μL of 10 mg/mL proteinase K at 56°C for 24 hours. After digestion, DNA was extracted with phenol-chloroform (1:1) and precipitated in ethanol and was then dissolved in 50 μL TE (Tris-EDTA) buffer (1 mmol/L Tris-HCl, 0.5 mmol/L EDTA, pH 8.0). DNA concentration was determined by spectrophotometry.
1.5. Bisulfite modification and methylation-specific PCR Genomic DNA (500 ng) was treated with sodium bisulfite using the EpiTect Bisulfite Kit (QIAGEN, Valencia, CA), according to the protocol recommended by the manufacturer. Two microliters of the final eluent was used for subsequent PCR amplification. We analyzed the methylation pattern of NRG1 exon 1, which has a region located between −136 to +79 (containing +1, the translational initiation site) rich in CpG sites. The primers used were as follows: methylated forward: 5′-GTTTTAGCGCGGTCGTTC-3′, methylated reverse: 5′-CGAACTCCGACTTCTTACCG-3′; unmethylated forward: 5′-GTAGTGTGAGTGTTTTAGTGTGGTTG-3′, unmethylated reverse: 5′-CAAACTCCAACTTCTTACCA3′ [19]. For both PCR reactions, a total of 100 ng of modified DNA was amplified in a 25-μL reaction containing 0.5 μM each of forward and reverse primer, 200 μM dNTPs, 1 × PCR buffer, and 1.25 U of Taq Hot Start DNA Polymerase (Takara Bio, Tokyo, Japan) under the following conditions: 5 minutes of denaturation at 94°C, followed by 40 cycles of 40 seconds at 94°C, 40 seconds at 61.3°C for methylated primers (58.8°C for unmethylated primers), 1 minute at 72°, and a final extension for 10 minutes at 72°C. The amplification products were separated on 2% agarose gels and stained with ethidium bromide. Most samples were subjected to 2 independent bisulfite treatments and analyzed from 2 independent PCR products. One control DNA sample methylated using SssI methyltransferase (New England Biolabs, MA, USA), according to the manufacturer's protocol, was used as a methylated positive control for those methylation-specific PCR (MSP) reactions.
W. Tang et al. using Stata 9.2 (StataCorp LP, College Station, Texas) and presented with GraphPad prism software (GraphPad Software, Inc, California, USA). For statistical analysis, 2sample Wilcoxon rank sum (Mann-Whitney) test was used to evaluate the differences between groups. Results were considered statistically significant when P b .05.
2. Results 2.1. Results of human sample data analysis The clinical data of 63 patients with HSCR and 35 normal controls were collected, including age, sex (male/female), and body weight. The ages of patients with HSCR and the controls were 3.4 ± 0.18 and 2.8 ± 0.24 months, the sex ratio was 51:12 and 26:9, and the body weight was 5.8 ± 0.28 and 5.1 ± 0.34 kg, respectively There were no significant differences between patients with HSCR and the control subjects regarding age, sex, and body weight.
2.2. Up-regulated mRNA and protein expression levels of NRG1 in HSCR To detect NRG1 expression levels in HSCR and control samples, we used TaqMan quantitative real-time PCR methods. The relative expression levels of NRG1 mRNA were normalized to β-actin expression levels. The medians of each group were 8.97 × 10−4 (HSCR-S), 3.71 × 10−4 (HSCR-D), and 4.89 × 10−5 (control). These results indicate that NRG1 mRNA levels in HSCR stenotic segment samples (P = 4.5 × 10−5) and dilated segment samples (P = 7.5 × 10−5) were both significantly higher than those in the controls. There was no significant difference between
1.6. Statistics and analysis For analysis, the results of reverse transcriptase PCR, we used the method of 2−ΔCt. Statistical analysis was performed
Fig. 1 Relative expression of NRG1 mRNA in HSCR and control samples. Data were presented as median values for NRG1 relative expression of tissues of HSCR and controls (*P b .01).
Aberrant high expression of NRG1 gene in HSCR
Fig. 2 The protein expression of NRG1 between patients with HSCR and the controls. NRG1 protein expression was determined by Western blot, and GAPDH was used as a loading control. HSCR, the stenotic segment of HSCR.
stenotic segment and dilated segment samples (P = .22) (Fig. 1). Such finding indicates that up-regulated NRG1 expression level may be associated with the development of HSCR. Moreover, Western blot analysis showed increased NRG1 protein levels in HSCR pathological segment than in the controls, which was consistent with the results obtained at the mRNA levels (Fig. 2).
2.3. Methylation pattern of the samples To investigate the possible cause of abnormal expression of NRG1, we analyzed the methylation pattern of the NRG1 exon 1 using MSP. The region targeted is located between −136 to +79 (containing +1, the translational initiation site) that is rich in CpG sites. Nevertheless, all patients with HSCR and the controls examined in this study exhibited a similar pattern, that is, the unmethylated allele (Fig. 3).
3. Discussion Hirschsprung disease is considered a disorder of the enteric nervous system (ENS), whereby the enteric ganglion precursors fail to migrate along the developing gut. Therefore, the pathogenesis of HSCR can only be conceptually understood in light of the molecular and cellular events occurring during the ENS development [20]. To date, there is strong evidence indicating that NRG1
1697 signaling is associated with the development and maintenance of the ENS [21-23]. It was previously reported that the overall NRG1 expression in the gut did not differ between patients with HSCR and the controls; they, however, only took full-thickness tissues from ganglionic portions of the bowel of those patients [7]. In the present study, we examined both ganglionic and aganglionic portions of a larger amount of samples. The results indicate that NRG1 mRNA levels in both HSCR stenotic segment and dilated segment samples were significantly higher than those in the controls. In addition, Western blotting analysis showed that increased NRG1 protein levels in patients with HSCR than in the controls, consistent with the changes observed at mRNA levels. Current studies are focused on discovering the role of NRG1 in HSCR from the point of genetic aspects [6-8], with few concerning about epigenetic factors that might be related to the regulation of NRG1 expression and the mechanism underlying this disease. DNA methylation is one of the most studied regulatory mechanisms in epigenetics. It is well established that hypomethylation is associated with transcriptional activation, whereas hypermethylation is associated with repression of transcription [14]. In this study, we hypothesized that the regulation of NRG1 expression in HSCR might be caused by aberrant DNA methylation of the gene. Although the alignment of NRG1 UniGene clusters to the assembled genomic sequence revealed 21 exons [24], the exon 1 region is the closest to the promoter and has the greatest impact on gene function. Moreover, the region located between −136 to +79 (containing +1, the translational initiation site) is rich in CpG sites. However, our results demonstrated complete unmethylation of the gene (exon 1) in all patients with HSCR and controls. Then, as it is known that microRNAs (miRNAs) might play a role in regulating gene expression by transcriptionally repressing mRNAs [25,26], we also used a bioinformatics method to predict miRNAs, which may regulate the expression of NRG1 gene. We applied miRanda (http://www.cbio.mskcc.org/ mirnaviewer), PicTar (http://pictar.mdc-berlin.de/), and TargetScan (http://www.targetscan.org) databases to predict related miRNAs of the NRG1 gene. The results of these 3 databases were inconsistent: miR-18a, miR-139,miR-130, miR-103, miR-107, miR-142, and miR-195 (miRanda); miR-18a, miR-18b, miR-34a, miR-34b, miR-139, and miR-
Fig. 3 Representative results of MSP analyses of DNA samples. M, methylated primers; U, ummethylated primers; P, positive control; S, stenotic segment samples; D, dilated segment samples; C, control samples.
1698 342 (PicTar); and miR-499, miR-876-5p, miR-494, and miR-376 (TargetScan). The results of the prediction were not coincident with each other in the bioinformatics software used above; therefore, further study of the correlated miRNAs of NRG1 is needed. Because the sample size of our study was still relatively small and we only chose one specific region of the gene to examine the methylation pattern in HSCR, it may not sufficient to conclude that the regulation of NRG1 expression is not caused by the methylation of the gene. Further studies with larger sample sizes and different ethnical populations, together with the analysis of the CpG sites at other NRG1 portions, are needed to gain more understanding of the role and mechanism of the NRG1 gene in the pathogenesis of HSCR.
Acknowledgments We thank Dr Bo Hang (Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA) for language editing and Dr Huan Chen and Changgui Lu (Nanjing Children's Hospital Affiliated to Nanjing Medical University) for sample collection. This study was supported by the grants SKLRM-KF-1104, Nanjing Medical Science and Technique Development Foundation (201108010), and Priority Academic Program Development of Jiangsu Higher Education Institutions.
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