Abstract
Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system characterized by aganglionosis in distal gut. In this study, we used two-dimensional gel electrophoresis (2-DE) technology coupled with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis to identify differentially expressed proteins in the aganglionic (stenotic) and ganglionic (normal) colon segment tissues from patients with HSCR. We identified 15 proteins with different expression levels between the stenotic and the normal colon segment tissues from patients with HSCR. Nine proteins were upregulated and six proteins downregulated in the stenotic colon segment tissues compared to the normal colon segment tissues. Based on the biological functions, we selected the Hsp27 upregulated proteins and the PRDX3 downregulated proteins to confirm their expression in 20 patients. The protein and mRNA expressions of Hsp27 were statistically higher in the stenotic colon segment tissues than in the normal colon segment tissues, whereas the protein and mRNA expressions of PRDX3 were statistically lower in the stenotic colon segment tissues than in the normal colon segment tissues. These findings of changes in mRNA and protein in tissues from patients with HSCR provide information which may be helpful in understanding the pathomechanism that is implicated in the disease.
Keywords: colon segment tissues, different expressions, Hirschsprung's disease, Hsp27 and PRDX3
Hirschsprung's disease (HSCR) or aganglionic megacolon, the most common congenital intestinal obstruction, is a developmental disorder of the enteric nervous system (ENS) characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut (Amiel & Yonnet 2001). HSCR represents the main genetic cause of functional intestinal obstruction with an incidence of l of 5000 live births. Males are affected four times more than females. There is a significant racial variation in incidence of the disease, and it is most often found among Asians (28 per 100,000 live births) (Haricharan & Georgeson 2008). However, as a disease characterized by motility disorder of aganglionic segment of the gut, less attention was paid to the pathophysiology underlying the motility dysfunction in HSCR which is still unclear, and the aetiology and pathogenesis of HSCR are still not completely understood (Kim et al. 2005).
The development of high-throughput proteomics approaches makes it possible for us to investigate the changes of cellular protein expression in a large scale which may provide more information for a better understanding of the pathogenesis of various diseases. As a powerful tool to investigate the mechanism of a complex biological process, the proteomics approach can reveal the protein alterations corresponding to certain pathological condition in an integrated way. Two-dimensional gel electrophoresis (2-DE) is one of those approaches which is widely used for its simplicity, reliability, high information content and ready accessibility to researchers (Rabilloud 2002; Chatterji & Borlak 2009; Lee et al. 2009; Zhao et al. 2010; Zhu et al. 2010). 2-DE allows the highest resolution of protein separation with up to 10,000 spots and is able to identify differences in protein composition between two populations of cells being analysed simultaneously on the same gel, thus minimizing difference in protein migration in a gel due to sample processing variations (Thiede et al. 2005). So far, there is no protein expression profile of stenotic colon segment tissues in HSCR, which may be very useful for the early diagnosis of HSCR.
In this study, we used 2-DE gel-based proteomics to compare the protein expression profiles of the stenotic (aganglionic) and the normal (ganglionic) colon segment tissues from patients with HSCR. We identified 15 proteins with different expression levels between the stenotic and the normal colon segment tissues from patients with HSCR using mass spectrometry (MALDI-TOF-MS). Considering the highly complex pathophysiology underlying the gut dysfunction in HSCR, we selected the Hsp27 upregulated and the PRDX3 downregulated proteins study in patients with HSCR, expecting to disclose the protein alterations related to bowel malfunction of HSCR.
Materials and methods
Patients and specimens
The age of the patients ranged from 6 to 36 months old, with an average age of 12.5 months. HSCR was diagnosed by a barium enema X-ray and suction rectal biopsy. All patients with HSCR showed aganglionosis restricted to the sigmoid colon. The aganglionic (stenotic) and ganglionic (normal) segments of the colon were immersed in liquid nitrogen immediately after surgery removal and then stored in −80 °C until use.
Ethical approval
This study was approved by Ethics Committee of China Medical University (Ethical review#: 2012PS17K). The stenotic and the normal colon segment tissues were obtained from 20 patients (including 17 male and three female patients) diagnosed as HSCR in the Department of Pediatric Surgery, Shengjing Affiliated Hospital of China Medical University.
Extraction of proteins
For 2-DE analysis, the stenotic and the normal colon segment tissues from patients with HSCR were homogenized in 1 ml lysis buffer [7 M urea, 2 M thiourea, 4% (V/W) CHAPS, 2% (V/V) IPG buffer (pH 3–10), 40 mM 1, 4-dithioerythritol, 1 mM PMSF and 1% (V/V) cocktail protease inhibitor; Sigma-Aldrich, St Louis, MO, USA], and sonicated in ice for 1 min with 0.2-s pulse on and 1-s pulse off to prevent overheating. To remove salt, samples were cleaned with a 2-D Cleanup kit (GE Healthcare, Amersham Biosciences). The protein concentration was determined by Bradford method. Protein lysates were stored in aliquots at −80 °C.
2-DE
2-DE (Pharmacia IPG-phor systems) was used to separate proteins. Total protein (80 μg) was run in the first dimension isoelectric focusing (IEF) with non-linear gradient IPG strips (13 cm) with pH 3–10 and a programmed voltage gradient. IPG strips were rehydrated at 30 V for 12 h. IEF was carried out using IPGphor IEF system (Amersham Bioscience, Freiburg, Germany) as follows: 100 V for 0.5 h, 250 V for 0.5 h, linear ramping to 4000 V for 1.5 h and finally 4000 V for 1.5 h with a total of 10 kV/h. Then the IPG strips were incubated in equilibration buffer [50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue] containing 1% (w/v) DTT for 10 min with gentle agitation, followed by incubation in the same equilibration buffer in which DTT was replaced with 2.5% iodoacetamide (IAA) to alkylate the proteins. After equilibration, the IPG strips were rinsed gently with water, then applied onto 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and overlaid with a solution of 0.5% agarose with a trace of bromophenol blue. The gel was run at 50 V for 1 h followed by 100 V for 1.5 h, using the mini gel system (Bio–Rad, New York Stock, NY, USA). The proteins were visualized with Coomassie Brilliant Blue (CBB) R-250.
Image acquisition and data analysis
Coomassie brilliant blue-stained gels were scanned by a PowerLook 2100XL image scanner (Umax, Taiwan, China). Spot detection, quantification and matching of the CBB-stained gels were performed using a 2-DE gel analysis software (ImageMaster 2-D platinum 6.0; GE Healthcare). The formula for the calculation of the protein spots is: the relative volume of spots (% vol) = (the sum of the intensities of the pixel units within the protein spot)/(the total volume of all the spots present in a gel) (%). A mean value of % vol for each spot was gotten from three parallel experiments. Spots with at least 1.5-fold changes in % vol (P < 0.05) were defined as differentially expressed proteins, and the corresponding spots were excised for further analysis.
The mass spectrometer (MS) and MS/MS spectra were searched against the International Protein Index (IPI) human database, version 3.18, with GPS Explorer TM v3.0 (AB Sciex, Dyna/Autodyn, GA, USA) and MASCOT database search algorithms (version 2.0). The search criteria trypsin specificity, cysteine carbamido methylation (C) and methionine oxidation (M) were used as variable modifications: 1 trypsin miscleavage allowed; 0.2-Da MS tolerance; and 0.3-Da MS/MS tolerance. Protein identifications were accepted with a Mowse score ≥ 58 and a P < 0.05.
Western blotting analysis
Equal amounts of total proteins (50 μg) from tissues were separated by 12.5% SDS–PAGE, electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk in 0.1% Tween 20 at 4 °C overnight, and subsequently incubated with the primary rabbit anti-human Hsp27 antibody (bs-0730R, 27 kDa) (1:500), rabbit anti-human GSK3β antibody (bs-0028R, 47 kDa) (1:500), rabbit anti-human Wnt1 antibody (bs-1739R, 38 kDa) (1:500) and rabbit anti-human PRDX3 antibody (bs-1874R, 28 kDa; Beijing Biosynthesis Biotechnology Co., Ltd, Beijing, China) (1:500), respectively, at 4 °C overnight. Then the membranes were washed, incubated with horseradish peroxidase-linked secondary antibodies (1:2000) for 1 h at room temperature. The signals were detected using an enhanced chemiluminescence (ECL) kit. Densitometry analysis was performed using the Gel-pro 4.0 software (Media Cybernetics, LP, Waltham, MA, USA). The relative density of the tested protein bands was calculated as the density of tested protein band normalized by the density of loading control (GAPDH) band.
Immunohistochemistry staining analysis
Immunohistochemistry staining was performed using DAKO EnVision Systems (DakoCytomation GmbH, Hamburg, Germany), according to the manufacturer's instructions. Consecutive paraffin-embedded tissue sections (4–7 μm) were dewaxed and dehydrated. Specimen was deparaffinized in xylene (Carl Roth GmbH+ Co. KG, Karlsruhe, Germany), rehydrated by a descending ethanol (Merck KGaA, Darmstadt, Germany) series and permeabilized with 0.5% Triton-X (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) in PBS for 10 min at room temperature. The specimens were boiled in 0.1 mM EDTA buffer (pH 8.0, Sigma Aldrich Chemie GmbH) or in Target Retrieval solution (pH 6.0, Dako Deutschland GmbH, Hamburg, Germany) for 15 min. After blocking for endogenous peroxidase using 0.4% H2O2 in PBS, the samples were incubated overnight at 4 °C with rabbit polyclonal Hsp27, GSK3β, Wnt1 and PRDX3 antibodies (diluted 1:50). Slides then were coated in a solution of goat anti-rabbit antibody (1:2000; Dako) for 30 min, and finally in a solution of streptavidin–horseradish peroxidase (LSAB2 System; Dako) at 10 min. Antibody detection was visualized using a substrate–chromogen solution (LSAB2 System; Dako) for 5–30 min that was counterstained with Mayer's haematoxylin (Merck) for 1 min. The density of the positively stained area was calculated at 400× magnification.
Real-time quantitative PCR analysis
Total RNA was extracted from the stenotic and the normal colon segment tissues from patients with HSCR using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA was synthesized with the TaKaRa RNA PCR kit (Takara Biotechnology, Shiga, Japan). Real-time PCR was performed on a Light Cycler (Roche Applied Science, Boehringer Mannheim GebH, Mannheim Area, Germany). Samples were prepared in duplicate and amplified over 45 cycles. Each cycle consisted of a denaturation phase of 15 s at 95 °C and a hybridization/elongation phase of 1 min (see Table1). The primers were listed in Table1. The housekeeping gene GAPDH (Takara, DR3783) was used as an endogenous control. The relative mRNA levels for each sample were calculated by the 2−△△ct method.
Table 1.
Primers for real-time PCR
| Target | Sequence (5′–3′) | Amplicon (bp) | Annealing temperature ( °C) |
|---|---|---|---|
| Hsp27 | F: CAA GGA TGG CGT GGT G | 136 | 56 |
| R: GGA CAG GGA GGA GGA AAC | |||
| GSK3β | F: GCG TCA GAT GCT AAT A | 124 | 50 |
| R: ACT CAA GTA ACT GGT GG | |||
| Wnt1 | F: CGG GCA ACA ACC AAA GTC | 109 | 58 |
| R: CAG CAG CAG CGT AGC AGA A | |||
| PRDX3 | F: TGT GAA GTT GTC GCA GTC | 138 | 51 |
| R: GTA GTC TCG GGA AAT CTG | |||
| GAPDH | F GGG AAG GTG AAG GTC G | 228 | 51 |
| R GAA GAT GGT GAT GGG ATT |
Statistical analysis
All experiments were performed in triplicate at a minimum. The significance of differences between groups was calculated by applying analysis of variance and Bonferroni's test, as indicated. All P values of <0.05 were considered statistically significant.
Results
Protein profiles from the stenotic and the normal colon segment tissues of HSCR
To validate the reproducibility, 2-DE was performed in triplicate. HSCR proteome of the stenotic and the normal colon segment tissues contained about 770–890 detectable proteins on a single mcCBB-stained 2-DE gel, with an average matching rate of 82.4% on total gels (Figure1). 2-DE images with high resolution and reproducibility were obtained, and representative images are shown in Figure1. We found 23 protein spots with at least 1.5-fold difference in vol % (P < 0.05) between stenotic and normal colon segment tissues and dissected them for further analysis.
Figure 1.
2-DE of protein profile in Hirschsprung's disease (HSCR). (a) Representative Coomassie-stained 2-DE gels of expression maps of proteins in stenotic colon segment tissues with HSCR. (b) Representative Coomassie-stained 2-DE gels of expression maps of proteins in normal colon segment tissues with HSCR.
Of the 23 dissected spots, we identified 15 proteins by MS as differentially expressed. Among the 15 spots, nine spots were upregulated and six spots were downregulated, and these proteins were identified as differently expressed (Tables2 and 3).
Table 2.
MALDI-TOF-mass spectrometer (MS) identification of differential protein spots
| No. | SWISS-PROT accession no. | Protein identified (up and down) | Mascot score | Mr (kDa)/PI | Peptide matched | Cover % |
|---|---|---|---|---|---|---|
| 1 | gi|4504517 | ↑ Hsp27 | 118 | 22.8/5.98 | 9/30 | 38 |
| 2 | gi|4502431 | ↑ BMPR1β | 149 | 72.3/5.77 | 23/78 | 33 |
| 3 | gi|4557325 | ↑ APOE | 127 | 43.8/6.13 | 15/41 | 31 |
| 4 | gi|119596536 | ↑ MYLIP | 76 | 11.3/4.79 | 7/48 | 63 |
| 5 | gi|31746 | ↑ GSTP1 | 95 | 25.1–4.5 | 12/70 | 47 |
| 6 | gi|48145803 | ↑ MYL9 | 74 | 19.89/4.94 | 11/48 | 56 |
| 7 | gi|119586097 | ↑ GSK3β | 45 | 24.58/5.03 | 15/30 | 7 |
| 8 | gi|33874637 | ↑ ACTN4 | 173 | 73.9/5.16 | 27/98 | 54 |
| 9 | gi|32189392 | ↓ PRDX3 | 125 | 22.1/5.66 | 9/17 | 40 |
| 10 | gi|2605811 | ↓ Wnt1 | 52 | 13.6/9.15 | 4/39 | 31 |
| 11 | gi|71297227 | ↓ DVL2 | 40 | 60.1/4.61 | 4/30 | 52 |
| 12 | gi|49168516 | ↓ ACTG2 | 146 | 42.3/5.31 | 23/95 | 63 |
| 13 | gi|182356 | ↓ FABP1 | 79 | 14.2/6.6 | 6/27 | 68 |
| 14 | gi|49168456 | ↑ TAGLN | 179 | 22.6/8.87 | 29/65 | 67 |
| 15 | gi|179032 | ↓ SM22 | 131 | 22.63/8.78 | 24/65 | 55 |
Table 3.
The function of proteins identified
| No | Protein name | Function* |
|---|---|---|
| 1 | Hsp27 | The protein encoded by this gene is induced by environmental stress and developmental changes. The encoded protein is involved in stress resistance and actin organization and translocates from the cytoplasm to the nucleus upon stress induction. |
| 2 | BMPR1β | BMPs are involved in endochondral bone formation and embryogenesis. BMPR1β binds ligands in the absence of type I receptors, but they require their respective type I receptors for signalling, whereas type I receptors require their respective type II receptors for ligand binding. |
| 3 | APOE | ApoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents. |
| 4 | MYLIP | Myosin regulatory light chain-interacting protein (MYLIP) is a novel ERM-like protein that interacts with myosin regulatory light chain and inhibits neurite outgrowth. |
| 5 | GSTP1 | Glutathione S-transferases (GSTs) are a family of enzymes that play an important role in detoxification by catalysing the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione. The GSTP1 variant proteins are thought to function in xenobiotic metabolism and play a role in susceptibility to cancer, and other diseases. |
| 6 | MYL9 | The protein encoded by this gene is a myosin light chain that may regulate muscle contraction by modulating the ATPase activity of myosin heads. |
| 7 | GSK3β | The protein is involved in energy metabolism, neuronal cell development and body pattern formation. |
| 8 | ACTN4 | Alpha-actinin is an actin-binding protein with multiple roles in different cell types. In non-muscle cells, the cytoskeletal isoform is found along microfilament bundles and adherens-type junctions, where it is involved in binding actin to the membrane. |
| 9 | PRDX3 | This gene encodes a protein with antioxidant function and is localized in the mitochondrion. Sequence comparisons with recently cloned mammalian homologues suggest that these genes consist of a family that is responsible for regulation of cellular proliferation, differentiation and antioxidant functions. |
| 10 | Wnt1 | The Wnt gene family consists of structurally related genes which encode secreted signalling proteins. It is very conserved in evolution, and the protein encoded by this gene is known to be 98% identical to the mouse Wnt1 protein at the amino acid level. |
| 11 | DVL2 | This gene encodes a 90-kD protein that undergoes posttranslational phosphorylation to form a 95-kD cytoplasmic protein, which may play a role in the signal transduction pathway mediated by multiple Wnt proteins. |
| 12 | ACTG2 | Actins are highly conserved proteins that are involved in various types of cell motility and in the maintenance of the cytoskeleton. This gene encodes actin gamma 2; a smooth muscle actin found in enteric tissues. |
| 13 | FABP1 | Fatty acid binding proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It is thought that FABP roles include fatty acid uptake, transport and metabolism. |
| 14 | TAGLN | The protein encoded by this gene is a transformation and shape change sensitive actin cross-linking/gelling protein found in fibroblasts and smooth muscle. Its expression is downregulated in many cell lines, and this downregulation may be an early and sensitive marker for the onset of transformation. A functional role of this protein is unclear. |
| 15 | SM22 | Same as above |
All the information about function are from the SWISS-PROT, NCBI database.
Western blot analysis of selected proteins in patients with HSCR
We selected four proteins (Hsp27, GSK3β, Wnt1 and PRDX3) for Western blot analysis, based on their biological functions, and confirmed their expression alterations in the colon tissue of HSCR. The protein levels of Hsp27 and GSK3β were higher in the stenotic colon segments than in the normal colon segments (Figure2), densitometry analysis showed statistically difference (P < 0.05 for both Hsp27 and GSK3β) (Table4). The protein levels of Wnt1 and PRDX3 were lower in the stenotic colon segments than in the normal colon segments (Figure2), densitometry analysis showed statistical difference (P < 0.05 for both Wnt1 and PRDX3) (Table3). In 20 patients were used for verification, patients with HSCR showed consistent results with 2-DE.
Figure 2.
Hsp27, GSK3β, Wnt1 and PRDX3 expression in Hirschsprung's disease (HSCR) by Western blot analysis. Protein extracts from the stenotic colon segment and the normal colon segment in HSCR were analysed by Western blot with Hsp27, GSK3β, Wnt1 and PRDX3 antibodies.
Table 4.
Densitometry analysis of Hsp27, GSK3β, Wnt1 and PRDX3 in the colon segment tissues of Hirschsprung's disease (mean ± SD)
| Protein name | Case (n) | Protein levels (relative density unit) | |
|---|---|---|---|
| Stenotic segment | Normal segment | ||
| Hsp27 | 20 | 255.19 ± 21.63 | 152.78 ± 19.31 |
| GSK3β | 20 | 317.65 ± 18.33 | 170.53 ± 15.47 |
| Wnt1 | 20 | 129.28 ± 20.41 | 233.15 ± 22.27 |
| PRDX3 | 20 | 156.13 ± 17.58 | 285.72 ± 23.64 |
Immunohistochemistry analysis of selected proteins in patients with HSCR
To further investigate the distribution and expression of the above four selected proteins in colon tissues, we performed immunohistochemistry staining. Hsp27, GSK3β, Wnt1 and PRDX3 were all located in the mucous layer and muscular layer of the colon segment tissues. The levels of Hsp27 and GSK3β were higher in the stenotic colon segment tissues (Figure3a) than in the normal colon segment tissues (Figure3b). The levels of Wnt1 and PRDX3 were lower (Figure4a) in the stenotic colon segment tissues than in the normal colon segment tissues (Figure4b). In 20 patients were used for verification, patients with HSCR showed consistent results with 2-DE.
Figure 3.
Distribution and expression of Hsp27 and GSK3β detected by immunohistochemistry in Hirschsprung's disease (HSCR). (a) Hsp27 and GSK3β staining in the mucous layer and muscular layer of the stenotic colon segment tissues (the arrow shows). (b) Hsp27 and GSK3β staining in the mucous layer and muscular layer of the normal colon segment tissues (the arrow shows). Original magnification ×400.
Figure 4.
Distribution and expression of Wnt1 and PRDX3 detected by immunohistochemistry in Hirschsprung's disease (HSCR). (a) Wnt1 and PRDX3 staining in the mucous layer and muscular layer of the stenotic colon segment tissues (the arrow shows). (b) Wnt1 and PRDX3 staining in the mucous layer and muscular layer of the normal colon segment tissues (the arrow shows). Original magnification ×400.
mRNA transcription level analysis of selected proteins in patients with HSCR
To test whether there were any changes at the transcriptional level of the four selected proteins, we compared the mRNA levels by performing real-time quantitative PCR. The mRNA levels of Hsp27 and GSK3β were 3.1-fold and 2.4-fold higher in the stenotic colon segments than in the normal colon segments (n = 20, P < 0.020; n = 20, P < 0.012), respectively. The mRNA levels of Wnt1 and PRDX3 were 3.9-fold and 2.7-fold lower in the stenotic colon segments than in the normal colon segments (n = 20, P < 0.0001; n = 20, P < 0.002), respectively (Tables4–6). In 20 patients were used for verification, patients with HSCR showed consistent results with 2-DE.
Table 6.
The relative quantity of Wnt1 and PRDX3 mRNA in two segments
| No. | Wnt1 Ct value | PRDX3Ct value | GAPDH Ct value | Times of gene (compared to stenotic segment) | P | |||
|---|---|---|---|---|---|---|---|---|
| Stenotic | Normal | Stenotic | Normal | Stenotic | Normal | |||
| 1 | 23.27 | 26.91 | 25.05 | 25.85 | 25.52 | 25.23 | Wnt1 = 3.868-fold | Wnt1 < 0.0001 |
| 2 | 24.01 | 25.97 | 24.73 | 25.61 | 26.32 | 26.07 | ||
| 3 | 24.29 | 26.92 | 25.09 | 25.93 | 28.11 | 27.88 | PRDX3 = 2.677-fold | PRDX3 < 0.002 |
| 4 | 25.47 | 27.27 | 25.21 | 27.85 | 28.32 | 26.11 | ||
| 5 | 23.61 | 25.05 | 24.98 | 25.23 | 24.14 | 25.65 | ||
| 6 | 24.02 | 25.31 | 25.49 | 26.87 | 26.67 | 27.23 | ||
| 7 | 23.68 | 24.31 | 23.89 | 24.14 | 25.13 | 25.06 | ||
| 8 | 24.06 | 26.22 | 25.26 | 27.08 | 27.88 | 25.55 | ||
| 9 | 24.72 | 26.08 | 24.96 | 26.15 | 25.01 | 25.28 | ||
| 10 | 24.62 | 25.94 | 25.31 | 26.86 | 26.52 | 25.11 | ||
| 11 | 25.23 | 27.29 | 25.18 | 27.03 | 26.16 | 25.66 | ||
| 12 | 24.88 | 26.03 | 24.85 | 26.55 | 27.77 | 28.14 | ||
| 13 | 23.81 | 25.06 | 24.59 | 25.69 | 24.44 | 24.97 | ||
| 14 | 24.99 | 26.51 | 25.37 | 26.91 | 25.08 | 26.78 | ||
| 15 | 24.07 | 26.64 | 25.77 | 26.41 | 26.56 | 27.13 | ||
| 16 | 24.31 | 26.61 | 24.92 | 25.83 | 28.11 | 26.12 | ||
| 17 | 24.09 | 25.77 | 25.08 | 26.25 | 26.89 | 25.33 | ||
| 18 | 24.35 | 25.54 | 25.11 | 26.04 | 25.41 | 26.76 | ||
| 19 | 23.66 | 25.68 | 26.07 | 26.99 | 26.87 | 25.66 | ||
| 20 | 25.01 | 26.59 | 25.12 | 27.01 | 27.66 | 26.54 | ||
Discussion
New molecular biomarkers or biomarker patterns found by proteomic high-throughput techniques will enable a more reliable early diagnosis of multifarious diseases and facilitate the prediction of their progression. Also the confirmatory experimental analysis is needed for translating the proteomic observations into clinical applications. It is well known that the onset of HSCR involves a series of complicated process including the disturbance of ganglion cells at different stages, and HSCR is caused by multiple factors (Amiel & Yonnet 2001; Chatterji & Borlak 2009). HSCR is a multigene disease caused by congenital absence of ganglion cells in the myenteric and submucosal plexuses of the gastrointestinal tract (aganglionosis). Many genes have been reported to be involved in the aetiology of HSCR and the pathomechanism of the disease remains unclear. In this study, we used 2-DE-based proteomics study to investigate the differential protein expression between the stenotic and the normal colon segment tissues from patients with HSCR to get more information about HSCR's aetiology. We identified 15 different expression proteins (Tables2 and 3). Furthermore, we selected two upregulated (Hsp27 and GSK3β) and two downregulated (Wnt1 and PRDX3) proteins from the 15 proteins, and tested their expressions by Western blotting, immunohistochemical staining and real-time quantitative PCR in 20 tissue samples from patients with HSCR. We found that the protein and mRNA levels of Hsp27 and GSK3β increased, while those of Wnt1 and PRDX3 decreased in the stenotic colon segment tissues compared to normal colon segment tissues. We demonstrated four proteins altered in stenotic segment comparing with normal control (Tables5 and 6). Our result might shed light on understanding HSCR disease as well as on the pathomechanism of physiological gut motility function.
Table 5.
The relative quantity of Hsp27 and GSK3β mRNA in two segments
| No. | Hsp27 | GSK3β | GAPDH | Times of gene (compared to normal segment) | P | |||
|---|---|---|---|---|---|---|---|---|
| Stenotic | Normal | Stenotic | Normal | Stenotic | Normal | |||
| 1 | 28.71 | 27.05 | 25.86 | 24.09 | 25.52 | 25.23 | Hsp27 = 3.131 fold | Hsp27 < 0.020 |
| 2 | 31.51 | 29.73 | 26.79 | 25.23 | 26.32 | 26.07 | ||
| 3 | 29.93 | 28.09 | 26.11 | 24.37 | 28.11 | 27.88 | GSK3β = 2.392 fold | GSK3β < 0.012 |
| 4 | 28.44 | 27.21 | 27.54 | 26.56 | 28.32 | 26.11 | ||
| 5 | 28.44 | 27.98 | 25.88 | 24.85 | 24.14 | 25.65 | ||
| 6 | 30.20 | 27.49 | 25.67 | 23.92 | 26.67 | 27.23 | ||
| 7 | 29.53 | 28.41 | 25.98 | 23.65 | 25.13 | 25.06 | ||
| 8 | 29.98 | 28.26 | 24.74 | 23.79 | 27.88 | 25.55 | ||
| 9 | 29.71 | 27.96 | 25.56 | 23.79 | 25.01 | 25.28 | ||
| 10 | 29.87 | 28.31 | 25.64 | 24.36 | 26.52 | 25.11 | ||
| 11 | 26.96 | 25.18 | 25.62 | 24.86 | 26.16 | 25.66 | ||
| 12 | 28.41 | 26.85 | 25.17 | 23.81 | 27.77 | 28.14 | ||
| 13 | 30.83 | 28.59 | 25.76 | 24.93 | 24.44 | 24.97 | ||
| 14 | 28.65 | 26.37 | 27.57 | 26.26 | 25.08 | 26.78 | ||
| 15 | 29.39 | 28.77 | 26.96 | 24.25 | 26.56 | 27.13 | ||
| 16 | 28.77 | 26.92 | 25.76 | 23.53 | 28.11 | 26.12 | ||
| 17 | 28.09 | 27.17 | 26.84 | 25.33 | 26.89 | 25.33 | ||
| 18 | 29.23 | 28.29 | 27.29 | 26.54 | 25.41 | 26.76 | ||
| 19 | 28.14 | 26.73 | 27.43 | 25.97 | 26.87 | 25.66 | ||
| 20 | 29.31 | 27.11 | 28.01 | 27.32 | 27.66 | 26.54 | ||
Hsp27 is a member of the heat-shock protein family, which are highly conserved molecular chaperones (Ellis & van der Vies 1991). Recently, studies found that increased Hsp27 levels are often found in the brain tissues of individuals with neurodegenerative disorders such as Parkinson's disease (PD) (Renkawek et al. 1999), amyotrophic lateral sclerosis (ALS) (Vleminckx et al. 2002) and the protein and mRNA levels of Hsp27 are substantially increased in the brains of patients and in the spinal cord of mutant mice with the rare Alexander disease (Head et al. 1993). In these publications, Hsp27 is reported to promote neuronal survival in rat pheochromocytoma PC12 cells (Zourlidou et al. 2004; Gorman et al. 2005), ND7 cells and B130/2 BHK cells. A combination of Hsp27 and Hsp104 can rescue striatal dysfunction in HSCR rat model and prevent cell death in primary neuronal cultures (Perrin et al. 2007). In our study, an increase of Hsp27 in mucous layer and muscular layer of stenotic colon segment tissues was detected.
Glycogen synthase kinase-3 beta (GSK3β) has a critical role in many cellular signalling pathways (Li et al. 2009). Physiologically, GSK3β phosphorylates and inactivates glycogen synthase to regulate the synthesis of glycogen. Pathologically, GSK3β has been recognized as a crucial modulator of apoptosis for over 10 years (Bijur et al. 2000). GSK3β was reported to be involved in Alzheimer's disease (Grimes & Jope 2001). Furthermore, GSK3β may be associated with PD (Nagao & Hayashi 2009). Studies targeting GSK3β gene by genetic approaches, such as GSK3β gene knock-down by shRNA, would be necessary to verify the role of GSK3β in the apoptosis (Mariappan et al. 2008).
Peroxiredoxins (PRDXs) are a family of peroxidases that are well conserved throughout evolution. The PRDX has been found to play a role in the regulation of intracellular H2O2 signalling (Zhang et al. 2009). Recently, PRDX3 knockout mice were shown to be more sensitive to lipopolysaccharide-induced oxidative stress (Li et al. 2007). Compared to GPX1, PRDX3 and PRDX5 are highly expressed in neurons (Jin et al. 2005). Interestingly, PRDX5 expression was found to be regulated in human osteoarthritis, degenerative tendon and Graves' thyroid (Wang et al. 2002; Gérard et al. 2005). The aforementioned studies all support a physiological importance and protective function of PRDXs.
Wingless-type MMTV integration site (Wnt) signalling pathway is important not only in embryonic development but also in adult intestinal homeostasis (Sancho et al. 2004; Katoh 2007). In the intestine, Wnt signalling is crucial for the proliferation and maintenance of intestinal stem cells and progenitor cells (Van de Wetering et al. 2002). Under normal conditions, Wnt pathways are exclusively required for maintaining intestinal stem cells (Nakamura et al. 2007). Therefore, the Wnt pathways may function cooperatively in intestinal epithelium. The Wnt signal pathway can be modified by promoting or inhibiting signalling through the cell surface receptors (Suzuki et al. 2002).
Colonic motility is a complex biological behaviour requiring delicate coordination and regulation. HSCR is a disease characterized by colonic motility disorder (Nemeth et al. 2002). Our study that the alterations of the four proteins reflected the disorder of mechanochemical signal in the affected bowel, may partially explain the tonic contraction of affected colon and may reflect the pathological changes of the colon segment tissues in HSCR. Besides, the nerve plexus of myenteric and submucosal and enteric smooth muscle cells may be involved in the pathomechanism for gut function disorder (Langer et al. 1994). Therefore, in our study, the altered expressing proteins and mRNA in Hsp27, GSK3β, Wnt1 and PRDX3 might contribute to the impaired development of enteric neurons involved pathogenesis of HSCR disease.
In conclusion, we provided an overview of proteomic changes in the stenotic and the normal segments of colon tissues from patients with HSCR. We found 15 differentially expressed proteins in the aganglionic segment to the aganglionosis. These proteins included Hsp27, GSK3β, Wnt1 and PRDX3. However, the precise role that they play in HSCR needs further investigation. Our study may provide more insights into developmental disorder of the enteric nervous system in HSCR disease. This information may help to develop new therapies for HSCR disease.
Acknowledgments
We thank Prof. Zhijie Li of the Central Laboratory, Shengjing Hospital Affiliated to China Medical University for her review of the manuscript.
Funding source
This work was supported by grants from the National Natural Science Foundation of China (no. 30772277 and 81170334).
Conflict of interest
The authors declare no conflict of interest.
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