Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Hepatology and Nutrition
Gene Expression Profiling Reveals Upregulated UCA1 and BMF in Gallbladder Epithelia of Children With Pancreaticobiliary Maljunction
Kaneko, Kenitiro*; Ito, Yoshinori†; Ono, Yasuyuki*; Tainaka, Takahisa*; Tsuchiya, Hironori*; Shimoyama, Yoshie‡; Ando, Hisami*
*Department of Pediatric Surgery, Japan
†Department of Pediatrics, Japan
‡Department of Pathology and Laboratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Received 8 December, 2010
Accepted 6 February, 2011
Address correspondence and reprint requests to Kenitiro Kaneko, MD, Department of Pediatric Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan (e-mail: firstname.lastname@example.org).
Funding was provided by a Grant-in-aid (21592275) for Scientific Research from the Japan Society for the Promotion of Science.
The authors report no conflicts of interest.
Background: Pancreaticobiliary maljunction is usually associated with choledochal cysts and often causes biliary carcinoma; however, the mechanism of carcinogenesis remains unknown. No study has analyzed overall changes in genetic expression beginning during childhood in gallbladder epithelia with pancreaticobiliary maljunction.
Patients and Methods: The genomewide expression of gallbladder epithelia was analyzed in 6 children with pancreaticobiliary maljunction and in 4 pediatric controls. Selected genes that were expressed differentially were further analyzed by the real-time reverse transcription-polymerase chain reaction (RT-PCR). The products of upregulated genes confirmed by real-time RT-PCR were immunohistochemically analyzed using gallbladders from 19 children with pancreaticobiliary maljunction, 5 pediatric controls, and 5 children with gallstones.
Results: Microarray analysis identified 188 upregulated and 160 downregulated genes. RT-PCR confirmed upregulation in 5 of 6 genes and downregulation in 1 of 5 genes, including UCA1, DUOX2, DUOXA2, ID1, BMF, and GP2. Immunohistochemistry showed a significantly higher expression of BMF in the pancreaticobiliary maljunction patients than in the controls and gallstone patients.
Conclusions: This study identified several deregulated genes in the gallbladder of children with pancreaticobiliary maljunction, which may contribute to the pathophysiology. UCA1, a noncoding RNA, is an oncofetal gene, and its upregulation may be important for biliary carcinogenesis. The elevated expression of BMF may function as an apoptotic activator in proliferative gallbladder epithelia.
Pancreaticobiliary maljunction is a congenital anomaly, defined as the aberrant union of the pancreatic and biliary ducts located outside the duodenal wall. Pancreaticobiliary maljunction is usually associated with choledochal cysts. Because Oddi sphincter is not involved in the union, pancreaticobiliary maljunction causes the 2-way regurgitation of bile and pancreatic juice, damaging these systems (1). During childhood, pancreaticobiliary maljunction produces characteristically intermittent symptoms including abdominal pain, vomiting, and jaundice (2). In adults, it causes malignant transformation in the gallbladder and a dilated bile duct (3). To prevent symptoms and carcinogenesis, choledochal cyst excision is the present treatment of choice. However, the surgery involves postoperative problems, such as hepatolithiasis and carcinogenesis at the residual bile ducts (4–6). New therapeutic strategies require knowledge of the mechanisms that cause symptoms and cancers. Recently, we elucidated the former mechanism: regurgitation of pancreatic juice into the biliary tract produces obstructive substances (mostly protein plugs and, rarely, fatty calcium stones), which increase transient pancreaticobiliary ductal pressure and cause symptoms (2,7,8). However, the latter mechanism of carcinogenesis remains unknown.
According to a nationwide survey of pancreaticobiliary maljunction in Japan, in 1239 patients with choledochal cysts, cancer occurred in 131 (11%), of which 85 (65%) had gallbladder and 44 (34%) had bile duct cancer. In 388 patients with pancreaticobiliary maljunction but without choledochal cysts, cancer occurred in 147 (38%), of which 137 (93%) had gallbladder cancer (3). This suggests that bile stasis may be related to carcinogenesis. Bile containing regurgitated pancreatic juice has been reported to produce substances that are hazardous to the biliary epithelium, including activated pancreatic enzymes, lysolecithin, secondary or unconjugated bile acids, and a mutagen with a molecular weight between 1500 and 3000 (9–12). Recent research results, however, have shown no changes in bile acids (13). Histopathological studies demonstrated increased cellular proliferation and subsequent hyperplasia of the gallbladder epithelium in pancreaticobiliary maljunction (14–18). Molecular abnormalities of the biliary epithelium include activating KRAS point mutation and TP53 inactivation. KRAS mutation occurs early in multistage carcinogenesis, and TP53 inactivation occurs relatively late (18–21). Speeding up of the cell cycle, hyperplasia, and KRAS mutation begin during childhood (16,17,22). The importance of KRAS mutation and hyperplasia contrasts with carcinogenesis caused by gallstones, where early TP53 inactivation and dysplasia are important, but KRAS is not significant (23); however, elucidation of the carcinogenic process is still limited. To clarify the pathophysiology that leads to carcinogenesis, instead of individual gene analysis that has been conducted so far, we analyzed whole human genes using a microarray to assess expressional differences beginning during childhood in gallbladder epithelia in the presence of pancreaticobiliary maljunction.
PATIENTS AND METHODS
Gallbladders from 6 patients with choledochal cyst and pancreaticobiliary maljunction removed between July 2008 and February 2009 were used. Pancreaticobiliary maljunction and dilation of the common bile duct were diagnosed using endoscopic retrograde cholangiopancreatography. Pancreaticobiliary maljunction was diagnosed when the bile and pancreatic ducts join above the notch of the common channel, which indicates the range of the sphincter action. The mean ages of the patients were 5.4 years (range 2–8 years). There were 2 boys and 4 girls. Another 10-year-old patient was encountered during this period, but this patient was excluded because of his more advanced age. Gallbladders resected at hepatectomy during the same period from 4 patients with hepatoblastoma were used as controls. Three of the 4 patients underwent hepatectomy after chemotherapy. The mean age was 2.8 years (range 1–4 years). There were 3 boys and 1 girl. The epithelia at the fundus of the gallbladders were macroscopically resected from the fibromuscular layer in the operating room, immediately frozen in liquid nitrogen, and stored at −80°C until evaluation. The study design and purpose, approved by the institutional review board of Nagoya University (#812), were fully explained to all of the patients, and informed consent was obtained.
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The quality of RNA was ensured using Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA).
One microgram of total RNA was used for aRNA synthesis using the MessageAmpTM II-Biotin Enhanced Kit (Ambion, Austin, TX). Ten micrograms of labeled aRNA sample was hybridized to the CodeLink Human Whole Genome Bioarray (Applied Microarrays, Tempe, AZ) according to the manufacturer's protocol. Posthybridization staining was carried out using Cy5-streptavidin for the microarray (GE Healthcare Bio-Sciences Corp, Piscataway, NJ). After washing, the arrays were scanned using the GenePix4000B (Molecular Devices, Sunnyvale, CA). The images were analyzed and the background was corrected with CodeLink TM Expression Analysis version 5.0 (Applied Microarrays). Microarray data from the 10 samples were Quantile normalized using the Microarray Data Analysis Tool version 3.0 (Filgen, Nagoya, Japan). Signal intensities were compared between the 2 groups with the Student t test using the Microarray Data Analysis Tool version 3.0 (Filgen). A gene was defined as being upregulated when the patient/control average intensity ratio was >2.0 with P < 0.05, and downregulated when the patient/control ratio was <0.5 with P < 0.05. Because data from spots with a low signal intensity are less reliable, upregulated genes of patients with an average intensity >400 and downregulated genes of the control with an average intensity >400 were selected.
Real-time Reverse Transcription-Polymerase Chain Reaction Assay
Among the genes selected above, several genes with higher/lower intensity ratios, or related to the cell cycle or inflammation, were further selected for verification by real-time reverse transcription-polymerase chain reaction (RT-PCR). cDNA was synthesized using the high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). mRNA expression was measured using real-time RT-PCR with TaqMan Gene Expression Assays on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). TaqMan gene-specific primer and probe sets are listed in Table 1. Relative quantification values were calculated according to the manufacturer's instructions. Briefly, the amount of the target gene is given by 2−ΔCt, where the threshold cycle (Ct) is the point at which the fluorescence of the TaqMan assay reaction exceeds the threshold limit. ΔCt is the difference in the Ct values between the target antigen and endogenous control glyceraldehyde-3-phosphate dehydrogenase. The target gene mRNA levels of patients and control subjects were compared with the Mann-Whitney U test using GraphPad Prism version 5.0c (GraphPad Software, La Jolla, CA). Differences were considered significant if P < 0.05.
Protein expressions of the upregulated genes confirmed using real-time RT-PCR were examined by immunohistochemistry. Gallbladders from 19 patients with choledochal cyst/pancreaticobiliary maljunction removed between June 2008 and February 2010 were used. These included the specimens used for microarray and real-time RT-PCR (the microarray study). The mean ages of patients were 5.0 years (range 5 months – 11 years). There were 5 boys and 14 girls. As controls, gallbladders from 5 patients with hepatoblastoma were used. Of these, 4 gallbladders were the same controls in the microarray study, and a gallbladder, resected during hepatectomy after chemotherapy from a 4-year-old boy, was added. For comparison (the gallstones is another factor predisposing to gallbladder cancer), 5 gallbladders resected because of gallstones were used. The mean ages of the 5 patients were 8.3 years (range 5–12 years). There were 2 boys and 3 girls. The specimens were rapidly immersed in 10% buffered formaldehyde solution, embedded in paraffin, and sliced into 4-μm sections. Sections were stained with hematoxylin and eosin for histology, and with a polyclonal antibody to DUOX2 (DUOX2 Y-15: sc-49939, Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:100), a polyclonal antibody to DUOXA2 (Anti-DUOXA2 HPA011085, Sigma-Aldrich, St Louis, MO; dilution 1:20), a polyclonal antibody to ID1 (Id1 C-20: sc-488, Santa Cruz Biotechnology; dilution 1:80), and a polyclonal antibody to BMF (PAB2443, Abnova, Taipei, Taiwan; dilution 1:50). Antigen retrieval was performed using Target Retrieval Solution, pH 9 (DAKO, Glostrup, Denmark), microwave treatment for DUOX2 Y-15; and citrate buffer, pH 6, and microwave treatment for Anti-DUOXA2 HPA011085, Id1 C-20, and PAB2443. Immunohistochemical staining was performed employing the ABC method, using the VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA). Peroxidase activity was developed with diaminobenzidine. As positive controls, human rectal tissue was used for DUOX2 and DUOXA2, pancreatic cancer tissue for ID1, and hepatocellular carcinoma tissue for BMF. For the negative controls, phosphate-buffered saline was used instead of primary antibodies. Expression in the epithelial cytoplasm was graded as follows: grade 0, negative or focally weak; grade 1, diffusely weak or focally intense; and grade 2, diffusely intense. When the nucleus was stained, the labeling index was defined as the percentage of positive cells, calculated in high-power fields of 2 or 3 foci containing about 100 contiguous epithelial cells. Frequency of hyperplasia was compared among the 3 groups with chi-square test using GraphPad Prism version 5.0c (GraphPad Software). Each expression and labeling index was compared among the 3 groups and between 2 groups with the Kruskal-Wallis and Mann-Whitney U tests, using GraphPad Prism version 5.0c (GraphPad Software). Differences were considered significant if P < 0.05.
The 57,347-element oligonucleotide microarray identified 188 upregulated and 160 downregulated genes. Among these genes, the criterion of an intensity >400 selected 20 upregulated and 31 downregulated genes (Tables 2 and 3). Further selected 6 upregulated and 5 downregulated genes were assayed by real-time RT-PCR, which confirmed upregulation in 5 genes and downregulation in 1 gene (Tables 2 and 3). Hyperplasia was found in 13 of 19 patients with pancreaticobiliary maljunction, but in none of the controls and patients with gallstones (P < 0.01). Immunohistochemistry detected proteins of DUOXA2, DUOX2, ID1, and BMF in the gallbladder epithelia (Fig. 1). Staining of DUOXA2 was observed in the cytoplasm, with a greater intensity on the apical side. DUOX2 was stained in the cytoplasm and apical membrane. Immunoreactivity of ID1 and BMF was present in the cytoplasm and, to a varying extent, in the nucleus. There was no difference in the staining pattern among the 3 groups of pancreaticobiliary maljunction, control, and gallstones. Expressions of BMF protein in both the cytoplasm and nucleus were significantly higher in the pancreaticobiliary maljunction patients than in the controls and gallstone patients (Fig. 1).
This is the first report of genomewide information on altered gene expressions in the biliary tract of children with choledochal cyst/pancreaticobiliary maljunction. The data revealed several genes already differentially expressed in children with pancreaticobiliary maljunction, including UCA1, DUOXA2, DUOX2, ID1, BMF, and GP2. The most interesting point regarding carcinogenesis is the upregulation of UCA1, urothelial cancer associated 1. UCA1 is an mRNA-like non–protein-coding RNA, which was independently found to be overexpressed in the bladder transitional cell carcinoma and doxorubicin-resistant squamous carcinoma cell lines (24–26). Although its role is not entirely clear, UCA1 is upregulated in embryonic and fetal tissues and many cancer cells; however, it is not expressed after birth in normal tissues except heart and spleen (25). This indicates that UCA1 is an oncofetal gene involving embryonic development and carcinogenesis. UCA1 has been demonstrated to promote cell proliferation and transformation (anchorage-independent growth), increase motility and invasion, and induce drug resistance in cancer cells (24–26). Tsang et al (26) demonstrated that UCA1-induced drug resistance was mediated by the suppression of apoptosis. Wang et al (24) reported that besides carcinomatous lesions, the upregulation of UCA1 is seen in chronic inflammatory proliferative lesions of the urinary tract, which are at high risk for transitional cell carcinoma. Similarly, UCA1 may be involved in the carcinogenesis of pancreaticobiliary maljunction, which produces chronic proliferative epithelia in the biliary tract. One candidate mechanism of UCA1 is resistance to apoptosis, along with the inactivation of TP53 in the late phase (18–21).
This study demonstrated that Bcl2 modifying factor (BMF) was markedly expressed at both mRNA and protein levels. BMF is a member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, which are grouped into 3 subfamilies based on the number of Bcl2 homology (BH) domains. BMF has a single BH domain, and belongs to the BH3-only proteins, which function as apoptotic activators. Each BH3-only family gene has specific apoptotic stimuli for activation (27). Because no other BCL2 family genes were differently expressed in the microarray analysis (data not shown), and also because BMF was not overexpressed in the gallbladder epithelia with gallstones (another factor predisposing to gallbladder cancer), the pathophysiology of pancreaticobiliary maljunction may involve specific stresses activating BMF. Previous reports implicated BMF in anoikis (ie, apoptosis induced by detachment from the extracellular matrix), along with the effects of arsenic trioxide, histone deacetylase inhibitors, transforming growth factor-β, and tumor necrosis factor (28). The stress pancreaticobiliary maljunction inflicts on the gallbladder epithelia is unknown, but BMF seems to be associated with increased epithelial cell turnover, characteristic of pancreaticobiliary maljunction (16,17,22). The elevated expression of BMF works contrary to carcinogenesis because BMF functions as an epithelial tumor suppressor (29). Schmelzle et al (29) reported that RAS suppresses BMF transcription in mammary cells. Gramantieri et al (30) demonstrated that microRNA-221 downregulates the expression of BMF posttranscriptionally in hepatocellular carcinoma. Similarly, KRAS mutation, reported to occur often in pancreaticobiliary maljunction, or some microRNAs are presumed to suppress BMF activation during carcinogenesis.
The upregulation of DUOXA2 and DUOX2 and downregulation of GP2 were reported in precancerous or cancer-related lesions such as colon epithelia in ulcerative colitis or airway epithelium in smokers, all of which are related to inflammation (31–35). ID1 reportedly enhances cell proliferation and inhibits cellular differentiation, both in vitro and in vivo, and can contribute to tumorigenesis (36). These genes also may be related to the pathophysiology in pancreaticobiliary maljunction, although a difference in expression was not confirmed or assessed at the protein level by immunohistochemistry.
We admit limitations of this study. We used gallbladders from patients with hepatoblastoma as controls. Interference of hepatoblastoma is possible. We could not clarify the overall process of the carcinogenesis using only the obtained data. No expressional changes relating to KRAS and TP53 genes were found in this study. KRAS mutation is infrequent during childhood (14%) (22). TP53 mutation occurs much later than KRAS mutation (23). The present data may represent altered gene expressions before KRAS and TP53 genetic abnormalities occur. However, it is significant to demonstrate that the pathophysiology of pancreaticobiliary maljunction involves a noncoding RNA and several genes related to inflammation and/or proliferation, which is presumed to be the main route of carcinogenesis caused by pancreaticobiliary maljunction. It is also important to note that these gene expressional differences begin during childhood.
We thank Ms Katayama, Mr Yamada, and Prof Nakamura at the Department of Pathology and Laboratory Medicine for technical assistance in immunohistochemistry.
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biliary carcinogenesis; choledochal cyst; microarray; noncoding RNA; pancreaticobiliary maljunction
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