Parasitol Res (2011) 109:657–673 DOI 10.1007/s00436-011-2298-3
ORIGINAL PAPER
Candidate genes involving in tumorigenesis of cholangiocarcinoma induced by Opisthorchis viverrini infection Zhiliang Wu & Thidarut Boonmars & Sirintip Boonjaraspinyo & Isao Nagano & Somchai Pinlaor & Anucha Puapairoj & Puangrat Yongvanit & Yuzo Takahashi
Received: 7 December 2010 / Accepted: 17 February 2011 / Published online: 5 March 2011 # Springer-Verlag 2011
Abstract Opisthorchiasis-associated cholangiocarcinoma (CCA) is one of main public health problems in Opisthorchis viverrini endemic areas. Although the definite relationship between prevalence of CCA and the parasite infection has been demonstrated, the molecular mechanism of tumorigenesis is still unknown. In the present study, by using animal model of opisthorchiasis-associated CCA, a kinetic analysis of cDNA microarray was performed to screen the candidate genes that involve in the development of opisthorchiasis-associated CCA. Microarray analysis revealed that the expressions of 131 genes were up-
Co-first author: Thidarut Boonmars Z. Wu (*) : I. Nagano : Y. Takahashi Department of Parasitology, Gifu University Graduate School of Medicine, Yanagido1-1, Gifu 501-1194, Japan e-mail:
[email protected] T. Boonmars : S. Boonjaraspinyo : S. Pinlaor Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand T. Boonmars : S. Boonjaraspinyo : S. Pinlaor : P. Yongvanit Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand A. Puapairoj Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand P. Yongvanit Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
regulated during the development of CCA, including the genes relative to cell proliferation, differentiation and transformation, cell growth and cycle regulation, apoptosis, DNA repair, and cytoskeletal structure. The expressions of 145 genes were down-regulated, including the genes relative to metabolic enzymes, tumor suppressor, apoptosis, and oxidative response and oxidation reduction. The present study listed up the candidate genes involving tumorigenesis, provided molecular information on the development of opisthorchiasis-associated CCA and the potential biomarkers for diagnosis and therapy, and suggested that the increased expression of cell differentiation, proliferation, transformation-related genes, and decreased expression of metabolic enzymes may play important roles in the tumorigenesis of CCA.
Introduction Cholangiocarcinoma (CCA) is a rare malignancy but highly fatal disease originating from the neoplastic transformation of biliary epithelium, which can be intrahepatic and extrahepatic (Blechacz and Gores 2008). As the second most common primary hepatic malignancy, its incidence and mortality rates are increasing worldwide in recent decades (Khan et al. 2005; Ustundag and Bayraktar 2008; Mosconi et al. 2009). Many risk factors contribute to CCA development, including primary sclerosing cholangitis (Abbas and Lindor 2009), congenital biliary cystic diseases, liver cirrhosis, and viral infections (Su et al. 1997), exposed thorotrast and nitrosamine (Mitacek et al. 1999a; Lipshutz et al. 2002), and parasitic liver diseases such as Opisthorchis viverrini (OV) and Clornorchis sinensis (IARC 1994; Kim et al. 2009). In southeastern Asia, CCA is becoming a major health problem, especially in northeastern of Thailand where is a
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heavy endemic area of O. viverrini with the highest prevalence of CCA in the world (Sriamporn et al. 2004; Poomphakwaen et al. 2009). The evidence from epidemiologic investigations and animal experiments has confirmed the definite relationship between O. viverrini infection and prevalence of CCA, combining with the traditional eating custom of fermented food containing potent carcinogens, for example, N-nitrosodimethylamine (NDMA; Sriamporn et al. 2004; Poomphakwaen et al. 2009; Mitacek et al. 1999b; Boonmars et al. 2009b). Efforts have been made for revealing the molecular mechanism of tumorigenesis of opisthorchiasis-associated CCA. Some genes or signaling pathway have been found to be likely involved in the tumorigenesis or potential as biomarkers, for example, KRAS and TP53 (Tangkawattana et al. 2008), RB1 signaling pathway genes (Boonmars et al. 2009b), annexin A2, MMP-9 (Yonglitthipagon et al. 2010), and IL-6 (Sripa et al. 2009). However, most of these approaches used patient tissues that are usually in late stage of CCA. The knowledge on the molecular mechanism during the tumorigenesis of CCA is still limited. Meanwhile, early diagnosis and treatment of CCA are now still unsatisfied because, in the majority of the cases, CCA is clinically silent with symptoms only developing at an advanced stage. The only effective treatment is the radical surgical resection, and survival rate is still quite low (Farley et al. 1995). Therefore, urgent efforts are needed to determine the gene expression profile and the molecular mechanism of tumorigenesis during CCA development induced by O. viverrini infection, which will be useful for identifying reliable biomarkers that will facilitate the early detection of OV-associated CCA, and for identifying the cancer-specific cellular targets that would provide the basis for novel therapeutic approaches. An animal model for opisthorchiasis-NDMA-associated CCA has been established (Thamavit et al. 1987, 1993) and this model is now only available one for OV infectioninduced CCA. In this model, infection with O. viverrini or administration with NDMA only does not induce formation of CCA, but combination of infection and NDMA administration dose cause the development of CCA. Global gene expression technology has become a powerful and efficient way to identify candidate genes with altered expression profiles in malignancies. In the present study, we employed OV-associated CCA animal model and investigated the kinetic expression of genes during tumorogenesis procedure (1, 3, and 6 month post infection) using cDNA microarray analysis (44 k, Agilent). By comparing the gene profiles among four groups (normal, OV infection, NDMA administration, and OV infection plus NDMA administration), the expression alterations of many genes related to cell growth, differentiation, proliferation, transformation, cell cycle, apoptosis, oxidative stress and antioxidation, and metabolism were observed. Some of these
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genes are well known and some have not been previously reported in CCA or other cancers, which may serve as potential diagnostic markers and therapeutic targets for OV infection-associated CCA.
Material and methods Parasite and infection Metacercariae of O. viverrini were collected as the previously described (Boonmars et al. 2007, 2009a, b). In brief, naturally infected cyprinoids fish captured from a fresh water reservoir in an endemic area of Khon Kaen, Northeast Thailand, were minced and digested with pepsinHCl, filtrated, and then washed with saline. Afterwards, the Opisthorchis metacercaria which was 204×145 μm, ovalshaped, with large, black excretory bladders were identified under a dissecting microscope. Sixty Syrian hamsters (6 weeks) were divided in to four groups (n=15 per group): (1) normal group; (2) infection group—15 hamsters were infected with 50 metacercariae; (3) NDMA group—15 hamsters were maintained with drinking water containing 12.5 ppm of NDMA (Wako, Japan) for 2 months; and (4) infection plus NDMA group—15 hamsters were infected with 50 metacercariae and maintained with drinking water containing 12.5 ppm of NDMA for 2 months. Hamster livers (five hamsters at each time point for each group) were collected at 1, 3, and 6 month post infection (p.i.) for total RNA isolation and histological observation. Histological examination One part of tissues from hilar region of liver was fixed with 10% formalin solution. Paraffin sections were prepared for hematoxylin and eosin (H&E) staining, and histological examination was performed for confirmation of pathology and CCA development in the animal model. RNA isolation Total RNA was extracted from the tissues (200 mg) at the same region (hilar region of liver) from the four groups mentioned above (normal, infection, NDMA, and infection plus NDMA) using TRIZOL (Invitrogen, Carlsbad, CA, USA) according to the manufacturer instructions. The isolated RNA was treated with DNase (RQ1 RNase-Free DNase, Promega, Co., Madison, WI, USA). The treated RNA was purified using conventional method and dissolved in RNase-free water. RNA purity were determined spectrophotometrically (1.8–2.0 in ratio of OD260/OD280) and by formaldehyde agarose gel electrophoresis (more than 1.6 in ratio of 28SrRNA/18SrRNA).
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Microarray and data analysis
which showed significant differential expression after O. viverrini infection and may be promising candidate biomarkers in CCA were selected. Out of these genes, four pair of primers failed to amplify and the other 14 pair of primers was used in the following real time PCR. The primer information was shown in Table 1. Total RNA was isolated and purified as mentioned above. Reverse transcription was performed using a Prime Reverse Transcriptase (TAKARA BIO Inc., Ootsu, Shiga, Japan) according to the manufacturer's instructions. Real time PCR was performed as previous described (Boonmars et al. 2009a). In brief, optimal conditions for all investigated genes were established using SYBR Premix Ex Taq Kit (TAKARA BIO) according to manufacturer's instructions. Twenty microliters of the reaction solution consisted of 2 μl of the template (appropriate dilution was determined by genes), 10 μl of SYBR Premix Ex Taq, 0.8 μl of 5 μM of each primer. PCR amplification was performed as follows: predenature for 1 cycle at 95°C for 30 s, and 40 cycles at
One-color microarray-based gene expression analysis was performed with Whole Mouse Genome (4X44K) Oligo Microarray (Agilent Technologies, USA) which contained 44,000 genes and transcripts with one 60-mer oligonucleotide probe representing each sequence in each chip, and four chips in one slideglass, according to manufacturer's instruction. In brief, cDNA synthesis and cRNA synthesis with incorporation of Cyanine 3-CTP were performed using the Agilent Low RNA Input Linear Amplification Kit (Agilent). After purification and quantization, the cRNA was fragmented and hybridized on the Agilent microarrays at 65°C for 17 h. The hybridized microarrays were washed with Gene Expression Wash Buffer (Agilent). The processed microarrays were scanned with the Agilent DNA microarray scanner (G2565BA, Agilent). Nine 4X44K array slides (one slide contains four array) were performed with 36 independent samples, including three samples from each group (normal, infection, NDMA, and infected plus NDMA) at each observed time point (1, 3, and 6 month p.i). One array slide was hybridized with four samples of uninfected normal, infection, NDMA, and infected plus NDMA at same time point. The microarray data were processed, normalized, and statistically analyzed with the Agilent G2567AA Feature Extraction Software (v8.5) using the setting for Agilent 4X44K array to the Agilent protocol. Files and images, including error values and P values, were exported from the Agilent Feature Extraction software. Spots that did not pass quality control procedures in this software were flagged and removed from further analysis. The fold change values for the differentially expressed genes were calculated from ratios of intensities between infection/ normal, NDMA/normal, infetction + NDMA/normal in same slide. The t test p values were utilized to detect the significance of differences between the two test groups. Log ratios of intensities for individual genes were determined for each test/control sample, from which the mean value of log ratios for each sample group was also obtained. The genes with an average fold change greater than 2.0 or less than 0.5 and P values
