Tumor Biol. (2015) 36:5831–5837 DOI 10.1007/s13277-015-3253-1
RESEARCH ARTICLE
Differential expression of the UGT1A family of genes in stomach cancer tissues Beyhan Cengiz & Onder Yumrutas & Esra Bozgeyik & Ersin Borazan & Yusuf Ziya Igci & Ibrahim Bozgeyik & Serdar Oztuzcu
Received: 6 January 2015 / Accepted: 10 February 2015 / Published online: 25 February 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Uridine 5′-diphospho-glucuronosyltransferases (UGT) are the key players in the biotransformation of drugs, xenobiotics, and endogenous compounds. Particularly, UDPglucuronosyltransferase 1A (UGT1A) participates in a wide range of biological and pharmacological processes and plays a critical role in the conjugation of endogenous and exogenous components. Thirteen alternative splicing products were produced from UGT1A gene locus designated as UGT1A1 and UGT1A3–10. A growing amount of evidence suggests that they have important roles in the carcinogenesis which is well documented by colon, liver, pancreas, and kidney cancer studies. Here, we report differential expressions of UGT1A genes in normal and tumor tissues of stomach cancer patients. Total numbers of 49 patients were enrolled for this study, and expression analysis of UGT1A genes was evaluated by the real-time PCR method. Accordingly, UGT1A1, UGT1A8, and UGT1A10 were found to be upregulated, and UGT1A3, UGT1A5, UGT1A7, and UGT1A9 were downregulated in stomach tumors. No expression changes were observed in Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3253-1) contains supplementary material, which is available to authorized users. B. Cengiz Department of Medical Biology, Faculty of Medicine, Gazi University, Ankara, Turkey O. Yumrutas : I. Bozgeyik Department of Medical Biology, Faculty of Medicine, Adiyaman University, Adiyaman, Turkey E. Bozgeyik (*) : Y. Z. Igci : S. Oztuzcu Department of Medical Biology, Faculty of Medicine, Gaziantep University, 27310 Gaziantep, Turkey e-mail:
[email protected] E. Borazan Department of General Surgery, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey
UGT1A4. Also, UGT1A6 transcription variants were significantly upregulated in stomach cancer tissues compared to normal stomach tissue. Additionally, UGT1A7 gene showed highest expression in both normal and tumoral tissues, and interestingly, UGT1A7 gene expression was significantly reduced in stage II patients as compared to other patients. In conclusion, UGT1A genes are differentially expressed in normal and tumoral stomach tissues and expression changes of these genes may affect the development and progression of various types of cancer including the cancer of the stomach. Keywords Gene expression . Stomach cancer . UDP-glucuronosyltransferases . UGT1A . UGT1A6
Introduction Uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes play crucial roles in the biotransformation of drugs, xenobiotics, and endogenous compounds. They are located on the endoplasmic reticulum membrane [1]. UDPglucuronosyltransferase 1A (UGT1A) includes a wide range of biological and pharmacological processes and plays a critical role in the conjugation of endogenous and exogenous components [1]. Xenobiotic metabolism is considered in two main stages; phase I is the process of hydroxylation by cytochrome p450, and phase II is the process of conjugation of the hydroxylated species with hydrophilic compounds [2]. Thus, the lipophilic compounds are removed from the body by becoming watersoluble [3]. Also, glucuronidation reaction takes place at the second phase and is the most common conjugation reaction [2]. UDP-glucuronic acid is a cofactor of UGTs which is required in the glucuronidation reaction. These reactions are catalyzed by the UDP-glycosyltransferases which are ubiquitously expressed at several tissues
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including the liver, kidney, colon, skin, brain, spleen, and nasal mucosa [4]. UTGs are consisted of two main subfamilies of genes. UGT2 is subdivided into two groups as UGT2A and UGT2B [5]. The UGT1A gene family is located at the 2q37 chromosomal region and comprises a total of 17 exons. Two to five exons are combined with the exon 1 to form the alternative UGT1A gene products [6]. There are 13 alternative splicing products encoded from UGT1 gene locus. Among these, nine of them (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10) encode functional protein products and the rest of them (UGT1A11P, UGT1A12P, UGT1A13P, UGT1A2P) are pseudogenes. Stomach cancer is one of the most common types of cancer and is responsible for the numerous cancer-related deaths worldwide [7]. Although the etiology of stomach cancer is not undoubtedly known, several factors including age, gender, diet, genetic predisposition, adenomatous polyps, familial polyps, some microorganisms (Helicobacter pylori), smoking, and alcohol habits increase the risk of developing disease. As well, a family history of cancer and any stomach disease also increases the risk of developing stomach cancer. A growing number of investigations suggest that UGT1A genes have important role in the gastric carcinogenesis [8–11]. Several studies assessed the role of the UGT1A gene family in colon [8], liver [11], pancreas [9], and kidney cancers [12], and differential expressions of UGT1A genes have been well reported. However, in studies conducted so far, the role of the UGT1A family of genes which participates in diverse biological and pharmacological processes has not been evaluated in stomach carcinogenesis yet. Moreover, as it is indicated above, UGT1A is widely expressed in gastrointestinal tract tissues. Accordingly, determining the differential expression of UGT1A genes in stomach cancer tissues is very important. Therefore, previous studies in this field have much focused on the polymorphic expression of UGT1A genes. Additionally, the relationship between UGT1A genes and stomach cancer has not been evaluated yet. The aim of the present study is to investigate the role of the UGT1A family of genes in the stomach carcinogenesis by evaluating the expression levels of the UGT1A family of genes.
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enrolled. Normal stomach tissue of patients was used as control. Demographic characteristics and clinical findings of patients are presented in Tables 1 and 2, respectively. Isolation of total RNA from fresh tissue samples Surgical tissue materials of patients were preserved in the liquid nitrogen until RNA isolations. Isolation of total RNA samples was achieved by using QIAamp RNA Kit (QIAGEN Sample and Assay Technologies, Germany) according to manufacturer’s instructions. To homogenize tissue samples, Qiazol reagent (QIAGEN Lysis Reagent, 79306) from QIAGEN was used. Concentrations of RNA samples were determined by using NanoDrop ND-1000 Spectrophotometer. RNA samples were subsequently converted to complementary DNAs (cDNAs). cDNA synthesis RNA samples were further converted into cDNAs by using ProtoScript® First Strand cDNA Synthesis Kit (New England Biolabs). Briefly, the following reaction mixture was prepared for each RNA sample: 0.5–1 μg total RNA, 2 μl Oligo d(T) 23 VN (50 μM) primer, and nuclease-free water. Reaction mixtures were incubated at 65 °C for the denaturation of secondary and tertiary structures of RNA. Following the incubation, reverse transcriptase buffer (10 μl) and enzyme mix (2 μl) were added to reaction tubes and subjected to the following thermal conditions: 1 h at 42 °C and 5 min at 80 °C. cDNA samples were stored at −20 °C in equal aliquots. Table 1
Demographic characteristics of patients
Parameter
Subtype
Patients n (%)
Age
65 Male Female No Rare No Gastritis Peptic ulcer Gastroesophageal reflux disease Hiatal hernia Other Yes No Yes No
7 (14.3) 24 (49.0) 18 (36.7) 33 (67.3) 16 (32.7) 43 (87.8) 6 (12.2) 21 (42.9) 12 (24.5) 8 (16.3) 5 (10.2)
Sex Alcohol intake Stomach disease
Material and methods Study groups and sample collection The study was approved by the local ethics committee in accordance with the Helsinki declaration. Also, all study participants gave an informed consent for participation. For the study, a total of 33 male (61.27±11.6) and 16 female (57.75± 20.17) patients diagnosed with the stomach cancers were
Gastrointestinal bleedings Stomach cancer in the family
0 (0.0) 3 (6.1) 45 (91.8) 4 (8.2) 46 (93.9) 3 (6.1)
Tumor Biol. (2015) 36:5831–5837 Table 2
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Clinical findings of patients
Parameter
Subtype
Patients n (%)
Diagnostic stage
Stage I
3 (6.1)
Stage II
7 (14.3)
Stage III Stage IV
8 (16.3) 31 (63.3)
Small curvature Antrum
6 (12.2) 13 (26.5)
Cardia, fundus
8 (16.3)
Greater curvature Multiple regions
5 (10.2) 15 (30.6)
Whole stomach Adenocarcinoma, outside signet ring Signet ring
2 (4.1) 30 (61.1)
Mix type
3 (6.1)
Evaluation of HER2 by IHC
Negative Positive
40 (81.6) 9 (18.4)
Serum CEA levels
10 ng/ml
32 (65.3) 17 (34.7)
Serum CA 19–9 levels
37 U/ml
36 (73.5) 13 (26.5)
Tumor localization
Histological type
16 (32.7)
CEA carcinoembryonic antigen, CA 19–9 cancer antigen 19–9
Real-time PCR (qRT-PCR) For the qRT-PCR quantifications, SYBR Green Mix from Thermo Scientific was used. Primers for the UGT1A gene locus were designed at NCBI database, and ACTB (β-actin) was used as internal control (Supplementary Table 1). A different set of primers was designed for the transcription variants of UGT1A6. All real-time PCR quantifications were held in LightCycler® 480 (Roche AB, Stockholm, Sweden). Statistical analysis Result of the qRT-PCR was obtained as Ct values. Ct values were analyzed according to the following formula: 2−ΔCt(ΔCt = CtTarget Gene − CtReference Gene) [13]. Statistical comparison was tested by Wilcoxon-Kruskal-Wallis H tests. For the comparison of multiple groups, ANOVA test was applied. All statistical test was two-tailed and p
