ShijiazhuangChina Published online: 16 May 2015.

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1 This article was downloaded by: [ ] On: 13 July 2015, At: 23:47 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: 5 Howick Place, London, SW1P 1WG Biomarkers Publication details, including instructions for authors and subscription information: Hsa-miR-196a2 functional SNP is associated with the risk of ESCC in individuals under 60 years old Na Wang a, Yan Li a, Rong-Miao Zhou a, Gui-Ying Wang b, Chun-Mei Wang c, Zhi-Feng Chen d & Wei Liu e a Department of Molecular Biology b Department of Surgery, The Fourth Hospital of Hebei Medical University ShijiazhuangChina c School of Life Sciences, Chinese University of Hong Kong Hong Kong SARChina d Department of Cancer Prevention and Control e Department of Medical Oncology, The Fourth Hospital of Hebei Medical University ShijiazhuangChina Published online: 16 May To cite this article: Na Wang, Yan Li, Rong-Miao Zhou, Gui-Ying Wang, Chun-Mei Wang, Zhi-Feng Chen & Wei Liu (2014) HsamiR-196a2 functional SNP is associated with the risk of ESCC in individuals under 60 years old, Biomarkers, 19:1, To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at

2 ISSN: X (print), (electronic) Biomarkers, 2014; 19(1): 43 48! 2014 Informa UK Ltd. DOI: / X RESEARCH ARTICLE Hsa-miR-196a2 functional SNP is associated with the risk of ESCC in individuals under 60 years old Na Wang 1, Yan Li 1, Rong-Miao Zhou 1, Gui-Ying Wang 2, Chun-Mei Wang 3, Zhi-Feng Chen 4, and Wei Liu 5 1 Department of Molecular Biology and 2 Department of Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China, 3 School of Life Sciences, Chinese University of Hong Kong, Hong Kong SAR, China, 4 Department of Cancer Prevention and Control, and 5 Department of Medical Oncology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China Abstract Background and aim: The mir-196a2 gene contains a C/T polymorphism (rs ). Its presence could change the conformation of secondary structure of mir-196a2 RNA, and directly affect the binding to target mrnas and the mirna maturation process. Both of which eventually alter protein expression and contributed to cancer susceptibility. This study assessed whether the rs single nucleotide polymorphism (SNP) could affect an individual s susceptibility to esophageal squamous cell carcinomas (ESCC). Methods: SNP rs was genotyped by polymerase chain reaction ligase detection reaction (PCR-LDR) in 597 ESCC patients and 597 control subjects. Results: Overall, there were no significant differences in the frequency of the mirna-196a2 SNP rs genotype between the ESCC cases and the controls ( 2 ¼ 1.395, p ¼ 0.498). The TT genotype, CT genotype and CT/TT combined genotype (dominant model) did not modify the risk of ESCC as compared with the CC genotype. Comparisons of the TT genotype to the CT/CC combined genotype did not reveal a significant association to ESCC, too. However, further analyses revealed an increased risk of ESCC in the dominant model (OR ¼ 1.56, 95% CI ¼ ) and the allele frequency comparison (OR ¼ 1.31, 95% CI ¼ ) in the 60-year-old group. Conclusions: These results suggest that the mirna-196a2 functional polymorphism rs might be an effective genetic marker for ESCC risk assessment in individuals younger than 60 years of age from a region of high ESCC incidence in northern China. Introduction Esophageal cancer is the eighth most common cancer and the sixth leading cause of cancer-related death worldwide. In 2008, an estimated new cases and deaths occurred (Jemal et al., 2011). The highest rates of esophageal squamous cell carcinomas (ESCC) over the world occur in Asian countries, especially China, Iran and Japan. The highest risk area, which ranges from northern Iran, across the central Asian republics to northern China, is referred to as the esophageal cancer belt. Although studies reported that poor nutritional status, low intake of fruits and vegetables and drinking beverages at high temperatures all contribute to the occurrence of ESCC (Islami et al., 2009a,b; Wu et al., 2009), inheritance is now also recognized as an important factor (Guohong et al., 2010). mir-196 is a non-coding microrna expressed in humans and mice (Lagos-Quintana et al., 2003; Lim et al., 2003). Address for correspondence: Wei Liu, Department of Medical Oncology, The Fourth Hospital of Hebei Medical University, Jiankang Road 12, Shijiazhuang , Hebei Province, China. Tel: Fax: , newhebeiliuwei@163.com Keywords Esophageal squamous cell carcinoma, mir-196a2, polymorphism, rs History Received 12 September 2013 Revised 11 November 2013 Accepted 12 November 2013 Published online 10 December 2013 The human mir-196 family includes three members (mir- 196a1, mir-196a2 and mir-196b), which are transcribed from three different genes (HOXB, HOXC and HOXA) located on chromosomes 17q21.3, 12q12-q13 and 7p15.2, respectively (He et al., 2011; Tanzer et al., 2005). High expression of mir-196a is associated with tumor recurrence, nodal metastasis and mortality (Liu et al., 2013). Studies have shown the oncogenic potential of mir-196a is due to its growth-promoting and anti-apoptotic ability in esophageal cancer cell lines (Luthra et al., 2008; Maru et al., 2009), and mirna-196a expression levels were significantly higher in esophageal adenocarcinoma (EA) than in their respective paired normal mucosal tissues (Luthra et al., 2008; Maru et al., 2009). Furthermore, several published studies (Haiart et al., 2011; Luzna et al., 2011; Maru et al., 2009) have also demonstrated that mir-196a expression levels were consistent with the lengths of the Barrett s esophagus (BE) segment and gradually increase in expression from normal squamous mucosa, to BE to EA. The hsa-mir-196a2 is composed of two different mature mirnas (mir-196a and mir196a*), which are processed from the same stem-loop (Hoffman et al., 2009). A common genetic variant (rs ) lies in the mature sequence of 2014

3 44 N. Wang et al. Biomarkers, 2014; 19(1): mir-196a* and is the result of a change from G:C to G:T in the stem region of the hsa-mir-196a2 precursor. Studies have shown that this polymorphism might influence mir-196a expression levels by affecting the processing of pre-mirna to its mature form (Hoffman et al., 2009). Previous studies reported positive associations between this variant and various types of cancers, including breast (Hu et al., 2009), colorectal (Lv et al., 2013; Zhan et al., 2011; Zhu et al., 2012), gastric (Peng et al., 2010), liver (Li et al., 2010a) and lung cancer (Tian et al., 2009) in the Chinese population. Therefore, single nucleotide polymorphism (SNP) rs is a strong candidate for a genetic marker of cancers. To date, three studies examined the association between rs polymorphisms and the susceptibility to esophageal cancer (Wang et al., 2010; Ye et al., 2008). Ye et al. (2008) first performed a case control study to assess the association between this SNP and esophageal cancer susceptibility in a population without restrictions on recruitment criteria for age, sex and ethnicity. Subsequently, two studies (Wang et al., 2010; Wei et al., 2013) reported an association between SNP rs and susceptibility to ESCC in individuals from a subset of the Chinese population with a much lower ESCC incidence. However, these findings were inconsistent and have not been confirmed in other independent studies in populations from high-incidence regions. Thus, we conducted a population-based case control study to investigate the association between SNP rs and the risk of ESCC in 597 ESCC patients and 597 control subjects of Chinese Han descent. Materials and methods Study subjects This case control study recruited 597 ESCC patients and 597 unrelated healthy control subjects during an endoscopic screening campaign between 2006 and All the study subjects were ethnically homogeneous of Han descent and permanent residents of Cixian county. The patients had histologically confirmed ESCC; self-reported, cancer-free subjects who were confirmed to be without upper gastrointestinal cancer (UGIC) by endoscopies were selected as the healthy controls. Two professional interviewers obtained information about sex, age, smoking habit and family history immediately following blood donation. Eventually, 597 healthy controls that were age-, gender- and smoking-matched to the ESCC cases were enrolled in the study. We defined smokers as having formerly been or currently smoking 5 cigarettes/day for at least 2 year. The definition of a family history of UGIC was individuals with at least one first-degree relative or at least two second-degree relatives with esophageal/cardiac/gastric cancer. The Ethics Committee of Hebei Cancer Institute approved the study and informed consent was obtained from all recruited subjects. DNA extraction Five milliliters of venous blood from each subject was drawn into Vacutainer tubes containing EDTA and stored at 4 C. After sampling, genomic DNA was extracted within 1 week by using proteinase K (Merck, Darmstadt, Germany) digestion followed by a salting out procedure according to the method published by Miller et al. (1988). Polymorphism genotyping The genotyping of mir-196a2 rs polymorphism (Shanghai Generay Biotech Co., Ltd. ( com.cn)) used polymerase chain reaction ligase detection reaction (PCR-LDR) method. The primers for amplification were F5 0 -TCTGTGGCTTAGGTAGTTTCATG-3 0 and R5 0 - CACTCACAGCTTGTCCTCCTT-3 0. PCRs were carried out in a total volume of 15 ml including 50 ng genomic DNA, 1.5 ml 10 PCR buffer, 1.5 ml of 25 mm MgCl 2, 0.3 ml of 10 mm dntps, 0.25 ml of 10 pmol/ml primers (Forward and Reserve) and 1.25 U of Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China). Cycling parameters were as follows: 94 C for 2 min, 35 cycles of 94 C for 15 s, 55 C for 15 s, 72 C for 25 s and a final extension step at 72 Cfor 3 min. Three LDR probes synthesized for each SNP locus included two SNP specific probes and one common probe. The two SNP specific probes used to discriminate the specific bases were 5 0 -TTTGAACTCGGCAACAAGAAACTGC-3 0 and 5 0 -TTTTTTGAACTCGGCAACAAGAAACTGT-3 0. The common probe was 5 0 -P-CTGAGTTACATCAGTCGGTTT TCGT-FAM-3 0, which was phosphorylated at the 5 0 end and labeled at the 3 0 end with 6-carboxyfluorescein (FAM). LDR reactions were performed in a 10-ml reaction volume containing 3 ml of PCR product, 1 ml of 10 Taq DNA ligase buffer, 0.01 ml of 10 pmol/ml each primers and 5 U Taq DNA ligase (New England Biolabs, Beijing, China). LDR parameters were as follows: 25 cycles of 94 C for 30 s and 58 C for 1 min. After the LDR reaction, 1 ml LDR reaction product was mixed with 10 ml loading buffer, which contained a DNA marker. The mixture was denatured at 95 C for 3 min, chilled immediately in ice water and analyzed on an ABI 3730XL DNA sequencer. Direct DNA sequencing the representative PCR products to confirm the accuracy of this method yielded 100% concordant results. Statistical analysis Statistical analysis was performed using the SPSS 20.0 software package (SPSS Company, Chicago, IL). Hardy Weinberg equilibrium analysis was performed using a goodness-of-fit 2 -test with one degree of freedom. p50.05 was considered representative of a departure from HWE. Comparison of allelotype and genotype distributions in the patients and healthy controls were performed by means of two-sided contingency tables using the 2 -test. The odds ratio (OR) and 95% confidence interval (CI) for genotypic-specific risk were calculated using an unconditional logistic regression model and adjusted by age, sex, smoking status and family history of UGIC accordingly. p50.05 was considered significant for all statistical analyses. Results Subject characteristics Table 1 presents the demographic characteristics of ESCC patients and healthy controls. The mean age of healthy controls and ESCC patients were years (range

4 DOI: / X Hsa-miR-196a2 functional SNP with risk of ESCC ) and years (range 32 85), respectively. The gender distribution in both groups was the same (390 men and 207 women). The proportion of smokers among healthy controls was comparable to that among ESCC patients (40.7% versus 43.0%). No statistically significant differences were found in the distribution of mean age, gender and smoking status between the ESCC patients and their healthy controls (p40.05), suggesting the frequency matching was adequate. In addition, the frequency of a positive family history of UGIC among ESCC (47.6%) patients was significantly higher than that in healthy controls (38.4%) ( 2 ¼ 10.34, p50.01), suggesting a family history of UGIC would increase the risk of ESCC (the age- and sex-adjusted OR ¼ 1.46, 95% CI ¼ , data were not shown). Association between the mir-196a2 rs polymorphism and the risks of ESCC The genotype distribution of mir-196a2 rs polymorphism in the healthy controls did not deviate from the Hardy Weinberg equilibrium ( 2 ¼ 0.001, p ¼ 0.97). The rs T-allele frequency of ESCC patients and Table 1. Demographic characteristics in ESCC and healthy individuals. Variables Control n (%) ESCC n (%) 2 -test p Value Gender Male 390 (65.3) 390 (65.3) Female 207 (34.7) 207 (34.7) Mean age (SD), Years a Smoking status Non-smoker 354 (59.3) 340 (57.0) Ex- or current smoker 243 (40.7) 257 (43.0) Family history of UGIC Negative 368 (61.6) 313 (52.4) Positive 229 (38.4) 284 (47.6) Genotyping of rs CC 145 (24.3) 128 (21.5) CT 298 (49.9) 307 (51.4) TT 154 (25.8) 162 (27.1) ESCC, esophageal squamous cell carcinoma; UGIC, upper gastrointestinal cancer. a t Test. the controls were 52.85% and 50.75%, respectively. There were no significant associations between rs polymorphism and ESCC risk in all the genetic models (T-allele versus C-allele: OR ¼ 1.09; 95% CI, ; CT versus CC: OR ¼ 1.17; 95% CI, ; TT versus CC: OR ¼ 1.20; 95% CI, ; dominant model: OR ¼ 1.18; 95% CI, ; recessive model: OR ¼ 1.07; 95% CI, ). To examine whether epidemiological factors modify the genetic variant, we performed stratified analyses based on gender, smoking status and family history of UGIC, and observed the similar results (Table 2). However, when stratified by age group, it was found that T allele was the dominant allele of ESCC patients in the groups of age 60 years (TT þ CT versus CC: OR ¼ 1.56; 95% CI, ) but not in the group of age 460 years (OR ¼ 0.82; 95% CI, ). Based on these OR results and 95% CI of the two age groups, we tested the gene age interaction (Altman & Bland, 2003; Wen et al., 2009) and estimated the odds ratio (OR ¼ 1.90; 95% CI, ), which offered good evidence to support a different risk effect in the people younger or older than 60 years. Discussion In this molecular epidemiological study of ESCC within a high-risk Chinese population, we observed no association between overall ESCC risk and SNP rs using logistic regression adjusted for age, gender, family history of UGIC and smoking status. To further explore the potential association, we performed subgroup analysis and found that the T allele was a dominant allele of ESCC patients in the group of age 60 years (TT þ CT versus CC: OR ¼ 1.56; 95% CI, ) but not in the group of age 460 years (OR ¼ 0.82; 95% CI, ). We tested the gene age interaction and found that SNP rs played a different role in these two age groups. These results indicate there may be a relationship between SNP rs and genetic predisposition of ESCC patients within different age groups. Studies have shown that ESCC patients with a family history of UGIC were significantly younger at onset and had a lower survival rate than in sporadic cases. These results suggest the Table 2. Genotype specific risks (ORs and 95% CI) of SNP rs for ESCC and controls. Group No. of case/controls T-allele versus C-allele CT versus CC TT versus CC Dominant model (TT þ CT versus CC) Recessive model (TT versus CT þ CC) Overall 597/ ( ) 1.17 ( ) 1.20 ( ) 1.18 ( ) 1.07 ( ) Age / ( ) 1.49 ( ) 1.69 ( ) 1.56 ( ) 1.30 ( ) / ( ) 0.86 ( ) 0.74 ( ) 0.82 ( ) 0.83 ( ) 1.51 ( ) 1.73 ( ) 2.28 ( ) 1.90 ( ) 1.57 ( ) Gender Male 390/ ( ) 1.30 ( ) 1.08 ( ) 1.22 ( ) 0.90 ( ) Female 207/ ( ) 0.98 ( ) 1.53 ( ) 1.13 ( ) 1.56 ( ) Smoking status Non-smoker 340/ ( ) 1.13 ( ) 1.22 ( ) 1.16 ( ) 1.12 ( ) Smokers 257/ ( ) 1.24 ( ) 1.19 ( ) 1.23 ( ) 1.03 ( ) Family history of UGIC Negative 313/ ( ) 1.36 ( ) 1.26 ( ) 1.32 ( ) 1.02 ( ) Positive 284/ ( ) 0.97 ( ) 1.16 ( ) 1.04 ( ) 1.17 ( ) ORs were all adjusted for sex, age, family history of UGIC and smoking status using logistic regression except allele frequency comparison (C-allele versus T-allele).

5 46 N. Wang et al. Biomarkers, 2014; 19(1): Table 3. Epidemiological studies of rs SNP and risk of esophageal cancer. First author Year Country Racial descent Genotyping Source of control Cases n (%) Controls n (%) Sample size (case/control) CC CT TT CC CT TT Ye 2008 USA Mixed SNPlex assay HB 307/ (27.0) 141 (46.0) 83 (27.0) 106 (31.4) 173 (51.2) 59 (17.5) Wang 2010 China Asian SNaPshot assay HB 458/ (32.3) 262 (57.2) 48 (10.5) 128 (26.2) 250 (51.1) 111 (22.7) Wei 2013 China Asian SNPscanÔ HB 367/ (17.7) 196 (53.4) 106 (28.9) 87 (23.5) 170 (45.9) 113 (30.5) Author 2013 China Asian PCR-LDR PB 597/ (21.5) 307 (51.4) 162 (27.1) 145 (24.3) 298 (49.9) 154 (25.8) PCR-LDR, Polymerase Chain Reaction-Ligation Detection Reaction; HB, Hospital-Based Case Control Study; PB, Population-Based Case Control Study. Figure 1. ORs and 95% CI of dominant model and recessive model between SNP rs and esophageal cancer. effect of genetic predisposition was stronger in younger cases (Wen et al., 2009). In our subgroup analyses, we found that a mismatch between family history of UGIC in ESCC patients and healthy control subjects only appeared in the groups of age 60 years, but not in the 460-year group (Table 4). These mismatched findings provide strong supporting evidence for a different effect of genetic predisposition in younger and older populations. Three published studies have explored the association between mir-196a2 rs polymorphisms and the risk of esophageal cancer (Table 3 and Figure 1). Ye et al. (2008) first reported the relationship between SNP rs and susceptibility to esophageal cancer, and found that this SNP was associated with an increased ESCC risk in recessive genetic model (TT versus CT þ CC). Our results showed a significant association (dominant genetic model, TT þ CT versus CC) only in the group of age 60 years. Although the results of these two studies both indicated that T allele might play a role in the development of esophageal cancer, the degree of the role was different. The variance could partially be explained by differences in race and histology. In of the study conducted by Ye et al., the samples were not restricted on recruitment criteria for age, sex and ethnicity. In addition, the histological type of the cases was almost all adenocarcinomas (85.5%), with only a small number of cases squamous cell carcinoma. It is worth mentioning that our results were the opposite to that of another published study (Wang et al., 2010), which evaluated the relationship between SNP rs and ESCC in the Chinese population and concluded that the C-allele was a dominant allele of ESCC patients. However, this conclusion could not be confirmed by the subsequent independent studies of Wei et al. (2013) or our study. However, the results of Wei et al. s study and our study are in good agreement. Although in these three studies, all subjects were of Han descent, and all the controls were frequency matched to the ESCC cases on age and sex, some differences were present. First, the source of controls was different (a hospital-based study and a population-based study); second, the ESCC incidence was different in southeast China, southwest China and north China (a low-risk area and a high-risk area). These two reasons may account for the inconsistent results found in the three studies. Our research held some key benefits in three aspects. Firstly, our study design was a population-based design, with a large number of cases and detailed information on multiple exposures in the high-incidence areas of ESCC. All the ESCC patients and controls were of Han descent and permanent residents of Cixian county (a high-risk area for ESCC in north China), which indicated that all the subjects were the same race and had the same genetic/environmental background. Compared with a hospital-based design, the population-based design in this study was more rigorous and the results more reliable. Secondly, we matched all the cases and controls on age, gender and smoking status using stratified random sampling method. As is well known, ESCC occurs most often in men over 50 years old, and tobacco smoking is one of the major environmental factors associated with an increased risk of ESCC (Radojicic et al., 2012; Wen et al., 2006, 2009). Studies have shown that gender differences in the proportion of ESCC cases were attributable to tobacco smoking. Therefore, we did an age-, sex- and smoking-matched case control study, which eliminated the effects of confounding factors as much as possible. Thirdly, family history of UGIC also plays a key role in ESCC risk (Zhang et al., 2011), which might be a risk factor for earlier onset and poorer prognosis of ESCC (Wen et al., 2009). Since the positive percentage of history family was significantly different between the two age groups (Table 4), we produced stratified analysis according

6 DOI: / X Hsa-miR-196a2 functional SNP with risk of ESCC 47 Table 4. The family history of UGIC of cases and controls in different age groups. Family history of UGIC Group Negative n (%) Positive n (%) Total 2 -test p Value OR (95% CI) 60 years Controls 194 (60.1) 129 (39.9) ( ) ESCC 158 (48.6) 167 (51.4) years Controls 174 (63.5) 100 (36.5) ( ) ESCC 155 (57.0) 117 (43.0) 272 to age-set. Stratified analysis in our study could enhance the stability of the results and the reliability of the conclusions. Several studies have investigated mir-196a expression levels in cancers. The expression levels of mir-196a are different in different cancers. For example, up-regulation of mir-196a was found in glioblastoma (Guan et al., 2010), ductal adenocarcinoma (Wang et al., 2009), esophagus adenocarcinoma (Maru et al., 2009), breast cancer cells (MDA-MB-231) (Li et al., 2010b) and colorectal cancer cells (SW480) (Schimanski et al., 2009), but mir-196a downregulation was found in melanoma (Mueller & Bosserhoff, 2011). Therefore, mir-196a appears to play various roles in tumors of different origins. On the other hand, studies have shown that mature mir-196a levels were increased 9.3-fold in breast cancer cells transfected with pre-mir-196a-c but only 4.4-fold with pre-mir-196a-t (Hoffman et al., 2009). The C allele of rs increased the expression of mature mir-196a2 in lung cancer tissues (Hu et al., 2008) and colorectal cancer tissues (Zhan et al., 2011). These results indicated that rs polymorphism might affect the processing of the pre mirna to its mature form. 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