A118G Polymorphism in l-opioid Receptor Gene and Interactions with Smoking and Drinking on Risk of Oesophageal Squamous Cell Carcinoma

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1 Journl of Clinicl Lbortory Anlysis 31: e22018 (2017) A118G Polymorphism in l-opioid Receptor Gene nd Interctions with Smoking nd Drinking on Risk of Oesophgel Squmous Cell Crcinom Xinfng Xu,* Boneng Mo, Lingdong Wu, Lei Liu, Juping Rui, nd Guochng Chen Deprtment of Gstroenterology, Yixing People s Hospitl, Jingsu, Chin Aim: To investigte the single nucleotide polymorphism (SNP) of A118G nd its interction with smoking nd drinking on oesophgel squmous cell crcinom (ESCC) risk. Methods: A totl of 960 subjects (545 mles nd 415 femles) with men ge of yers were selected, including 490 ESCC ptients nd 470 norml control subjects. A logistic regression model ws used to exmine the ssocition between A118G nd ESCC nd its interction with A118G nd current smoking nd drinking. The odds rtio (OR) nd 95% confident intervl (95%CI) were clculted. Results: The frequency for the A llele of A118G ws significntly higher in ESCC cses, OR (95%CI) = 1.22 ( ). Logistic regression nlysis showed significnt ssocition between the A llele in A118G nd incresed ESCC risk. The ESCC risk ws significntly higher in crriers of the A llele of the A118G polymorphism thn those with GG (AG + AA vs. GG, djusted OR (95%CI) = 1.20 ( )). We found tht current smokers with AG or AA of the A118G genotype hve the highest ESCC risk compred with never smokers with GG genotype; the OR (95%CI) ws 2.57 ( ). Current drinkers with AG or AA of the A118G genotype hve the highest ESCC risk compred with not currently drinking subjects with the GG genotype, OR (95%CI) = 2.36 ( ), fter djusting for covrites. Conclusion: The A llele of A118G nd ESCC nd dditionl interction between the A llele of A118G nd smoking or drinking were ssocited with incresed ESCC risk. 31:e22018, Wiley Periodicls, Inc. Key words: A118G; lcohol consumption; interction; oesophgel squmous cell crcinom; polymorphism; smoking INTRODUCTION Oesophgel squmous cell crcinom (ESCC) is the sixth most common cuse of cncer-relted deth worldwide. ESCC is lso predominnt histologicl subtype of oesophgel cncer nd is chrcterised by high mortlity rte in Chin (1, 2). It is usully dignosed t reltively lte stge, nd tretment options re limited; the 5-yer survivl rte for ESCC ptients hs been very low (3). Previous studies hve determined severl genetic fctors for ESCC (4 6); however, the precise moleculr mechnisms of crcinogenesis nd progression re not well known. Thus, the identifiction of trget genes for the determintion of tumour development is urgently required for ESCC dignosis nd tretment. Epidemiologic studies hve suggested tht ptients who receive generl nesthesi with opioids rther regionl nesthetics re t higher risk of cncer recurrence (7, 8). Furthermore, in ddition to the nlgesic effect, l-opioid receptor ctivtion my influence tumour growth nd cncer progression (9). Severl genetic vrints hve been identified within the l-opioid receptor gene (10). The most common nd well-known genetic vrint is the single nucleotide polymorphism (SNP) A118G. However, no previous studies hve focused on the impct of A118G polymorphism; dditionl interctions with environmentl fctors, such s smoking nd lcohol consumption on ESCC risk, lso remin to be conducted. Thus, the *Correspondence to: Xinfng Xu, Deprtment of Gstroenterology, Yixing People s Hospitl, Tong Zhen Gun Rod No. 75, Jingsu, Chin. E-mil: xuxinfng33@163.com Received 6 My 2016; Accepted 7 June 2016 DOI /jcl Published online in Wiley Online Librry (wileyonlinelibrry.com) Wiley Periodicls, Inc.

2 2 of 6 Xu et l. im of this study ws to investigte the impct of SNP of A118G nd dditionl interctions with current smoking nd drinking on ESCC risk in Chinese popultion. MATERIALS AND METHODS Subjects This ws cse-controlled study on ESCC. Chinese ptients with ESCC were consecutively recruited between Mrch 2010 nd September 2013 from Yixing People s Hospitl. A totl of 960 subjects (545 mles, 415 femles) with men ge of yers were selected, including 470 ESCC ptients nd 490 norml control subjects. All cses were confirmed vi histopthologicl dignosis. The subjects who received chemotherpy or rdiotherpy prior to surgery were excluded from this study. Helthy controls were rndomly selected from popultion screening progrmme for risk fctors of ESCC in the sme regions nd mtched to cses on the bsis of ge (3 yers) nd gender (Fig. 1). Blood smples were collected from ech prticipnt. Detiled personl informtion on demogrphic chrcteristics nd smoking nd drinking sttus were collected vi interview. At recruitment, written informed consent ws obtined from ech prticipnt. Body Mesurements Dt on demogrphic informtion, diet, smoking, nd drinking informtion for ll prticipnts were obtined using stndrd questionnire dministered by trined stff. We defined current lcohol consumption s more thn one drink of ny type per month or not currently drinking s <1 drink of ny type per month (11); current smokers were defined s those who hve smoked t lest 100 cigrettes nd still smoked t the time of the interview; individuls with no history of cigrette smoking were considered to be never smokers (12, 13). A low-fibre diet nd high-ft diet were defined ccording to the Dietry Pyrmid (14). Body weight, height, nd wist circumference (WC) were lso mesured ccording to stndrdised procedures (15). Body mss index (BMI) ws clculted s weight in kilogrms divided by the squre of the height in metres. Blood smples were collected in the morning fter t lest 8 hr of fsting. All plsm nd serum smples were frozen t 80 C until lbortory testing. Genomic DNA Extrction nd Genotyping Approximtely 2 ml of whole blood ws collected from ll prticipnts in sterile EDTA-coted vcutiners. Genomic DNA from prticipnts ws extrcted Fig. 1. A flowchrt of study popultion selection.

3 A118G Polymorphism, Smoking Drinking nd ESCC 3 of 6 from EDTA-treted whole blood using the DNA Blood Mini Kit (Qigen, Hilden, Germny) ccording to the mnufcturer s instructions nd stored t 80 C until used for genotyping. Genotyping of A118G SNP (rs ) ws performed using the TqMn pltform produced by the BGI Compny, Chin. Genotyping ws repeted on 10% rndom smple of study prticipnts, nd the results were 100% concordnt. ABI Prism7000 softwre (Applied Biosystems, Crlsbd, CA) nd n llelic discrimintion procedure were used for genotyping. A 25-ll rection mixture included 1.25 ll SNP Genotyping Assys (209), 12.5 ll Genotyping Mster Mix (29), 20 ng DNA, nd conditions s follows: initil denturtion for 10 min nd 95 C, denturtion for 15 sec nd 92 C, nd nneling nd extension for 90 sec nd 60 C (50 cycles). Sttisticl Anlysis The men nd stndrd devition (SD) were clculted for normlly distributed continuous vribles, nd the t-test ws used for comprison between cses nd controls; percentges were clculted for ctegoricl vribles, nd the v 2 test ws used for comprison between the cse nd control group prticipnts (version 19.0; SPSS Inc., Chicgo, IL). A Hrdy Weinberg equilibrium (HWE) ws performed using SNPStts (vilble online t SNPstts). A logistic regression model ws used to exmine the ssocition between A118G nd ESCC nd the interction between A118G nd smoking or lcohol consumption. The odds rtio (OR) nd 95% confident intervl (95%CI) were clculted. Odds were djusted for gender, ge, high-ft diet, nd low-fibre diet. RESULTS A totl of 960 subjects (545 mles nd 415 femles) with men ge of yers were selected, including 490 ESCC ptients nd 470 norml control subjects. Prticipnt chrcteristics strtified by cses nd controls re shown in Tble 1. The distribution of current smokers, lcohol consumption, both smoking nd drinking, nd high-ft diet significntly differed between the cses nd controls. The men WC ws higher in the ESCC cses thn the controls. The genotypes of A118G were distributed ccording to Hrdy Weinberg equilibrium (P > 0.05). The frequency for the A llele of A118G ws significntly higher in the ESCC cses, OR (95%CI) = 1.22 ( ). Logistic regression nlysis showed significnt ssocition between genotypes of the A llele in A118G nd incresed ESCC risk fter djusting for gender, ge, high-ft diet, nd low-fibre diet. ESCC TABLE 1. Generl Chrcteristic of Study Prticipnts in Cses nd Controls Vribles ESCC Cses (n = 490) Controls (n = 470) P-vlues Age (yer) Mles N (%) 256(52.2) 269(57.2) Alcohol 213(43.4) 166(35.3) consumption N (%) Current smokers N (%) 203 (41.4) 154 (32.8) Both smoking nd 118 (24.1) 86 (18.3) drinking N (%) High-ft diet N (%) 159(32.4) 124 (26.4) Low-fibre diet N (%) 123 (25.1) 109 (23.2) BMI (kg/m 2 ) WC (cm) FPG (mmol/l) TG (mmol/l) TC (mmol/l) HDL (mmol/l) Mens stndrd devition for ge, FPG, TC, HDL-C, TG, WC, nd BMI. TC, totl cholesterol; HDL, high density lipoprotein; FPG, fst plsm glucose; TG, triglyceride; WC, wist circumference; BMI, body mss index. risk ws significntly higher in crriers of the A llele of the A118G polymorphism thn those with GG (AG + AA vs. GG, djusted OR (95%CI) = 1.20 ( ); Tble 2). We lso conducted n ssocition nlysis mong smoking, drinking, nd ESCC using logistic regression. We found tht the OR (95% CI) ws 1.48 ( ) for lwys smokers compred with never smokers nd 1.69 ( ) for lwys drinkers compred with not current drinkers (Tble 3). To obtin the odds rtios nd 95%CI for the joint effects of A118G nd smoking nd lcohol consumption on ESCC, we conducted n interction nlysis between A118G nd current smoking nd drinking using logistic regression. We found tht current smokers with AG or AA of the A118G genotype hve the highest ESCC risk compred with never smoking subjects with the GG genotype (OR (95%CI) of 2.57 ( ) fter covrite djustment; Tble 4). We lso found tht current drinkers with AG or AA of the A118G genotype hve the highest ESCC risk compred with not current drinking subjects with the GG genotype (OR (95%CI) of 2.36 ( ) fter djusting for covrites; Tble 5). DISCUSSION In this study, we found tht A118G polymorphism ws significntly ssocited with ESCC risk in Chinese popultion, the A llele of A118G ws significntly higher in ESCC cses nd ESCC risk ws lso higher in the A llele of A118G crriers thn tht in

4 4 of 6 Xu et l. TABLE 2. Genotype nd Allele Frequencies for A118G in Cses nd Controls Frequencies N (%) Genotypes nd lleles Controls (n = 470) ESCC Cses (n = 490) OR (95%CI) P-vlues HWE test GG 40 (8.5) 23 (4.7) AG 170 (36.2) 163 (33.3) 1.18 ( ) AA 260 (55.3) 304 (62.0) 1.29 ( ) AG + AA 210 (44.7) 186 (38.0) 1.20 ( ) A 690 (73.4) 771 (78.7) G 250 (26.6) 209 (21.3) 1.22 ( ) Adjusted for gender, ge, high-ft diet, low-fibre diet, nd WC. TABLE 3. The Assocition Between Smoking nd Drinking nd ESCC Risk Vribles Frequencies N (%) Controls (n = 470) ESCC Cses (n = 490) OR (95%CI) P-vlues Smoking Never 316 (67.2) 287 (58.6) Currently 154 (32.8) 203 (41.4) 1.48 ( ) Drinking Not 304 (64.7) 277 (56.5) b currently Currently 166 (35.3) 213 (43.4) 1.69 ( ) Adjusted for gender, ge, high-ft diet, low-fibre diet, WC, nd drinking. b Adjusted for gender, ge, high-ft diet, low-fibre diet, WC, nd smoking. TABLE 4. Interction Between A118G nd Smoking on ESCC Risk A118G Smoking OR (95% CI) P-vlues GG Never 1.00 GG Currently 1.36 ( ) AG + AA Never 1.15 ( ) AG + AA Currently 2.57 ( ) <0.001 Adjusted for gender, ge, high-ft diet, low-fibre diet, nd WC. TABLE 5. Interction Between A118G nd Alcohol Consumption on ESCC Risk rs Alcohol consumption OR (95% CI) P-vlues GG Not currently 1.00 GG Currently 1.27 ( ) AG + AA Not currently 1.18 ( ) AG + AA Currently 2.36 ( ) <0.001 Adjusted for gender, ge, high-ft diet, low-fibre diet, nd WC. subjects with the GG llele of A118G fter djustment. Although A118G polymorphism nd its reltionship with cncer susceptibility hve been investigted in mny studies, few hve focused on n ssocition between A118G polymorphism nd ESCC. To our knowledge, this is the second epidemiologic study to evlute the ssocition between the A118G of the l-opioid receptor gene nd ESCC risk. Up to now, we found only one humn popultion study (16) investigting this spect. In tht study, Wng et l. conducted cse-controlled study in Chinese Hn nd TuJi popultions in the Enshi region, Chin. They found tht the A118G polymorphism in the l-opioid receptor gene my be ssocited with risk of ESCC; compred with the GG genotype, the AA genotype exhibited significntly elevted risk for ESCC. Interest in the direct effect of opioids nd l-opioid receptors on tumour progression hs grown in recent yers. There is ccumulting experimentl evidence of direct effect of l-opioid receptors on tumour progression or recurrence (8, 17 20), prticulrly for brest cncer (5, 20). Direct mechnisms (8) for direct effect of l-opioids on cncer progression were demonstrted. The l-opioid receptor is the min trget for opites such s morphine, fentnyl, nd heroin nd hs binding ffinity two orders of mgnitude greter thn the ffinity of other opioid receptors; in ddition, direct effect of opioids on tumour progression ws observed during the development of the peripherl opioid ntgonist methylnltrexone for opioid-induced constiption. Becuse methylnltrexone does not cross the blood brin brrier, centrlly medited nlgesi is preserved (21). Tobcco smoking nd lcohol consumption were widely reported importnt environmentl risk fctors for ESCC (22 26). Some studies (27, 28) suggested significnt interction between gene polymorphism (such s P21, XRCC1, GSTT1, nd ERCC2) nd current smoking or lcohol consumption in determining ESCC risk. In this study, we lso found significnt interction between the A llele of A118G plus current smoking or lcohol consumption in determining ESCC risk. Current smokers or drinkers with the A llele hve the highest ESCC risk. A study by Wng et l. (16) reveled

5 A118G Polymorphism, Smoking Drinking nd ESCC 5 of 6 tht the A118G polymorphism my be n importnt inherited genetic vrible ssocited with ESCC risk, nd their results implied n interction with environmentl fctors (such s smoking) in individuls with more G lleles. They lso found tht non-smoking my be more protective ginst ESCC occurrence, which ws consistent with the results of our study. Evidence points to the endogenous opioid system (nd the l-opioid receptor in prticulr) in mediting the rewrding effects of drug use, including those ssocited with nicotine, nd OPRM1 A118G hs been shown to lter the receptor protein level in preclinicl models nd smoking behviour in humns (29). The limittions of this study should be considered. First, lthough the number of study prticipnts met the requirement for nlysis, the present smple size ws reltively smll. The smoking nd drinking rtes in women were very low. These limited rtes led to insufficient dt for gender difference nlysis. Second, the gene gene interction with other SNPs of OPRM1 or other genes nd interctions with other environmentl risk fctors (such s diet) should be investigted in future studies. Thirdly, OR vlues were reltively low in the correltion nlysis, so this reltion need to be verified in others popultion. In conclusion, we found tht A118G polymorphism ws significntly ssocited with ESCC risk in our study, tht the rte of the A llele of A118G ws significntly higher in the ESCC cses thn the controls, nd tht the ESCC risk ws lso higher in the A llele of A118G crriers thn in subjects with the GG genotype. We lso found significnt interction between the A llele of A118G nd current smoking or lcohol consumption. Current smokers or drinkers with the A llele hve the highest ESCC risk. ACKNOWLEDGEMENTS The writing of this study ws supported by the Deprtment of Gstroenterology, Yixing People s Hospitl. We thnk ll the prtners nd stffs who helped us in the process of this study. REFERENCES 1. 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