RESEARCH ARTICLE. Wen Li 1, Jing Deng 2 *, Shuang-Shuang Wang 1, Liang Ma 1, Jiang Pei 1, Xiao-Xi Zeng 1, Jian-Xin Tang 1. Abstract.

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1 DOI: Methyltion of the RAR-β Gene nd Cigrette Smoking in NSCLC in Southern-Centrl Chinese RESEARCH ARTICLE Assocition of Methyltion of the RAR-β Gene with Cigrette Smoking in Non-Smll Cell Lung Cncer with Southern-centrl Chinese Popultion Wen Li 1, Jing Deng 2 *, Shung-Shung Wng 1, Ling M 1, Jing Pei 1, Xio-Xi Zeng 1, Jin-Xin Tng 1 Abstrct Pthogenesis of lung cncer is complicted biologicl process including multiple genetic nd epigenetic chnges. Since cigrette smoking is confirmed s the most min risk fctor of non-smll cell lung cncer (NSCLC), the im of this study ws to determine whether tobcco exposure plys role in gene methyltion. Methyltion of the RAR-β gene were detected using methyltion-specific polymerse chin rection in DNA from 167 newly dignosed cses with NSCLC nd corresponding 105 controls. A significnt sttisticl ssocition ws found in the detection rte of the promoter methyltion of RAR-β gene between NSCLC nd controls (x 2 =166.01; p<0.01), nd hypermethyltion of the RAR-β gene ws significntly ssocited with smoking sttus (p=0.038, p<0.05). No reltionship ws found between RAR-β gene methyltion nd pthologic stging including clinicl stge, cell type, gender nd drinking (p>0.05), nd the methyltion of RAR-β gene rte of NSCLC ws slightly higher in stges Ⅲ+Ⅳ (80.0%) thn in Ⅰ+Ⅱ (70.8%). Similr results were obtined for methyltion of the RAR-β gene between squmous cell crcinom (77.9%) nd other cell type lung cncer (73.9%). These results showed tht the frequency of methyltion incresed grdully with the development of clinicl stge in smoking-ssocited lung cncer ptients, nd tobcco smoke my be ply potentil role in RAR-β gene methyltion in the erly pthogenesis nd process in lung cncer, prticulrly squmous cell crcinom. Aberrnt promoter methyltion is considered to be promising mrker of previous crcinogen exposure nd cncer risk. Keywords: Methyltion - non-smll cell lung cncer - RARβ gene - cigrette exposure Asin Pc J Cncer Prev, 15 (24), Introduction Lung cncer is the leding cuse of cncer deths in Chin nd worldwide (Siegel et l., 2013; Cheng et l., 2012). Five-yer survivl rte of lung cncer ptients is less thn or equl 15%, Non-smll cell lung cncer ccounts for 80% of ll lung cncer. An importnt reson for the ptients is tht lung cncer is often detected when it hs spred t the time of dignosis, nd the long-term prognosis is poor (Li et l., 2014). Therefore, erly dignosis of lung cncer is relistic pproch to reduce the mortlity relted with lung cncer. Although new instruments pper promising such s CT, PET nd X-ry, which cn not precisely relize erly detection of lung cncer. The development of dignostic tools is necessry for the erly detection of lung cncer (Hssnein et l., 2012; Suzuki et l., 2010). Aberrnt DNA hymethyltion re involved in prticipting in the pthogenesis nd progression of tumorigenesis nd plyed n importnt role in non-smll cell lung cncer (Gsche et l., 2012; Frks et l., 2014; Suzuki et l., 2013; Chen et l., 2011; Dniunite et l., 2011; Losi-Guembrovski et l., 2007; Missoui et l., 2010; Zhng et l., 2011). Some scientific reserch hve shown tht RAR-β ws bsent in vrying degrees in ll kinds of mlignnt toumer (Chen et l., 2011; Dniunite et l., 2011; Losi-Guembrovski et l., 2007; Wng et l., 2012; Zhng et l., 2011). It is well known tht decrese RAR-β expression is ssocited with the formtion of tumor nd berrnt promoter methyltion of the RAR-β gene is detected in NSCLC. However, genetic studies hve not been ttempted to identify the role of berrnt promoter methyltion of the RAR-β gene tht my be ssocited with NSCLC ptients. The present reserch is imed t investigting the potentil role of berrnt promoter methyltion of the RAR-β gene in NSCLC risk nd its effects on clinicl chrcteristics in southern-centrl Chinese popultion. 1 Key Lbortory of Green Pckging nd Appliction of Biologicl Nnotechnology of Hunn Province, 2 College of Pckging nd Mteril Engineering, Hunn University of Technology, Zhuzhou, Hunn Province, P.R. Chin *For correspondence: dengjing0102@ yhoo.com Asin Pcific Journl of Cncer Prevention, Vol 15,

2 Wen Li et l Mterils nd Methods Study subjects The study object included 167 newly dignosed ptients with NSCLC nd 105 cncers-free controls. Finl dignoses of cses were confirmed by routine histopthologicl exmintion. All subjects were recruited between Jnury 2010 nd June 2013 between the Hunn provincil tumor hospitl in Chngsh (Chngsh, Chin) nd the Centrl Hospitl of Zhuzhou City (Zhuzhou, Chin). At recruitment, written informed consents bout the study were obtined from ll the ptients nd controls. Ech prticipnt ws then interviewed to collect informtion on demogrphic chrcteristics. The clinic pthologicl fetures of smples re shown in Tble 1. All study subjects provided written consents nd were ethnic South-Centrl Chinese popultion Hn, Ech prticipnt ws interviewed to collect informtion on demogrphic chrcteristics. And the reserch protocol ws pproved by the Institutionl Review Bord of the hospitl. DNA extrction nd bisulfite tretmen Genomic DNA from peripherl blood lymphocytes ws extrcted using the stndrd kit-bsed method (Gentr Systems, Minnepolis, MN). Genomic DNA (0.5-1 μg) ws treted with sodium bisulfite vi the EZ DNA methyltion-gold kit (Zymo Reserch, USA). Bisulfite tretments chnged unmethylted cytosines into urcils while leving methylted cytosines unmodified. The bisulfite-modified DNA ws used immeditely for PCR or stored t -70 C. the PCR products were nlyzed in 2% TBE grose gels. Sttisticl nlysis Sttisticl nlyses were performed using Sttisticl softwre SPSS13.0. The ssocition between the methyltion sttus of RAR-β gene nd clinicopthologicl prmeters ws nlyzed using χ 2 test. The ssocition between the methyltion of RAR-β gene nd NSCLC ws determined using the logistic regression method to ssess odds rtio (ORs) nd 95% confidence intervls (95%CI). Results Chrcteristics of study subjects The ssocition with clinicl fetures between lung cncer ptient nd helthy control smple re listed in Tble 1. The fctors of ge nd sex consumption hd no significnt difference between ptients nd controls. Conversely, there were significnt sttisticl between smoking nd drinking sttus, which indicte tht smoking nd drinking re risk fctor for lung cncer in our study popultion. RAR-β promoter methyltion profile Aberrnt promoter methyltion of the RAR-β gene ws detected to 80.23% (134/167) of cses, nd none of the 105 controls shown methyltion in promoter methyltion of the RAR-β gene. Tble 2 shown distribution of methyltion sttus in ptients nd controls nd its ssocition with risk of lung cncer. There ws significnt sttisticl ssocition of the promoter methyltion of the RAR-β gene with lung cncer risk (p<0.01). Positive control for methyltion Lung cncer ptient DNA ws treted in vitro with excess SssI methyltrnsferse (New Englnd Biolbs), to generte completely methylted DNA t ll CpGs nd ws used s positive control for methylted lleles of ech gene. DNA from helthy control smple ws used s the control for unmethylted lleles. And Genomic DNA ws treted with sodium bisulfite nd stored t -70 C. Methyltion-specific PCR Modified DNA ws then used in MSP for the detection of the methyltion sttus of RAR-β promoter. The primer sequences (Mruym et l., 2002; Li et l., 2014), designed to mplify specificlly methylted (M) or unmethylted (U) forms of the RAR-β promoter were: M1 (forwrd) 5 - TCGAGAACGCGAGCGATTC G-3 ; M2 (reverse) 5 -GACCAATCCAACCGA AACGA-3 ; U1 (forwrd) 5 -TTGAGAATGTG AGTGATTTGA-3 ; U2 (reverse) 5 -AACCAAT CCAACCAAAACAA-3. The PCR mixture contined 12.5μl TqMix (Dongsheng, Biotech), 2μl of bisulfitemodified DNA, 1μl 10 pm ech primer nd 8.5 μl sterile wter. The PCR profile ws consisted of n initil denturtion t 94 C for 4 minutes, followed by 35 steps cycles of 94 C for 45 second, 62 C for 45 second, nd 72 for 60 second, nd finl extension step of 72 C for 10 minutes. Wter blnk ws used s negtive control. Both M nd U primer set generted 169 bp products nd Asin Pcific Journl of Cncer Prevention, Vol 15, 2014 RAR-β promoter methyltion profile nd clinicopthologicl chrcteristics Tble 3 depicted the reltionship between the methyltion of the RAR-β gene nd clinicopthologicl chrcteristics of lung cncers. Hypermethyltion of the RAR-β gene ws significnt sttisticl ssocition with smoking sttus (p=0.038, p<0.05). No reltionship ws found between RAR-β gene methyltion nd other pthologic stging including clinicl stge, cell type, gender nd drinking (p>0.05). Those indicted cigrette smoking is mjor risk fctor of lung cncer. Tble 1. Demogrphic Chrcteristics of Cncer Ptients nd Controls Chrcteristics Cses (%) Controls (%) x 2 -vlue p-vlue Totl Age Men+SD 58.2± ±13.2 Gender Mle (Ref) 104 (62.3) 58 (55.2) Femle 63 (37.7) 47 (44.8) Smoking sttus No (Ref) 44 (26.3) 82 (78.1) Yes 123 (73.7) 23 (21.9) Drinking sttus No (Ref) 98 (58.54) 77 (73.33) Yes 69 (41.9) 28 (26.67)

3 Tble 2. Distribution of Methyltion Sttus in Ptients nd Controls nd Its Assocition with Risk of Lung Cncer RAR-β methyltion of smoking lung cncer ptients in pthologicl type nd clinicl stg Tble 4 ws showed the ssocition of RARβ methyltion with pthologicl type nd clinicl stg in smoking lung cncer ptients. No sttisticl difference ws observed in the detection rte of RAR-β gene methyltion between pthologicl type nd clinicl stg (p>0.05). Discussion Totl methyltion unmethyltion (%) (%) lungcncer (Ref) (80.23) 33 (19.77) Controls (100%) Pthogenesis of lung cncer ws complicted biologicl process including multiple genetic nd epigenetic chnges (Lokk et l., 2012; Zho et l., 2012). The role of the genetic mechnisms hd become n incresing concern to globl investigtors in recent reserches. DNA methyltion profiles of lung cncer hd suggested tht the methyltion ws n importnt mrker in genome modifiction, the berrnt promoter methyltion DOI: Methyltion of the RAR-β Gene nd Cigrette Smoking in NSCLC in Southern-Centrl Chinese p-vlue p<0.001 Tble 3. Frequencies of Methyltion of RARB in 167cses of Lung Cncer of tumor suppressor genes ws ssocited with mechnism in ll kinds of cncer (Frks et l., 2014; Suzuki et l., 2013; Chen et l., 2011; Dniunite et l., 2011; Losi- Guembrovski et l., 2007). Although the rnge the methyltion of some genes ws low to high frequency (Hwes et l., 2010; Shw et l., 2006), Methyltion mechnism is still not very cler in process of cncers. Severl studies showed seprtely tht methyltion of CpG islnds of RARβ genes hd significnt role in the development of lung cncer (Jin et l., 2009; Zho et l., 2012; Chung et l., 2011). A number of reports hd proven tht methyltion of the RAR-β promoter ffected numerous tumor-suppressing genes, which were subsequently silent nd not expressed (Li et l., 2014). Other reserches hd indicted the bnorml methyltion of RAR-β ws ssocited with erly pthogenesis of lung cncer, induced by correltive environmentl fctor exposure (Mruym et l., 2002; Lokk et l., 2012; Shw et l., 2006; Jin et l., 2009; Zho et l., 2012). In the present study, the detection rte of methyltion of the RAR-β gene exhibited significnt differences between lung cncers s compred with control, there ws significnt sttisticl ssocition between lung cncer ptients with controls. But no sttisticl differences were found in clinicopthologicl chrcteristics except for cigrette prmeter with lung cncer ptients, which ws consistent with previous studies. These results suggest tht RAR-β methyltion nd cigrette smoking were closely Vribles N methyltion (%) unmethyltion (%) x 2 -vlue p-vlue OR 95%CI Gender Mle (Ref) (75.96) 25 (24.04) Femle (87.30) 8 (12.70) Pthologicl type Squmous cell crinom (Ref) ( (20.03) Adenocrcinom nd other cell type (81.48) 15 (18.52) Clinicl stg Ⅰ+Ⅱ (Ref) (73.85) 17 (26.15) Ⅲ+Ⅳ (84.31) 16 (15.69) Cigrette No (Ref) (90.90) 4 (9.10) Yes (76.42) 29 (23.58) Drinking sttus No (Ref) (82.65) 17 (17.35) Yes (80.30) 16 (19.70) b OR, odds rtio; CI, confidence intervl. c including the smll cell, lrge cell, nd mixed cell crcinoms or undifferentited crcinoms Tble 4. RAR-β Methyltion of Smoking Lung Cncer Ptients in Pthologicl Type nd Clinicl Stge Vribles N methyltion (%) unmethyltion (%) x 2 -vlue p-vlue OR 95%CI (76.42) 29 (23.58) Clinicl stge Ⅰ+Ⅱ (Ref) (70.83) 14 (29.07) Ⅲ+Ⅳ (80.00) 15 (20.00) Pthologicl type Squmous cell crinom (Ref) (77.92) 17 (22.08) Adenocrcinom nd other cell type (73.91) 12 (26.09) b OR, odds rtio; CI, confidence intervl. c including the smll cell, lrge cell, nd mixed cell crcinoms or undifferentited crcinoms Asin Pcific Journl of Cncer Prevention, Vol 15,

4 Wen Li et l relted to the development process of NSCLC. Tobcco smoking ws responsible for substntil morbidity nd mortlity worldwide. And the methyltion rte ws closely correlted with the smoking, which might ply role in vriety of smoking-relted phenomen (Breitling et l., 2011; Cheng et l., 2012; Wu et l., 2014). Previous science reserches reported different results on the ssocition nlysis between RAR-β gene methyltion nd smoking sttus. A number of reserchers hd reported tht the RAR-β gene ws specificlly trgeted by crcinogens in cigrette smoke nd the frequency of berrnt methyltion of RAR-β gene incresed in smokers (Zho et l., 2012). In opposition, some uthors filed to demonstrte potentil role of the RAR-β promoter methyltion in lung crcinogenesis (Scesnite A et l., 2012). In the present study, the detection rte of methyltion of the RAR-β gene exhibited significnt reltionship between lung cncers nd controls in smoking sttus, nd ws significntly higher in the non-smoking group tht in the smoking group, Which incresed times greter risk of NSCLC compred with smoker. Those findings might suggest tht tobcco smoking is risk fctor in the development process of NSCLC nd tobcco plys potentil role in gene methyltion. RARβ methyltion ws nlyzed in clinicl stge nd pthologicl type prmeters with lung cncer ptient, the results shown tht no sttisticlly significnt difference ws found. We were unble to conclude from this experiment whether RAR-β gene methyltion could be used s n indictor of dignosis of lung cncer on ccount of our smll smple size. but the methyltion of RAR-β gene rte of NSCLC is slightly higher in stges Ⅲ+Ⅳ (80.0%) thn in Ⅰ+Ⅱ (70.8%). And similr results were obtined for the methyltion RAR-β gene between squmous cell crcinom (77.9%) nd other cell type lung cncer (73.9%). the frequency of methyltion ws incresed grdully with the development of clinicl stge in smoking lung cncer ptients, nd tobcco smoking plyed domin roles in NSCLC with hypermethyltion of RAR-β gene prompter (Breitling et l., 2011; Scesnite et l., 2012; Jin et l., 2010; Zeilinger et l., 2013; Wu et l., 2014). Although it ws uncler tht environmentl fctor underlie the trgeting of specific genes promoters for hyperthyltion, the niml experiment hd shown the crcinogens cn induce some genes methylted in mice (Jin et l., 2010; Scesnite et l., 2012; Zeilinger S et l., 2013; Wu et l., 2014). Those dt mybe suggest tht tobcco ply potentil role in RAR-β gene methyltion in the erly pthogenesis nd process in lung cncer. The methyltion of RAR-β gene prompter ws considered to be novel nd promising mrker of previous crcinogen exposure nd cncer risk (Leong et l., 2011; Wlter et l., 2014), especilly in squmous cell crcinom. Accordingly, it needs further work to increse smple size nd understnd the function of RAR-β gene t the moleculr level. In conclusion, we believed tht RARβ methyltion ws ssocited with higher susceptibility nd hs prognostic significnce in erly stge to NSCLC, nd my be promising mrker in erly dignosis with southerncentrl Chinese popultion Hn. Although the results my Asin Pcific Journl of Cncer Prevention, Vol 15, 2014 be chnce findings they nevertheless highlight the need to investigte interctions with tobcco smoke in studies on the promoter methyltion. Acknowledgements This study ws supported by the Ntionl Nturl Science Foundtion of Chin ( ), the Nturl Science Foundtion of Hunn Province of Chin (12JJ4082, 14JJ2049), Outstnding Youth Project Supported by Scientific Reserch Fund of Hunn Provincil Eduction Deprtment (14B050), nd the Nturl Science Foundtion Hunn University of Technology, Chin (No.2013HZX04). References Mruym R, Toyook S, Toyook KO, et l (2002). Aberrnt promoter methyltion profile of prostte cncers nd its reltionship to clinicopthologicl fetures. Clin Cncer Res, 8, Shw RJ, Liloglou T, Rogers SN, et l (2006). Promoter methyltion of P16, ARbet, E-cdherin, cyclin A1 nd cytoglobin in orl cncer: quntittive evlution using pyrosequencing. BrJ Cncer, 94, Losi-Guembrovski R, Kusne H, Guembrovski AL, et l (2007). DNA methyltion ptterns of the CDH1, RARB, nd SFN genes in choroid plexus tumors. Cncer Genet Cytogenet, 179, Jin M, Kwkmi K, Fukui Y, et l (2009). 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