Association on polymorphisms in LncRNA HOTAIR and susceptibility to HNSCC in Chinese population

Transcription

1 Europen Review for Medicl nd Phrmcologicl Sciences 2018; 22: Assocition on polymorphisms in LncRNA HOTAIR nd susceptibility to HNSCC in Chinese popultion B. WU 1, J. LIU 2, B. WANG 2, X. LIAO 1, Z. CUI 1, N. DING 1 1 Deprtment of Stomtology, Beijing Luhe Hospitl Cpitl Medicl University, Beijing, Chin 2 Deprtment of Stomtology, Beijing Tongren Hospitl, Beijing, Chin Bin Wu nd Jingming Liu contributed eqully to this work Abstrct. OBJECTIVE: More nd more evidence hs shown tht the criticl functions of long non-coding RNA (lncrna) polymorphism in the crcinogenicity mechnism of vriety of cncers. The ssocition between lncrna HOX trnscript ntisense intergenic RNA (HOTAIR) polymorphism nd the risk of hed nd neck squmous cell crcinom (HNSCC) in Chinese popultion hs not been reported. To investigted the effects of HOTAIR polymorphism on cncer susceptibility, the influence of HOTAIR vrints on the risk of HNSCC ws nlyzed in this study. PATIENTS AND METHODS: In this cse-control study, the tgging SNPs (rs874945, rs , nd rs ) in HOTAIR gene were genotyped in Chinese popultion consisting of 366HNSCC cses nd 732 controls. RESULTS: It ws found tht rs ws ssocited with significntly incresed risk of HNSCC in Chinese popultion [GG vs. AA: djusted odds rtio (OR) = 1.23, 95% confidence intervl (CI) = ; dditive model: OR = 1.21, 95%CI = ]. However, there were no significnt ssocitions of rs nd rs with HNSCC risk. CONCLUSIONS: HOTAIR rs my influence HNSCC susceptibility nd serve s dignostic biomrker. Key Words LncRNA, HOTAIR, Polymorphism, HNSCC. Introduction Hed nd neck cncer (HNC), predominntly hed nd neck squmous cell crcinom (HN- SCC), includes cncers of orl cvity, orophrynx, hypophrynx, nd lrynx. It is the sixth most common cncer in the world 1. 45,780 new individuls will be dignosed with cncer of the orl cvity nd the phrynx. 8,650 deths of these diseses will occur in 2015 in the United Sttes ccording to the most recent vilble estimtes 2. Applictions of tobcco nd lcohol hve been widely considered to be mjor risk fctors of HN- SCC 3. However, not ll individuls with such risk fctors ultimtely develop HNSCC, suggesting tht genetic susceptibility my ply n importnt role in the development of HNSCC. Non-coding RNAs longer thn 200 nucleotides re defined s long noncoding RNAs (lncrnas) 4. The ENCODE project suggests tht lncrnas ply roles in 76% of the humn genome trnscription 5. Growing evidence indictes tht lncrnas could interct with DNA, RNA, nd protein t trnscriptionl nd post-trnscriptionl level so s to regulte the expression of protein-coding genes, nd ffect the rnge of biologicl processes, such s cell growth, survivl, migrtion, nd invsion 4. The HOX trnscript ntisense intergenic RNA (HOTAIR) is member of the lncrnas generting from the HOXC locus locting on the 12q13.13 of humn chromosome. HOTAIR prticiptes in the development nd progression of vriety of mlignncies including brest cncer 6, colorectl cncer 7, nd gstric cncer 8. A systemtic review nd met-nlysis 9 reveled tht HOTAIR represents potentil biomrker of prognosis in ptients with HNSCC. Considering the impliction of HOTAIR in cncers nd the function of single-nucleotide polymorphisms (SNPs) within lncrna genes on the expression or structure of lncrnas, incresing studies were conducted to explore the ssocitions between SNPs within HOTAIR nd cncer risks. Assocition between HOTAIR SNPs nd cncers were found 10,11. However, there is no study on the reltionship between HOTAIR polymorphism nd HNSCC risk. Thus, this investigtion, cse-control study including 366 HNSCC cses nd 732 controls, ws conducted to exmine the effect of tgsnps in HOTAIR on HNSCC risk in Chinese popultion. 702 Corresponding Author: Bin Wu, MM; e-mil: doupi965732@163.com

2 HOTAIR polymorphisms in HNSCC Ptients nd Methods Ethics Sttement The institutionl Review Bord of Beijing Luhe Hospitl Cpitl Medicl University pproved this cse-control study. The design nd performnce involving humn subjects were clerly described in reserch protocol. All prticipnts were voluntry nd signed the informed consent before this reserch. Study Ptients A totl of 366 HNSCC ptients confirmed by two pthologists were consecutively recruited from Beijing Luhe Hospitl Cpitl Medicl University from Jnury 2009 to My Subjects with second orl cvity cncer primry tumors, primry tumors of the nsophrynx or sinonsl trct, metstsized cncer from other orgns, or ny histopthologic dignosis other thn orl cvity cncer were excluded. 732 helthy controls were rndomly selected from cohort of more thn 12,000 prticipnts in community-bsed screening progrm for non-infectious diseses in Beijing, Chin. Control subjects were mtched to the cses by ge (±5 yers old) nd sex. All prticipnts were geneticlly unrelted, ethnic Hn Chinese popultion. Dt on demogrphic chrcteristics (e.g., ge nd gender) nd environmentl exposure (e.g., smoking nd drinking sttus) were collected in fce-to-fce interview. Ech prticipnt donted 5 ml of venous blood nd provided written informed consent. SNPs Selection nd Genotyping TgSNPs in the HOTAIR genes were selected on the bsis of the HpMp dtbse nd literture review. We used Hploview softwre to serch tgsnpsmaf (minor llele frequency) >0.05. A linkge disequilibrium vlue of r 2 <0.8 ws tken into ccount for SNP selection. Ultimtely, three cndidte SNPs were chosen: , rs nd rs in the HOTAIR gene. Genomic DNA ws extrcted from leukocyte pellet by proteinse K digestion, followed by phenol-chloroform extrction nd ethnol precipittion. TqMn llelic discrimintion ssy on the pltform of 7900HT Rel-time polymerse chin rection (PCR) system ws performed to distinguish the SNP genotype. Two negtive controls (no DNA) were included in ech 384-well rection plte, nd the genotyping results were determined by using SDS2.3 Allelic Discrimintion Softwre (Applied Biosystems, Wlthm, MA, USA). Sttisticl Anlysis The goodness of fit χ 2 ws used to test Hrdy-Weinberg equilibrium of SNPs mong the control subjects. Two-sided χ 2 -tests were used to evlute differences in the distributions of demogrphic chrcteristics, selected vribles, nd genotypes between the cses nd controls. The ssocitions of SNPs with HNSCC risks were estimted by computing the crude nd djusted odds rtios (ORs) nd their 95% confidence intervls (CIs) using logistic regression nlyses. All the sttisticl nlyses were performed with the Sttisticl Anlysis System softwre (v.9.1.3; SAS Institute, Cry, NC, USA). Two-sided tests were used for sttisticl nlysis nd p<0.05 represented tht the difference ws sttisticlly significnt. Results As shown in Tble I, there were no significnt differences in the distributions of ge nd gender between the cses nd controls (p=0.202 nd 0.637, respectively), suggesting successful mtching. However, the proportion of drinkers in the cses ws higher thn tht in the controls (39.0% vs. 22.0%, p=0.011). The minor llele frequency (MAF) nd the genotyping rte of the three selected SNPs re presented in Tble II. The observed genotype frequencies for these three SNPs were ll in Hrdy-Weinberg equilibrium in the controls (p=0.183, nd for rs874945, rs , nd rs , respectively). The genotype frequen- Tble I. Selected chrcteristics in HNSCC cses nd controls. Vribles Cse Control p N (%) N (%) All subjects 366 (100) 732(100) Age < (40.4) 267 (36.5) (59.6) 465 (63.5) Gender Femles 159 (43.4) 329 (44.9) Mles 207 (56.6) 403 (55.1) Smoking Ever 144 (39.3) 253 (34.6) Never 222 (60.7) 479 (65.4) Drinking Ever 106 (39.0) 161 (22.0) Never 260 (71.0) 571 (78.0) Two-sided chi-squred test. 703

3 B. Wu, J. Liu, B. Wng, X. Lio, Z. Cui, N. Ding Tble II. Primry informtion nd minor llele frequencies (MAFs) of selected SNPs. SNP Bse MAF in our HWE chnge controls rs G>A rs A>G rs C>G HWE = Hrdy-Weinberg equilibrium. MAF = minor llele frequency. cies of the three SNPs in the cses nd the controls re summrized in Tble III. After the djustment for ge, sex, smoking, nd lcohol sttus, multivrite logistic regression nlysis reveled tht rs ws significntly ssocited with n incresed risk of HNSCC (GG vs. AA: djusted OR=1.23, 95%CI= , dditive model: OR=1.21, 95%CI= ). No significnt ssocitions of vrint genotypes with the other two SNPs nd HNSCC risk were observed. The strtifiction nlysis on the ssocitions between rs nd HNSCC risk from ge, sex, smoking, nd drinking were further conducted. As shown in Tble IV, the significnt ssocition of rs with HNSCC risk ws found mong the older group (djusted OR=1.32, 95%CI= , p=0.040) nd drinkers (djusted OR=1.50, 95%CI= , p=0.040). Tble III. Logistic regression nlysis for ssocitions between selected SNPs nd risk of HNSCC. SNP Genotype Cse (%) Control (%) Adjusted OR p-vlue (95%CI) rs GG 191 (52.2) 358(48.9) 1.00 AG 152 (41.5) 317(43.3) 0.91 ( ) AA 22 (6.0) 55(7.5) 0.87( ) Additive model 0.88( ) rs AA 113 (30.9) 268(36.6) 1.00 AG 188 (51.4) 362(49.5) 1.24( ) GG 65 (17.8) 101(13.8) 1.23( ) Additive model 1.21( ) rs CC 140 (38.3) 241(32.9) 1.00 CG 167 (45.6) 369(50.4) 0.77( ) GG 59 (16.1) 122(16.7) 0.90( ) Additive model 0.87( ) Logistic regression with djustment for ge, sex,smoking nd drinking. Significnt vlues (p<0.05) re in bold. Tble IV. Strtified nlysis for ssocitions between vrint genotype of rs nd HNSCC risk. Vribles rs Adjusted b p b OR(95%CI) Cses Controls AA/AG/GG AA/AG/GG Age, yr <60 46/65/37 98/102/ ( ) /123/28 170/260/ ( ) Gender Femles 63/67/29 140/151/ ( ) Mles 50/121/36 128/211/ ( ) Smoking Ever 34/80/30 79/130/ ( ) Never 79/108/35 189/232/ ( ) Drinking Ever 26/55/25 53/88/ ( ) Never 87/133/40 215/274/ ( ) Mjor homozygote/heterozygote/rre homozygote between cses nd controls; b Logistic regression with djustment for ge, sex, smoking nd drinking. Significnt vlues (p<0.05) re in bold. 704

4 HOTAIR polymorphisms in HNSCC Discussion In this study, the ssocitions of 3 selected SNPs in HOTAIR with HNSCC risk were evluted in cse-control study, which showed tht rs ws ssocited with HNSCC risk in this Chinese popultion. As fr s it concerns, this is the first study to evlute the ssocitions between HOTAIR polymorphism nd HNSCC risk. As reported, HOTAIR cn function s moleculr scffold, trgeting Polycomb Repressive Complex 2 (PRC2) 6 nd Lysine Specific Demethylse 1 (LSD1) complexes 12 to specific genes on genome-wide level nd led H3K27 to methylte nd H3K4 to demethylte nd silence the expression of relted genes. Additionlly, HOTAIR cn influence the suppression of trget mirnas by cting s mirna sponge 13. Recently, incresing evidence hs reveled berrnt expression of HOTAIR in wide rnge of cncers, nd the expression level of HOTAIR my serve s potentil biomrker for dignosis, metstsis, nd prognosis, indicting HO- TAIR is involved in the etiology of relted cncers 6-8. Efforts were mde to explore whether polymorphisms could influence the expression or the structure of HOTAIR, thus ffecting individul susceptibility to cncers. The rs polymorphism is locted in the first intron of HOTAIR. Du et l 14 found tht rs contributes to n incresed risk of gstric cncer. This study reveled tht rs ws ssocited to HNSCC risk. A recent met-nlysis 15 including 28,527 cses nd 37,151 controls hs concluded tht rs in high LD (r 2 =1) with rs ws relted to HNC susceptibility. However, the ssocition between rs nd HNSCC risk ws not found in this study. This difference my due to the reltively smll smple size or the complex function of HOTAIR. Further studies on the interction effect of rs C llele nd rs T llele on the biologicl behvior of CC cells re wrrnted. The rs polymorphism is locted in the 3 ner the gene of HOTAIR. Wng et l 16 reported tht there is no ssocition between rs nd noise-induced hering loss. Jin et l 17 found no ssocition between rs nd cervicl cncer risk. Another study 18 indicted tht rs mkes no contribution to colorectl cncer susceptibility. Conclusions rs is in the first intron of HOTAIR, which is significntly ssocited with incresed risk of HNSCC. Further functionl studies on the effect of rs on biologicl behvior re needed to confirm nd extend our findings. Conflict of Interest: The uthors declre no conflict of interest. References 1) Liu B, Ding JF, Luo J, Lu L, Yng F, Tn XD. Seven protective mirna signtures for prognosis of cervicl cncer. Oncotrget 2016; 7: ) Siegel RL, Miller KD, Jeml A. Cncer sttistics, CA Cncer J Clin 2015; 65: ) Mier H, Dietz A, Gewelke U, Heller WD, Weiduer H. Tobcco nd lcohol nd the risk of hed nd neck cncer. Clin Investig 1992; 70: ) Zhng CG, Yin DD, Sun SY, Hn L. The use of lncrna nlysis for strtifiction mngement of prognostic risk in ptients with NSCLC. Eur Rev Med Phrmcol Sci 2017; 21: ) Gerstein MB, Kundje A, Hrihrn M, Lndt SG, Yn KK, Cheng C, Mu XJ, Khurn E, Rozowsky J, Alexnder R, Min R, Alves P, Abyzov A, Addlemn N, Bhrdwj N, Boyle AP, Cyting P, Chros A, Chen DZ, Cheng Y, Clrke D, Estmn C, Euskirchen G, Frietze S, Fu Y, Gertz J, Grubert F, Hrmnci A, Jin P, Ksowski M, Lcroute P, Leng JJ, Lin J, Monhn H, O Geen H, Ouyng Z, Prtridge EC, Ptcsil D, Puli F, Rh D, Rmirez L, Reddy TE, Reed B, Shi M, Slifer T, Wng J, Wu L, Yng X, Yip KY, Zilbermn-Schpir G, Btzoglou S, Sidow A, Frnhm PJ, Myers RM, Weissmn SM, Snyder M. Architecture of the humn regultory network derived from ENCODE dt. Nture 2012; 489: ) Gupt RA, Shh N, Wng KC, Kim J, Horlings HM, Wong DJ, Tsi MC, Hung T, Argni P, Rinn JL, Wng Y, Brzosk P, Kong B, Li R, West RB, vn de Vijver MJ, Sukumr S, Chng HY. Long non-coding RNA HOTAIR reprogrms chromtin stte to promote cncer metstsis. Nture 2010; 464: ) Kogo R, Shimmur T, Mimori K, Kwhr K, Imoto S, Sudo T, Tnk F, Shibt K, Suzuki A, Komune S, Miyno S, Mori M. Long noncoding RNA HOTAIR regultes polycomb-dependent chromtin modifiction nd is ssocited with poor prognosis in colorectl cncers. Cncer Res 2011; 71: ) Li T, Mo X, Fu L, Xio B, Guo J. Moleculr mechnisms of long noncoding RNAs on gstric cncer. Oncotrget 2016; 7: ) Troino G, Cponio V, Boldrup L, Gu X, Muzio LL, Sgrmell N, Wng L, Nylnder K. Expression of the long non-coding RNA HOTAIR s prognostic fctor in squmous cell crcinom of the hed nd neck: systemtic review nd met-nlysis. Oncotrget 2017; 8: ) Guo W, Dong Z, Bi Y, Guo Y, Shen S, Kung G, Xu J. Associtions between polymorphisms of 705

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