BLfdC eAU

HOI NGH! KHOA HOC CONG NGH? SINH HOC Wi

BLfdC eAU PHAN TiCH MO'C DO METHYL H 6 A TAI VUNG PROMOTER CCiA

GEN DNMT3L TR^N BENH NHAN UNG THU" C6 TC CUNG «

Trtnyng Kim Phuong, Le Thi Tnic Linh, Lao Dire Thugn, L§ Huyen Ai Thuy*

Tnj^g Dai hgc Ma Tp. Ho Chl Minh T 6 M TAT

Sy methyl h6a DNA hifn dang du^c n h i ^ nha nghien curu tren th6 gidi quan tam theo khuynh hudng su dyng chung nhu diu chiing sinh hpc (biomarker) trong ung thtr. Trong s6 5 gen da dugc c6ng bo cho dfin nay thuoc hg g«i mS h6a cho enzyme DNMn i Eukaryote bao g6m DmfTl, DNMT2, DNMT3 (DNMTSA, DNMT3B, DNMT3l),g^ DNMT3L lSjn$t gen tiem nang do ho?t dgng bat thuimg cua gen nay lit co thi c6 lien quan dka benh ung thu. Thyc nghiSm tien hanh tren 8 mau trong dd c6 4 njau b?nh phim (gom 2 mau djch phit tfi b ^ am djio vi 2 mlu sinh thiSt mo tii cac b?nh nhSn ung thur c6 tir cung) vd 4 mau djch phit ti bio ngudi linh cho kit ( ^ budc diu: tinh chit giii methyl hoa (hypomethylation) t^i 11 vi tri CpG quan trgng thu^c viing promoter ciia gca DNMT3L rit c6 kha nang li mgt dilm d|ic tnmg ciia cac te bio ung thu. Nghien ciiu se dugc tiip tyc mo rgng tren co miu lon hon.

Tir khoa: BSP, DNMTs. epigenetics, gen DNMT3L, ung thu c6 tii cung.

MdDAU

Sv methyl hda - la mOt bien ddi cOng hoa tri dan den si/ b6 sung nhdm -CH3 vao vi trf cartwne s6 5 cua Cytosine trong c|p nucleotide CpG nho sv xiic t^c cua hd enzyme DNA methyltransferase (DNMTs), \d bi^n dii epigenetics chinh trong bt> gen d0ng v$t co vu (Laird, 2003). Trong nh&ng nSm g^n day, hi§n tu-ong nay dang avKfC nhiSu nha nghien ctru trfln th6 gidi quan tdm theo khuynh hi/dng SLP dgng chung nhir d4u chiing sinh hoc (biomarker) vd thyc tk cho th^y c6 mOt sy lifin quan h^t siic ch^t che giira hi?n tuong epigenetic vol nhilu logi ung thu.

Nhdm enzyme DNMTs bao gfim DNMT1, DNMT2, DNMT3a, DNMT3b vd DNMT3L (Das vd Singal, 2004). Trcing dd, vijc giCr vai tr* quan trong trong vi$c hlnh thanh sy methyl h6a de novo vd duy tri sy methyl h6a nSy Id nhd- 3 enzyme DNMT1, DNWT3a, DNMT3b. DNMT3a va DNMT3b Id cdc de novo DNA methyltransferase ci5 nhi?m vy thiet Igp DNA methyl hda 6du tien bang cdch gan nhOng goc methyl miri vdo Cytosine trong cap CpG md \ns6e dd khdng bj methyl h6a, DNMT1 Id methyltransferase c6 nhi#m VM duy tri g6c methyl ir nhiing vj trf Cystosine trSn sg'i don DNA vira dugc t6ng hgp trong qud trinh tdi bdn DNA. Do do sy methyl hda DNA duvc di truyen cho cac t h i h$ tk bdo tiip theo nhis vdo ca thk methyl hoa DNA mgch khuOn (Gozuacik va Kimchi, 2006). Cdc enzyme ndy cOng gdp phin vao sy methyl hda bit thi/frng va dan den sy phdt tnen cua ung thir. Sy biiu hi$n qud miic cua gen DNMTs ir mirc 6g mRNA da dirge nh^n thiy ir mOt vai bgnh ung thu, vd sy bieu hien cua DNMT1 tang trong te bdo blnh thu6ng thi co the gay nen s\f methyl hda de novo t>it thirdiig ir ddo CpG. Miic dp bieu hi^n ciDa cdc mRNA cua DNMTs Id khdc nhau trong suot chu trinh t i bdo, va sy biiu hign DNMTs khong phD hgp c6 thi gdp phin thay do! sy methyl h6a trong cdc te bdo una Ihir (NarayanefaA,2003). Thyc t i nhiiu nghifin ciiu da chiing minh cho thiy c6sy methyl hda bitthy&ngtren mOtsoger dd ndn khii u vd gen gdy ung thy dud'i xuc tac cua hp enzyme DNA methyltransferase cd Hdn quan ch^t che vir\ ung thu ndi Chung vd ung thu co tCr cung ndi rieng (Hirst va Marra, 2009; Brooks ef al., 2009; Duenas-Gonzalez et al.. 2005). VI vdy. nhung bien dSl bit thudng trfin hg gen DNMTs nhu sy bit hogt hay hogt hda bat thud'ng trfin gen DNMTs se giy nfin sy gidm t«eu hign hay sy biiu hien qua miic cua cac protein DNMTs. diiu ndy cd t h i Id nguyfin nhan dan din tinh trgng methyl hda bit thudng cOa t i bao. Trong so 5 gen da dtfgrc cdng bo cho din nay thu$c hg gen ma hda cho enzyme DNA meth^transferase 6 Eukaryote bao gom DNtATI, DNMT2. DNMT3 (DNMT3A, DNMT3B, DNMT3L) (Bestor, 2000; Nakayama efa/., 2001), nhCrng gen ndo hogt dgng binh thudng hay bat thudng ciia nd id thyc sy c6 liSn quan din b^nh ly ung thu. Nghien ciiu vl vdy se dugc bit diu bing vific khai \hae dir li$u nham nit ra co sd khoa hpc cho thyc nghigm sau ndy. Cdc ndi dung chfnh md bdi bdo se tdp trung bao gdm: (1) Khai thdc dO li$u, lya chon gen (cdc gen) thu$c hg DNMTs md hogt dQng (cd t h i binh thudng hodc bit thudng) cua nd cd lifin quan din b$nh (ung thy). (2)^

Lya chgn phuong phap khdo sdt tlnh chit metfiyl hda cda gen (cdc gen) ndi trfin trong thyc nghi#m: a i u ndy hodn toSn phg thugc vdo tlnh chit di truyin phdn tu cCia gen se duoc khdo sdt. (3) Khao sat vd phdn tfch tlnh chit methyl hda ciJa gen (cdc gen) ndy tren mgt sd miu b^nh pham.

NGUYEN Lieu VA PHU-ONG P H A P NguySn lifu

Miu DNA chung du^rc cung d p b ^ hang Qiagen bao gim hai nguin DNA ngudi: 1/ DNA da bj methyl hda vd 2/ DNA chua bj methyl hoa. Sii dyng nguin DNA chiing nay se giiip chiing tdi ddnh gid dug'C d$ d$c hi§u cda cap mii trong phdn ling Bisulfite Sequencing PCR (BSP). Cac mdu b#nh phSm bao gom hai miu djch phit t i bdo am dao dd dircc chin dodn nhllm HPV type die (miu so 31 nhiim type 16, mSu s6 545 nhiim type 18), 2 mlu sinh fhiit md dupc phju thudt cit bd tir bdnh nhdn ung thu co tu cung (mau s i Ml vd M2) vd 8 mau djch phfit t i bdo khac dupc cho Id 'blnli thuvng" (bdi chdng dupc thu nhdn ti> nhiing ngudi den khdm phy khoa vdi k i t qud Pap' smear binh thudng vd hodn todn khdng nhiem HPV) dupc cung cip tir phdng khdm Au Igc, cfing ty CP Cdng ngh§ Vi#t A vd B^nh vifin Himg Virong TP. HCM. Cac mau dupc bdo qudn ir nhifit do -30°C trudc khi tiln hdnh tdch chiit DNA.

Phu'O'ng phap Bisulfite Sequencing PCR (BSP)

DNA sau tdch chiit bang phuong phdp phenol/chlorafonn dupc biin dii bisulfite bing bd kit DNA modificatkw Ml (Epitect Kit. Qiagen). Sau dd. DNA da biin doi bisulfit ducrc tinh sach vd thyc hien PCR vdi bO mii DNMT3L-SF (5-

;^GAGGGTTTTAI I I I I I GAATTTTA-y) vd DNMT3L-SR (5'-AAAAATCCAAACGCACCTAAAAC-3'). San pham PCR fly^ giai trmh ty cQng vdi edp moi DNMT3L-SF vd DBMT3L-SR.

KfeT QUA VA THAo LUAN Khai thdc d(r ll$u vd khao sdt in silica

Trong s i 27 cdng trinh thyc nghigm (si ligu cdp nhdt din 4/2013) d i cap tdi hifin tirong methyl hda DNA cd li§n quan mn hg enzyme DNA Methyl Transferase, chung tfli thiy rang chi cd 5 cdng trinh cd khao sdt miic 6(f methyl hda vd mii lidn quan cua hif n tupng ndy den cdc bgnh ung thu: c i & cung (Gokul et al.. 2007), dg ddy (Lima et al.. 2007), t i bao

^ m d t (Mandenwad et al.. 2010), gan (Parita ef al., 2006); t i t ca cac cong binh nghifin ciru ndy deu thyc hi§n sau ndm Qua khdo sdt/n silico, diiJng tdi^ghl nhan; nhdm cdc gen DNMTs (DNMT1, DNMT2, DNMT3A, DNfJfTSB. DNMT3L) khong phan bd tren cung mdt nhi§m sac thi, vd dac diem chung cda t i t ca cdc thdnh vien &iuOc hg gen DA//W7s Id vung promoter cua chung diu thiiu trlnh l y hgp TATA a gin vj tri bdt dau phien ma vd chi>a khd nhieu vj trf bam cua nhdn t6 didu noa phien ma Spi, dupc dieu hda bdi hai loai promoter mot logi nhieu CpG vd mpt logi ft CpG (dii lifiu khdng trinh bay).

DNMT1 CO kfch thudc khod^ng 59.129 kb. nim trdn NST s i 19 d vj tri 19p13.2 gim 40 exon vd 39 intron. duoc dieu khifin bo;i mgt promoter nhieu CpG vd 3 promoter ft CpG. Chiic ndng cda enzyme DNMT1 Id methyl hda DNA heml- methylation. tuc la nhung DNA da dupc ddnh diu, ddm bao cho su sao chep chfnh xdc tinh trgng methyl hda DNA trong qud trinh phdn chia tfi bao. Chiing tdi chua tim thiy tdi lidu nghien ciiu nao (ca co bdn vd ung dyng), d i cdp den gen nay vdl (*uc ndng methyl hda lien quan din cdc bdnh ly, nhat Id ung thu DNMTZ eo kich thudc khoang 135.535 kb, nam trdn NST JO tgi vjtri 10p15.1 gdm 11 exon vd 10 intron. DA/MT2 dupc cho Id thdnh vifin cOa hg DNMTvi nd cd trinh ty bao thu cao vdi nhiing DNMT khdc. Tuy nhifin, nhiing nghien ciiu gin day cho thiy vai t ^ chu y i u ciia DNfi^T2 Id methyl hda tRNA ciia acid aspartic. Vi vdy enzyme nay da duoc doi ten thdnh tflNA aspartic acid methyltransferase 1 (TRDMT1). Vdl nhung thdng tin tim dupc vd phdn tich nhu vCra trlnh bdy d tren. hai gen DNMT1 vd 2 khfing phdi id nhOng d-ng ci> vien thudc gia dinh gen DNMTs de chiing tdi lya chgn phdn tlc^

Gen DNMT3A (DNA 5-Cytosine Methyltransferase 3a) cd kfch thudc khoang 243 kb gom 23 exon vd 22 Intron nam d vj W 2p23.3 (Robertson ef al., 1999). Trong khi vi trf bit dau djch ma thupc viing exon 2, gen cd 4 vj tri bat diu phidn md vdi 3 exon 1 (vimg nim sau vj tri bit dau phidn ma) dupc tgm ggi Id exon 1" (ddi khodng 39bp); exon l'' (vCing 1 ddi SObp, vung 2 ddi 52bp) cdch exon 1'khoang 520bp; exon 1"= (ddi 3a2bp) cdch exon l " khodng 26kb. Cd hai exon 1'vd 1 ndm trong vCing gidu CpG (sdn phim phifin ma dupc diiu khiin bdi promoter thuQc nhdm 1) trong khi exon 1" nim trong viJng It CpG (sdn pham phifin m i cua promoter thudc nhdm 2) (Yanagisawa et al., 2002). Thyc t i khi sii dyng phin niini Methprimer d i ttm ddo CpG trong vung ndy, chdng tdi cung nhdn thay promoter thuOc nhdm mdt, exon 1", exon 1 nam trong ddo CpG trong khi preimoter, exon cdn Igi nam trong viing ft CpG; ngodi ddo CpG cd kleh thudc khoang 880bp (chtia trinh ty promoter, exon 1^, l") cdn mdt dao CpG nhd nam v i phfa ddu 5'cd kfch thudc 91 bp (dO' lifiu khdng trinh bdy).

Ndm 2007, Lima vd ddng tac gid khdo sat miic di> methyl hda d vdng promoter ciia gen DNMT3A bang phuong phdp Methylation Specific PCR (MSP) h^n doi tupng bgnh nhdn ung thu dg ddy (Lima etal., 2007). Phan tfch bO mil (dii lifiu khdng trinh bdy) cung nhu viing gen md nhdm tac gid chgn khdo sdt, chdng tdi nhgn thiy cap mil methyl vd khdng methyl dupc thiet k i giup rd sodt tlnh trgng methyl hda (hogc khong) cua hai vj tri CpG nam d hai diu ciia ddo CpG nhd (91bp); cac thdng so vd dd d^c hifiu cDa mii khi kiim tra diu r i t tot. Kit qud khdo sdt ciia Lima vd ddng tdc gid trfin 66 miu (20 miu tCr ngudi binh thudng. 46 miu con Igi dupc chan dodn td ung thu) cho thiy vi tri CpG khdo sdt d tlnh trgng methyl hda 100% trong t i t cd cdc mlu. Nguyfin nhdn nhdm nghifin ciiu khdng tim thiy sy khdc bi$t vk mire do methyl hda giua ngudi binh thudng vd ngudi bdnh ung thu theo chiing tdi cd ie do vj tri CpG lya chgn chua phu hpp;

moi thiit k i cho phdn iing MSP chua thgt sy hpp ly. Cu thi trong nghien cCru ndy, moi dupc thiet k i chi d i rd sodt mdt vi tri CpG (trdn moi mil). Tuy nhifin, thyc t i cho thay rat khd de hai mil chl khdc nhau mgt nucleotide nam giO'a trlnh ty moi cd t h i phdn bigt dup'c chfnh xdc nhung trinh ty gen chl khdc nhau mgt nucleotide ay. Gen DNMT3A bxing t i bdo binh thu'dng dupc biiu hidn d mgt miic d$ nhlt djnh va gia tang biiu hign trang mdt sd t i bdo ung thu (bdng quang, rufit kit, thdn, tuyin tyy) da dupc chiing minh (Robertson et al., 1999). Sy gia tdng biiu hifin ndy, xfit v i khfa cgnh epigenetics, cd thi do sy gidi methyl hda (hypomethylation) trong vCing promoter cOa gen. Nhu dd d i cgp a trfin, cdng dt ve phfa dau 5', cang cd nhieu trinh ty nhgn biit cua cdc nhdn to lie che phifin ma. Do vgy, vdng lya chgn d i khao sdt sy gidi methyl hda lam tdng biiu hign phii hop cd le Id vung promoter mgnh nhlt cda gen md cy thi ir day Id hai viing tir - 428 din +640 (vj trf 1+ Id base diu tien cua exon 1') vd hi -334 din +1021 (vj trf 1+ la base diu tifin cda exon 1"^). Sau dd. Gokul va dong tdc gia (2007) cQng tien hdnh khdo sdt miic dg methjri hda d viing promoter cOa gen DNMT3A trfin bgnh nhdn bj ung thu co td" cung bing ky thudt bisulfite sequencing (Gokul ef al.. 2007). Nhdm tdc gid so sdnh miic do meth^ hda ciia 12 vj tri CpG tren 7 mlu ung thu c i tu- cung (tir giai dogn I din IV) vdi 3 mlu blnh thudng thiy ring khdng cd sy khdc bi$t giiia nhdm t i bdo ung thu vd t i bao binh thudng; miic dp methyl hda cda 12 vj trf CpG nay trong khodng tii 0 - 34. Phdn tich 12 vj tri CpG md nhdm tdc gld chgn khdo sdt. chiing t i i nhan thiy 12 CpG ndy nam d phin dau ciia dao CpG Id'n (880bp); UiuOc viing ttr -428 din +640 (vj trf 1+ id base ddu tifin cua exon 1°). Nhu da phdn tich ir tren, ddy Id viing khd t i t trfin gen DNMT3A de lya chgn khi mudn khdo sat miic dp methyl hda nhung kit qua hau nhu khdng the hifin sy khdc bifit ddng k l giu-a ngudi binh thudng va ngudi ung thu bdi vi 12 vj tri CpG vdi C khdng methyl hda. Do vdy cd le hogt dong cOa gen DNMT3A khdng lien quan den hien tupng epigenetics trong ung thu co tu cung.

Chiing tdi cung se khdng chpn gen ndy de phdn tich trong nhiing thyc nghifim dau tifin cua minh.

Gen DNMT3B (DNA 5-Cytosine Methyltransferase 3B) cd kfch thudc khoang 63 kb. nim trfin NST s i 20 d v\ tri 20q11 2, cd 23 exon vd 22 intron (Robertson ef al.. 1999 ). Gen dupc diiu khien bdi hai pnjmoter: mgt promoter nam trong vung nhiiu CpG trong khi promoter cdn lai ndm trong vimg ft CpG (Yanagisawa ef al.. 2002). Tuong ty nhu gen

HQI NGH! KHOA HOC CONG NGHE SINH HQC •

DNMT3A, kit qud khdo sdt tinh trgng methyl hda cua 30 vi tri CpG d viing promoter cda gen DNMT3B fren 8 mau u ^ Uiu c i ti> cung vd 3 mlu binh thudng cho thiy khdng cd sy khdc bigt vk tlnh trgng methyl hda gi&a l i bdo binh thudng va te bdo ung tfiu, chi cd mgt s6 vi tri CpG xudt hidn meth^ hda (du ligu khdng trlnh bdy) nen DNMT3B cung khdng phai Id lya chon cua chiing tdi cho budc khao sdt trong thyc nghidm k i tiip.

Gen DNMT3L ndm trfin nhiim sac t h i s6 21, thugc chudi ddi d' vj tri 21q22.3, cd kich thudc gan 16 kb gim 12 exon vd 11 intron. Viing promoter aia gen ndy cd chiiu ddi khoang 477 nucleotide vd viing 5' promoter cda gen nay khdra chira ddo CpG. Ddc bi§t gen ndy cd viing promoter thieu trlnh ty hdp TATA ir gin vi trf bit diu phifin md va chda nhieu vj lrl gan yfiu t i phifin md Sp1. Gen ndy ma hda cho protein DNA methyltransferase 3L. Protein DNMT3L cd chiiu ddi 387 amino acid chiia cac miin chiic ndng khac nhau nhu: vung gidu cystein ATRX (a-thalassemia/mental retardation syndrome, X-lmked), viing PHD (polybramo homology domain), miin -N tuong iing trong DNMT3A vd DNMT3B (Kim vd Zhao, 2005). miin -C chira cdc vung bdo tdn cao I, IV. Vi, VIII. Protein ndy dupc bieu hifin cy the d cdc t i bdo mint trong thdi k^ hlnh thdnh giao ti> vd glal doan phat trien phdi thai (Bourchisef a/.. 2001).

Protein DNMT3L khfing dupc cho Id DNA methyltransferase diing nghTa bdi vl trong phdn tCr cda protein ndy thiiu nhiing amino acid quan trgng can cho hogt ddng ciia chfnh nd (Bourchis ef al., 2001) nhung DNMT3L ddng mgt val tr6 quan trong cho sy diiu chinh miic dg epigenetics cOa t i bdo. Chdng cung cd vai trd methyl hda de novo vd se dugc hogt hda khi tuong tdc vdi DNA methyltransferase 3 alpha (DNMT3a)/DNA methyltransferase 3^beta (DNMT3|i) (Kareta ef ai, 2006). Protein DNMT3L giiip hoat dOng de novo methyltransferases bing cdch lidn kit vdi cdc miin -C ciia DNMT3A vd DNMT3B vd Idm tang miic dg hogt dgng cua cdc e n : i ^ e ndy din din Idm tdng khd nang bdm vdo DNA ciia DNMT3A vd DNMT3B. DNMT3L cd t h i dug'C xem nhu la mgt yiu to trao d i l chit nln cho cdc chiic ndng cOa DNMT3A vd DNMT3B vd Id mdt yeu to kich thich tmng gian chung cho tit cd hogt ddng meOiyl hda ete novo cua DNMT3A vd DNMT3B. Ngodi ra, pnstein DNMT3L cung cd chiic ndng ngdn chgn sy phifin ma khi nd lidn kit vdi (HDAC1). Cdc VLing bdo t i n PHD cua DNMT3L tuang tdc vd kich hogt H0AC1 da cho thiy ring protein nay cung tham gia gdp phin cung vdi de-acetylation histone, tu bo chit nhiem sic vd iic chi phidn md (Deplus ef al. 2002).

M$t s i nghidn ciiu in vdro cho ffiiy rang sy methyl hda pramoler DNMT3L lifin quan den hoat dOng (^ien ma cua gen (Hu ef al.. 2008). Ngodi ra, nhiing biin dii tren gen DNMT3L cd tiie xdy ra nhu sy gidm hodc tdng cudng sy methyl hda trfin promoter DNh/fTSL. cd thi Idm tang hay gidm mtPc dp biiu hifin ciia gen trong cdc te bdo bit thudng.

Gokul vd ding tdc gid (2007) da khang djnh sy giai methyl hda (hypomethylation) trfin vdng promoter cua DNMT3L cd thk xem nhu Id mit biomariier hiru higu cho bgnh ung thu co tCr cung. Trong nghifin ciiu ndy, nhdm tdc gia da khao sdt tlnh trgng methyl hda cda cdc CpG ir vung promoter cda 16 mau ung thu c i tir cung, 6 mlu binh thudng dii vdi gen DNMT3L bing ky thudt Bisulfite Sequencing PCR Ndm 2010, Mandenwad vd ding tdc gia cung khdo sdt tinh tr^ng methyl hda cua 11 vj trf CpG d vdng promoter cua gen DNMT3L d t i bdo u vdy mat. K i t qud thu dupc tuong ty nhir d t i bdo c i tli cung, hie diu cd hifin tupng gidi methyl hda d 11 vj tri CpG ndy (Hlnh la). Kit qud ciia Mandenivad vd ding tdc gid hodn todn khing djnh vd Cing hg cho nhgn ^nh cOa nhdm Gokul vd ding tdc gid (2007). Nhung kit quS ndy dupe mfi td chi tiit nhu sau: d nhirng t i bao binh thudng, ty lfi methyl hda d 11 vj tri CpG ndy khd cao tir 66 - 100% (mdu xanh chuyin sang vdng vd mdu dd Id chiem chd yiu) (Hlnh lb, 1c). Nhung d cdc t i bao ung thu thi l^ IS methyl gidm xuing nhiiu (mdu xanh Id chu yiu) (hign tupng hypomethylation) (Htnh lb, 1c). Trong t i bdo btnh thudng, gen DNMT3L bj methyl hda gin nhu tuygt doi nfin hogt ddng phifin ma cua gen bj lic c h i dan din biiu hi$n oia enzyme DNMT3L gidm ho$c bj i>c chi Nhung khi sy methyl hda bj gidm di (hypomethylation), nghTa Id vCing promoter dupc gi^i phdng. gen cd t h i dupc ty do phifin ma vd bieu hign thdnh protein (enz^e). Md chiic ndng cda enzyme ndy Id chuyen gic methyl gdy ndn hifin hfpng metiyl hda DNA, vi vgy hogt d$ng metiiyl hda tinh trfin tong thi bd gen at han bi thay dii, trong dd mfit s i gen khdc se bj methyl hda dan den ptilfin ma bj irc chl. Neu hifin tuong ndy roi vdo cdc gen cfi nhifim vy d'c chi khii u thi rd rdng ddy Id can nguyfin din din bfinh.

Phdn tich k i t qud Bisulfite Sequencing PCR (BSP)

Kit qud thyc nghifim dupc tiin hdnh tuan ty: 1) Thd nghidm mil trong phdn dng BSP su dung DNA chdng, 2) Tii ini hda m0t s i cht tidu phdn dng BSP nhu nhifit dp lai, ham lupng DNA, nong dp moi, 3) Thyc hign BSP va giai trinh ty 8 miu bdnh phim (xem phin vdt ligu).

Phdn tich kit qud gldi trinh ty 8 sdn pham BSP cho thay: cd 11 ^^ frf CpG diu dupc dgc rd rdng, chinh xac (dd peak huynh quang d 4 mlu bfinh ly blnh thudng thap hon 4 mau bfinh). Nhiing kit qud ndy dupc md td chi tet nhu sau (Hinh 2): d nhu-ng t i bdo binh thudng, ty lfi metiiyl hda d 11 vj trf CpG ndy rdt cao, mdu dd xuit hifin la ehfl yiu (tit cd 4 mau diu ed 9/11 vj tri methyl hda). Nhung d nhdm mlu cac te bdo ung thu vd/hogc cd sy bien doi te bdo bit thudng cung xdc dinh nhiim HPV type ddc. ty Id methyl gidm xuing nhiiu (mdu xanh Id chd yiu). & hai mlu djch phit: 11/11 vj fri CpG diu gidi methyl hda, trong khi hai mdu md, mau Ml cd9/11 vj trf CpG gidi methirt hda va mlu M2cd 10/11 vjtri CpG gidi methyl hda

^ C C O N G N G H f SINH HOC T O A N Q U O C 2013

Hlnh 1. Gen DNMT3L Chii th[ch:(a) So db gen DNMT3L. vj fri promoter, 5UTR, cdc exon, ddo CpG. v[ tri bat diu phifin mi (+1). (b) Mirc dft methyl hda cua cdc vi tri CpG khdo sdt so sSnh glira t i bdo thudng vd ung thir c i tir cung (b), ung thu mdng kit vd u hlnh viy ir mat (c)

vnrrr^M'i fc< •

' isr£r.GAi'>i£t»i?reT.-i>u'>rt; r r . m Mb. OMWtllwHPV

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GiMiivii* HPV

Hlnh 2. Kil qud khdo sdt muc d$ methyl hda tgM 1 vj tri CpG thugc vdng promoter gen DNMT3L Chu thich: (a) 11 vj tri CpG khdo sdt ndm glOa hai mil trong phSn iing BSP; (b) mirc d$ methyl hda cua 11 vi fri CpG khdo sdt so sanh giira ung thu cd ti> cung (31,545. M1, M2) vd t i bdo thudng (miu 1, 3,4, 5)

Thdi gian gin ddy, trong nude dd cd mOt s i cdng frlnh xuit bdn v i viec khdo sat hign tupng methyl hda bit thudng trong cdc bfinh dgc biet Id ung thu vd nhdm nghidn ciru chflng tdi cung Id mOt trong s i dd, tuy. nhidn tat cd diu tdp trung nghifin cfl-u ve methyl hda vupt miic (hypermethylation). Ddy Id cdng bo dau tifin chuing tdi ^ cgp din gidi methyl hda bit thudng (hypomethylation). Tuy so lupng mdu sfl- dyng trong nghifin ciiu Id hgn chi, nhung tfnh chit giai methyl hda bit thudng ir nhdm mau bfinh so vdi mlu Idnh cdng Id mgt ghi nhdn rit ddng quan tam va vl thi nghifin ciiu se dupc tfip tgc mS r$ng v i cd mlu.

K ^ LUAN

Qua frlnh thyc hifin d i tdi, mOt s6 kit ludn dupc nit ra nhu sau: (1) Gen DNMT3L thupc hg gen DNMTs vdi hogt ddng bit thu'dng cfla nd rit cd t h i cd lidn quan din bgnh ung thu. (2) Vdi dgc tfnh di tmyin Id gen DNMT3L khdng chda ddo CpG trong vilng promoter nen phuong phdp Bisulfite Sequencing PCR (BSP) dupc lya dign d i khdo sdt mdc dg methyl hda tgi 11 vj frf CpG quan trong thugc vflng promoter cua gen ndy Id phu hpp. (3) Thyc nghidm tiln hdnh trdn 8 mau trong dd ed 4 miu binh phim (gom 2 mlu djch phit t i bdo dm dgo vd 2 mlu sinh thiit md tu- cdc bfinh nhdn ung thu c i tCr eung) vd 4 mlu djch phit t i bdo ngudi lanh cho kit qud budc diu: tfnh chit gldi methyl hda (hypomethylation) tgi 11 v\ tri CpG quan trong thudc wng promoter gen DNMT3L rit ed kha ndng Id mdt diim dgc trung ciia cdc t i bdo ung thu.

Nghidn ciiu se dupc tiip tyc md rQng trfin ed mau Idn hvn trong thdi gian tdi LAi cam ffn

Nghien aiu dirpc ihirc hifn btri sif ldi Irp kmh phi cua Sp Gido dvc va Ddo ttfo vd Dai hpc Md. IT. HCM

HOI NGH! KHOA HOC CONG NGHE SINH HQC

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ANALYSING OF THE METHYLATION STATUS OF PROMOTER DNMT3L GENE ON CERVICAL CANCER PATIENTS

T r u o n g Kim P h u o n g , Le Thi T r u e L i n h , Lao D u e T h u a n , Le H u y e n A i T h u y * Faculty of Bhtechndogy, Ho Chi Minh city Open University

SUMMARY

In recent year, methylation is concerned t>y lots of reseachers over the world as biomadters for cancers. Among five genes otDNMh femily genes of Eukaryote organisms includes DimTl, DNI\dT2, DNMT3 (DNMT3A, DNMT3B, DNIidT3L), DNMT3L ^ e il l potential madter because its abnoimal activities should he occurred to cancer. The experimental process were canied out of I samples including 4 specimens (2 vaginal specimens and 2 tissue biopsy samples of cervical cancer patients) and 4 vagiMl specimens of healthy person. From the initial results, it can be shown that hypomethylation at 11 important CpG sites of DNMTll promoter is a considered maricer for cancer cells. This study should be continuted with a large sample sizes.

Keyword: BSP, cervical cancer, DNMTs, DNMT3L gene, epigenetics.

'Author for corresspondence. Email: lhathuy@gmail.com