Y Hpc VI$T NAM T H A N S B • 2>2012
NGHIEN din PHAT HIlN SURVIVIN MRNA, HMAM MRNA TUrCAC TE BAO UNG THITVU TRONG MAU
Nguyin Minh Hien*, Phgm Thi^n Ng^c**
Le Quang Hu^n***, Le Thi ^'nft Phiic***, Phgm Thj Thanh Thiiy**
TOM TAT:
Ung thu vij (UTV) Id logi ung thu r^t thudng gdp 0' phy nu, vi$c chdn dodn vd d i l u trj sdm UTV se gflp phdn gidm ty Ifl tdi phdt vd ty l | t u vong cho ngud'i b|nh Trong nhCrng ndm g^n ddy cdc nhd khoa hpc dd chung minh dup'c cde te bdo ung thu efl ngufln gflc tir khfli u xdm nhdp vd di chuyen rdt sd'm vdo mdu ngoai vi gpi Id t l bdo ung thu di chuyen (CTC) Phdt hi$n cdc gen ddc hi$u t u CTCs cd t h l hO'u Ich trong sdng lpc, tien luo'ng.
theo dfli sau d i l u tn Mgc tieu cua nghi6n cuu ndy Id. Phdt hi^n hWIAM mRNA, Sun/ivin mRNA trong mdu t)$nh nhdn UTV, tim moi \\in quan giOa s y bieu hifln eiia hMAM mRNA, Survivin mRNA vd mflt so yeu to khde trong benh UTV D6t tup-ng va p h i r v n g phap: NghiSn cuu t r i n mdu mdu cua 24 benh nhdn d u a c chdn dodn xdc dinh Id ung thu vu, 5 ngud'i khoe manh t u nguyfln tham gia vdo nhdm nghidn cuu S u dyng k? thudt RT-PCR, gtai trinh t u gen, de phdt hien mRNA Survivin vd hMAM Kf dflng te bdo ung thu VLI, mfl ung thu vu vd eac te bdo ung t h u vu trong mdu K i t qua, Ty Id phdt hidn hMAM trong mdu Id 14/24 (58,3%), Survivin Id 11/24 (45,8%), Khdng thay bieu hi$n eua hMAM vd Survivin trong mdu ngud'i khoe manh (0/5), Ty le bieu hien cua 2 gen ndy c6 hdn quan den kich thud'c u cijng nhu giat dogn bflnh vdl (p<0,05). K i t luan, Phdt hi$n dup'c gen dde trung t u cac te bdo ung thu vu trong mdu.
Tip khoa RT- PCR, Survivin, hMAM. ung thuvu
ABSTRACT:
DETECTION OF SURVIVIN MRNA, BMAM, MRNA GENES FROM BREAST CANCER
CELLS IN PERIPHERAL BLOOD
Breast cancer is a very common cancer in vi'omen, the diagnosis and the therapy of early stage tumors have the potential to decrease morbidity and mortality rate of patients vi^ith breast cancer In recent years, scientists have demonstrated that the cancer celts derived from tumor infiltrating and moving in peripheral blood from a very early stage known as Circulating Tumor Cell (CTC) The detection sensitive genes from CTCs may prove useful for screening, prognostication, monitoring of response to therapy The goal of this study Detection hMAM mRNA, Sun/ivin mRNA in penpheral blood of breast cancer patients, correlation between the expression of hMAM mRNA, Survivin mRNA and some other factors in them Patients and methods Blood sample obtained from 24 breast cancer patients, 5 healthy volunteers. Using RT- PCR, gene sequencing techniques to discover Survivin, hMAM gene m breast cancer cell lines, cancer tissues and in CTCs, Results: Detection rates hMAM mRNA and Survivin are 14/24 (58 3%), 11/24 (45 8%) in blood, no expression of hMAM and Survivin in healthy human blood (0/5) The expression rate of two genes related to tumor size and stage of disease (p <0.05).
Conclusion, The findings were special genes from breast cancer cells in penpheral blood
Keyv/ords:. RT-PCR, Sun/ivin. hMAM.
breast cancer
I. D A T V A N DE:
Ung thu vu la loai ung thu pho bien a phu nu' nhieu nuac tren the gioi, dae biet la cac nuac dang phat trien. Hang nam tren toan the giai co khoang 1.3 trieu phu nu mac benh
• Binh viin Thanh Nhin Ha Ndi.
"* Viin Cdng nghi Smh hoc
Trudng Dai hoc Y Hi Noi.
Hpl NGHI KHOA HQC cCiA Hpl H 6 A SINH Y papC H A N Q I VA CAC TJNH P H I A B A C L A N THlT XVHI
ung thu vu mdi dugrc ch4n dodn vd c6 tAi tran [10]. hMAh4 Id m$t glycoprotein g6m 93aa, 450.000 ca tu vong vi edn b§nh ndy [I ]. Theo
nhChig \</ thuyet ve ung thu hpc tru6c ddy, s\r di cdn cOa cdc t^ bdo ung thu chi xdy ra 6 giai do^n muOn. Tuy nhiSn, theo cdng trinh mdi nhat ciia cdc nhd khoa hpc MJf. cdc t^
bao ung thu di chuyen (circulating tumor cells - CTCs) c6 thl thdm nh§p vdo mdu ngo^i vi ngay tir giai do^n r^t s6m. S^r xu^t hi^n vd di chuyen cOa cdc CTC l£i kh6i u vdo mdu ngo^i vi bao gom nhieu giai do^n. Ban dau cdc te bdo ung thu phdt trien nhanh, can nhieu oxy nen phdt trien xam lan. Cac te bdo nay c6 mgt dp thu the tren be m^t thap dan t6i sy gidm lien ket v6i nhau va ch6ng Igi qud trinh chet theo chuong trinh. K^t qud la cae te bdo ndy c6 khd ndng song s6t cao han va de dang xam nhap vdo mau. Sau khi vao mau, cac CTC khong con kha ndng bi?t hda va di ehuyen tdi cdc ca quan khae. Tren mo hinh nghien cuu ung thu d dpng vat, ngudi ta CO the phat hien 1 te bao di cdn trong 10'' d^n lO' TB bach cau [4] Ket qua nay chi ra y nghTa cua viec phat hien ra cac CTC mang nhung gen chi thi dde trung cho khoi u trong mau, gop phan xac dinh sdm, tien lupng va theo doi dieu trj ung thu vii. Tren the gidi dd cd mpt so gen chi thj UTV dup'c phdt hien va nghien cuu, trong do hMAM va Survivin la nhung gen co dp nhay vd dp dde hi?u cao.
Survivin (baculoviral inhibitor of apoptosis repeat-containing 5 - BIRC5) la protein dupc ma hda bdi gen B1RC5. Survivin la mpt protein uc che hoat hod easpase la enzyme thuy phan protein tir do dan d6n ijre ch^ qua trinh apoptosis. Survivin mRNA co chieu dai 15kb, nam d vi tri NST 17q25. Survivin bi^u hien cao nhat trong cac ddng te bao UTV va thap han nhieu trong cae ung thu khac [5,6,7], bieu hien eiia Survivin Hen quan nhieu den mirc dp di can va tai phat benh
thdnh vien ciia hp uteroglobin. hMAM mRNA dupc md hda bdi mpt gen SCGB2A2 (sccretoglobin family 2A member2) ndm trSn nhiem sdc th^ 11 q 13. Ca che gdy ung thu ciia gen ndy Iiln quan d^n thay ddi te bao bi^u md tuyen vu, kich thich tdng trudng, tdng ty 10 phdn bdo. nd d^c hi^u cho ung thu dang bieu md. Bi^u hipn hMAM dd du^c cho Id chi gidi hgn trong tuy^n vu binh thudng vd ung thu vu [3,4]. Day Id ca sd de chiing toi tien hdnh nghien curu vdi 2 muc tieu: 1) Phai hi^n hMAM mRNA. Survivin mRNA a mau va mo b^nh nhan UTV: 2) Ttm mdi lien quan giUa su bieu hi$n ciia hMAM mRNA. Survivin mRNA va mgt so yeu to khac trong b^nh UTV.
11. 0 6 l TU0NG VA PHUONG P H A P NGHIEN CCrU:
1. Doi tvp^ng nghien cihi:
- 24 b?nh nhan duqrc chan doan xac djnh la UTV. trong dd cd 9 benh nhan <50 va 15 benh nhan >50 tuoi.
- Tieu chuan lua chpn: b?nh nhan dupc chan dodn lam sang va mo bfnh hpc Id UTV.
Ty nguydn tham gia vao nhdm nghien cuu.
Cdc b?nh nhan dupc phan loai d\ra vdo tieu chuan phan logi giai dogn b?nh TNM [1].
Trong dd T Id kich thudc u, N Id muc do di cdn hgch, M: tinh trang di cdn xa. Tieu chuan logi trie: Cd bat ky mpt khdi u hay ung thu mpt CO quan ndo khac ngodi UTV.
- 5 phy mi khde manh lam nhdm chung.
2. Nguyen li^u va phu-ong phap Nguyen lieu: Khdi bach cku dupc tdch ra tCr 5ml mdu cua doi tucmg nghien cim. 30gr md vu tuai dugc ldy ra tir benh nhan UTV dam bdo lay va bdo quan mau dung quy trinh ky thuat.
Ddng t^ bao UTV: MCF7, KPL4, BT 474, MDA-MB 23-1
Y HQC Vl$T NAM T H A N G 8 - 2/2012
Ddng tl bdo t h ^ binh thudng HEK 29- 3: lam chung dm
Hda chat de tdch chiet RNA t6ng s6 tir mdu va mo ciia hdng QIAGEN
Ay thugl: Cdc budc thyc nghi$m xdc dinh gen Survivin va hMAM nhu sau:
- Tdch RNA tong sd tir ddng te bdo UTV, theo phuang phdp tdch kit QlAamp RNA, ket qud tdch chiet du(?e di^n di kiem tra tren gel agarose. Dp tinh sgch va hdm lupng RNA dupc xac dinh bdng pho h^p thy
trfin mdy do NanoDrope -1000. Dp tinh s^h phy thupc vdo ty sd OD260nm/OD28onm. Mpt acid nucleic dupe gpi Id s^ch khi ty so ndy Id 1,8 - 2,0.
Khi cdc m§u nghien cuu ddm bdo dp tinh sgch mdi tien hdnh cdc quy trinh kj' thugt tiep theo.
Tgo cDNA: Sir dyng k>^ thugt RT-PCR (Reverse Transcription-PCR) de tong hpp cDNA tir mRNA.
- PCR nhan bdn gen vdi primer ddc hieu cd trinh ly nhusau:
Tdn primer GAPDH F GAPDH R hMAMFI hMAM Rl hMAM F 2 hMAM R2 Sun/ivin F Survivin R
Trinh t y 5'-CGG AGT CAA CGG ATT TGG I C G TAT-3' 5'- AGO CTT CTC CAT GGT GGT GAA GAC-3 5'- GAA GTT GCT GAT GGT OCT CAT GCT GGC-3' 5'- CTC ACC ATA CCC TGC AGT TCT GTG AGC-3' 5'- CTC CCA GCA CTG CTA CGC AGG CTC-3' 5'- CAC CTC AAC ATT GCT GAG AGT TTC ATC CG-3' 5'- AGAACTGGCCCTTCTTGGAGG- 3'
6' - CTTTTTATGTTCCTCTATGGGGTC-3'
T m 65,3 67 71 3 7 1 3 72,1 70 4 63 54
K T ( b p ) 307
326
203
170
- Dien di tren Agarose de kiem tra ket qua - Giai trinh ty mau duong tinh so sdnh vdi cau true eiia gen hMAM va Survivin 6 ngan hang gen.
- Sau khi nhan ban thdnh cdng hMAM mRNA va Survivin mRNA d it nhdt mpt ddng t6 bdo ung thu via, se dp dyng quy trinh chuan de nhan ban trong mau vd md.
- Ky thuat dinh luang CA-15-3: ban chdt la mot glycoprotein khdng ddc hieu nhung nhay cam ddi vdi UTV. Binh thudng cd trong huySt thanh ngudi vdi ndng dp < 30 U/ml. dinh lupng dua vdo ky thuat miln djch hda phdt quang thyc hi?n tren mdy mien djch COBAS 601
- Phuang phap: Nghien cuu md td cdt ngang. Su dung Chi-Square, Fishers exact test d§ so sanh cac ty le.
III. KET QUA NGHIEN CUU:
1. Phat hien HMAM mRNA va Survivin mRNA trong nhom nghien ciru
LL Kit qua phat hi^n hMAM mRNA va Survivin mRNA & ddng te bao UTV
Hinh I: Ddng te bao MCF 7
HCI NGHI KHOA HQC CCiA Hpl H6A SINH Y DUqC HA NQI VA CAC TINH PHIA B^C U N THCf XVIII g 1: Ket qua hMAM va Survivin o
Ddng ti bio KPL4
MDA-MB23-1 BT474 MCF7 HEK 29-3
dfinn TB hMAlU
+ + +
-
Survivin + + + + Nh^n xit: Trudc khi ti^n hdnh thi
-
nghi?m trfin mSu b^nh phdm chdng tdi dd thyc hi?n quy trinh phdt hi$n cdc mRNA tii bon ddng te bdo UTV (MCF7, MDA-MB 23-
1, KPL4, BT474) vd ddng to bdo binh thudng (HEK 29-3) vdi myc dich chuk hda k?
thu^t, xdc dinh sy bieu hi$n ciia gen nghiSn euu trong it nhat mpt ddng TB UTV. Chiing tdi tim thay sy phien md ciia hMAM d 3/4 ddng TB UTV. Survivin d 4/4 ddng te bdo UTV. Khdng thay sy bieu hi$n ciia hMAM mRNA vd Surviviiy mRNA d ddng te bdo than thudng (HEK 29-3). Dieu ndy chiirng td
hMAM vd Survivin cd bi^u hi§n cao d ddng TB UTV. Ddy Id co sd de ti^n nhdn bdn gen nghiSn ciru d mau b^nh phdm.
L2, Kit qua phdt hifn hMAM mRNA va Survivin mRNA & nhdm nghiin cmt (NC):
Sau khi tdch RNA, sd dyng ky thudt RT- PCR tgo cDNA chung tdi tien hdnh phdn ijrng PCR nhan bdn moi GAPDH. Tat cd cdc mau NC deu duong tinh khi kiem tra bdng GAPDH Id gen npi chuan, dieu ndy chung to chat lupng RNA vd qud trinh tgo cDNA ddp img cdc dieu ki^n can thiet cho thi nghidm xdc djnh gen nghien cim.
Hinh 2: Ket qud dipn di sdn pham RT-PCR vdi mdi GAPDH
1,2,3: mau mdu BN UTV; 4,5,6 mau md BN UTV; 7,8,9 mau mdu khde mgnh; 10: nudc cdt, 11: ddng tg bdo MCF7;M: Marker lOOObp.
Y Hpc VigT NAM T H A N G 8 • 2/2012
Hinh 3: Kk qui di?n di sdn phim RT-PCR v6i mbt hMAM
TCr 1-6 md UTV (BN mi s6 2,3,4,5.7,8), tir 7-12 miu UTV (BN mi 2,3,4,5,7,8); 13: ddng TB MCF7; 14: ddng TB KPL4; 15: ddng TB BT474, 16 ddng TB MDA-MB 23-1, Marker lOOObp,
Hinh 4: Ket qua di$n di san pham RT -PCR voi moi Survivm va GAPDH a mo BN UTV.
1: Nudc cat; 2,3,4: Ket qua bieu hi?n duong tinh vdi mdi GAPDH; 5: Ket qua bieu hi?n duong tinh vdi mdi Survivin; 6: ket Ket qua bieu hien am tfnh vdi mdi Survivin; M: marker lOOObp
Hinh 5: KSt qua difn di san pham RT -PCR vdi mdi Survivin trong mau BN UTV.
1,2,3,6,7,8,9: duong tinh (Bn ma 6,7,8,11,12,13,14); 4,5: am tinh (BN ma 9,10), M;
Marker lOOObp.
H^l NGHj KHOA HQC cClA Hfy H 6 A SINH Y Dt/qC HA N^I VA CAC TINH PHIA BAC LAN TM XVIII
gang 2: Ty 1^ bi^u hijn hMAM mRNA, .Survivin mRNA d mlu benh pham Miu b4nh phim
MSu UTV MS UTV M i u khde m^nh
n 24 22 5
hMAM 14 (58,3%) 20(90,9%) 0 (0%)
SurvMn 11 (45,6%) 17 (77,2%) 0 (0%)
Nfi^n xit: Ty I? bieu hi?n hMAM mRNA vd Survivin mRNA d md UTV cao han trong mdu. Dieu ndy phii h<7p vi te bdo ung thu cd nguAn g6e tir md ung thu xam nhgp vdo mdu.
Cdc te bdo ung thu trong mdu khdng cd khd ndng bi^t hda di chuydn d^n cdc ca quan khde.
Bieu hi?n ciia hMAM trong mdu ciia b?nh nhdn UTV Id 58,3% vd Survivin Id 45,8% cao han so vdi nghien cuii ciia Shen Changxing 36.2%, 33% [8]. Khdng ihky ban sao ciia hMAM\k Survivin d m§u chung binh thudng. Dieu ndy phii h(7p vdi nhieu nghien cuu khdng thay bieu hi$n ciia hMAM vd Survivin d nhdm khde mgnh tinh nguySn tham gta nghifin ciru[4,5,7.8].
2. Moi lien quan giira sy bieu hl^n ciia hMAM mRNA, Sur\i\in mRNA trong mau ngoai vi vd mpt so yeu to khde trong hynh UTV.
C i c y6u td l i i n quan UTV TuAi
2 50
< 5 0
Kich thu'd'C u
<2cm 2-5cm
>5cm
Di can xa MO M l
H^ch khdng cd
Giai doan b$nh 1
2 3 v i 4
K § t q u 4 C A 1 5 - 3 Tang Binh thu'dng
Th^ mo b^nh hpc The ong xSm nh$p Th^ tieu thuy T h l nhay
n
15 9
4 12 8
22 2
7 17
4 12 8
4 20
16 4 4
hIVIAM (%)
8/15(63%) 6/9 (67%) P- 0,52
3/4 (75%) 4/12(33.3%)
7/8 (85%) P= 0,04
12/22(64,6%) 2/2 (100%)
P= 0,21
6/7(71,4%) 9/17(52,9%) P=0,42
3/4 (75%) 4/12 (33,3%)
7/8 (87.5%) P=0,04
4/4(100%) 10/20(50%) P=0,06
8/16 (50%) 2/4 (50%) 4/4(100%) P=0,*8
Survrvin {%)
9/15(60%) 2/9 (22%) P= 0,07
0/4 (0%) 5/12 (42%)
6/8 (75%) P=0,04
9/22 (40,9%) 2/2 (100%)
P=0.1
0/7 (0%) 11/17(64,7%)
P=0.01
0/4 (0%) 5/12(41,7%)
6/8 (75%) P=0,04
3/4 (75%) 8/20 (40%)
P=0,2
8/16 (50%) 1/4 (25%) 2/4 (50%) P=0,78
Y Hpc VigT NAM THANG B - 2/2012 Nh^n xit: Khdng thdy sv khde bi$t v^ ty
1^ bieu hi$n cua hMAM mRNA vd Survivin mRNA vdi tudi tren vd dudi 50. tinh tr^ng di cdn xa, cung khdng thay cd sy khde bi?t ve ty 1? bieu hi^n cua cdc gen ndy vdi cdc the md b?nh hpc trong nhdm UTV du(;rc nghien cdu.
Ty 1$ bieu hi?n hMAM va Survivin cd lien quan vdi kich thudc u, giai dogn b?nh (p<0.05). Ty 1? bieu hi?n ciia Survivin thap hem hMAM nhung cd sy lien quan giO:a bifiu hi?n Survivin vdi tinh trgng di can hgch (p=0,01). cdn sy bieu hien ciia hMAM khdng cd sy khac bi?t g\Qa nhdm cd vd khdng cd di cdn hgch. Trong so 24 b?nh nhan UTV ma chung tdi nghien cuu cd 4 trudng hop cd ket qud CA15-3 tdng. chifim ty I? 16.7%. Ty I?
bieu hien ciia gen Survivin vd hMAM khdng cd sy khac bi?t d nhdm cd ket qud CAI5-3 tang hay binh thudng vdi (p>0.05).
IV. KET LUAN:
4.1. Da xac dinh duac hMAM mRNA vd Survivin mRNA tir te bdo ung thu vu trong mau. Ty le bieu hien ciia hMAM mRNA d md: 20/22 (90,9%), d mau Id 14/24 (58,3%).
Ty le bieu hien cua Survivin a md la 17/ 22 (77,2%), d mdu Id 11/24 (45,8%). Khdng thiy sy biSu hien cua hMAM vd Survivin d mdu ngudi khde manh.
4.2. Ty le bi^u hien eiia hMAM, Survivin trong mdu lien quan d^n kich thudc u va giai dogn benh vdi (p<0,05). Ty 1? bieu hien ciia Survivin lien quan den muc dp di can hach (p
=0,01), trong khi ly le bieu hi?n ciia hMAM khong lien quan den di can hach cua benh nhan UTV (p=0,42). Ty ie bi^u hien cua hMAM va Survivin khdng eo sy khac biet d benh nhan tren va dudi 50 tudi, tinh trang di can xa, muc dp bieu hien CAI5-3 (p>0,05) va thl md benh hoc trong nhdm nghien euu.
TAI LIEU THAM K H A O :
1. Nguyin Bd Dire (2004). "B?nh ung thu vii", Nhd xuat bdn y hpc 13-458
2. Chung- Chi Chen et al (2006),"Combination of Multiple mRNA Markers (PTTGI. Survivin, UbcHlO and TKl) in the Diagnosis of Taiwanese Patients with Breast Cancer by Membrane Array"
3. Kurt Grunewald et al, (2000),
"Mammaglobin gene expression: a superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth- factor receptor and cytokeratin-19", lab Invest, 80. 1071 -1077
4. Otto Zach et al (1999), " Detection of circulating Mammary carcinoma cells in peripheral blood of breast cancer patients via a nested reverse transcriptase polymerase chain reaction assay for Mammaglobin mRNA", journal of clinical oncology, vol 17. (July), 2015 - 2025 5. Paul N. Span, Fred C.G.J. Sweep et
al.(2004), '"Survivin Is an Independent Prognostic Marker for Risk Stratification of Breast Cancer Patients", Clinical Chemistry; 50,1986-1993.
6. SHIN-ICHl VAMASHITA,YOSHIKO MASUDA(2007), " Survivin Expression Predicts Early Recurrence in Early-stage Breast Cancer", Anticancer Res.;27(4C):
2803-8.
7. Tamm I, Wang V,et al (1998). "lAP- family protein Survivin inhibits easpase activity and apoptosis induced by Fas (CD95), Bax. caspases, and anticancer drugs" . Cancer Res. 58 (23): 5315-20.
PMID 9850056.
8. Shen Changxin et al (2009), "The detection of circulating tumor cells of breast cancer patients by using multimarker (Survivin, hTERT and hMAM) quantitative real-time PCR", Clinbiochem vol 42, issue 3, 194 - 200.
