KHẢO SÁT TÌNH TRẠNG METHYL HÓA GEN p76/XK“2 TẠI CÁC ĐẢO CpG THUỘC VÙNG PROMOTER TRÊN MỘT SÓ BỆNH UNG THƯ

B TR GIÁO D C VÀ ÀO T O NG IH CM TP.HCM BÁO CÁO KHÓA LU N T T NGHI P Tên đ tài: KH O SÁT TÌNH TR NG METHYL HÓA GEN p16INK4 T I CÁC O CpG THU C VỐNG PROMOTER TRểN M TS B NH UNG TH KHOA CỌNG NGH SINH H C CHUYểN NGÀNH: Vi sinh - Sinh h c phơn t CBHD: PGS.TS Lê Huy n Ái Thúy ThS Lao c Thu n SVTH: Lý Th Tuy t Ng c MSSV: 1053012493 Khóa: 2010 - 2014 Tp H Chí Minh, tháng n m 2014 17 L IC M N Tôi xin bƠy t lịng kính tr ng vƠ bi t n chơn thƠnh đ i v i PGS.TS Lê Huy n Ễi Thúy, ng i đ y nhi t huy t v i khoa h c đƣ truy n cho ng n l a đam mê nghiên c u vƠ ln giúp đ tơi lúc khó kh n Xin chơn thƠnh c m n ThS Lao c Thu n, ng i th y đƣ ln t n tình h ng d n, đ ng viên vƠ giúp đ su t trình th c hi n đ tƠi Tôi c ng xin c m n ThS.Tr ng Kim Ph Thí Nghi m Sinh H c Phơn T - Tr ng vƠ t t c thƠnh viên phòng ng H M TP.HCM đƣ h tr , giúp đ su t th i gian v a qua Cu i xin bƠy t lòng bi t n sơu s c đ n Ba M vƠ em đƣ ln u th ng, ch m sóc, đ ng viên vƠ t o u ki n t t nh t cho Tp H Chí Minh, Tháng 5, 2014 Tuy t Ng c DANH M C CÁC B NG BI U PH N T NG QUAN TÀI LI U PH N V T LI U VÀ PH NG PHÁP B ng 2.1 B ng m i gen p16INK4 B ng 2.2 Các b c tách chi t phenol/chloroform B ng 2.3 ThƠnh ph n dung d ch lysis buffer B ng 2.4 Các b c ch n b Ct conversion reagent (EZ1) B ng 2.5 ThƠnh ph n ph n ng PCR PH N K T QU VÀ TH O LU N B ng 3.1 Khái qt tình hình methyl hóa gen p16INK4 m t s b nh ung th B ng 3.2 Ph ng pháp đ c s d ng 24 cơng trình nghiên c u B ng 3.3 Lo i m u b nh ph m đ c s d ng 24 cơng trình nghiên c u B ng 3.4 B ng trung bình t n s có tr ng s m t s b nh ung th B ng 3.5 Khái qt tình hình methyl hóa gen p16INK4 b nh ung th vú B ng 3.6 Khái qt tình hình methyl hóa gen p16INK4 b nh ung th c t cung B ng 3.7 B ng trung bình t n s có tr ng s TBUT, TBBT vƠ ung th theo giai đo n b nh ung th vú B ng 3.8 B ng trung bình t n s có tr ng s TBUT, TBBT vƠ ung th theo giai đo n b nh ung c t cung B ng 3.9 K t qu kh o sát đ c tính v t lỦ c a m i B ng 3.10 B ng n ng đ vƠ ch t l B ng 3.1 B ng k t qu s b ph ng DNA tách chi t ng pháp MSP B ng 3.13 B ng mô t đ c m m u b nh ph m ung th vú B ng 3.13 M i t ung th vú ng quan gi a t l methyl hóa t i vùng promoter gen p16INK4 v i B ng 3.14 M i t ng quan gi a t l methyl hóa t i vùng promoter gen p16INK4 v i y u t lơm sƠng b nh nhơn ung th vú B ng 3.15 M c đ liên quan gi a tính ch t methyl hóa t i vùng promoter p16INK4 v i ung th vú gen DANH M C HÌNH V VÀ S PH N T NG QUAN TÀI LI U Hình 1.1 T l m c b nh vƠ t l t vong toƠn th gi i Hình 1.2 T l m c b nh ung th vú toƠn th gi i Hình 1.4 Mơ t ung th vú Hình 1.5 Ung th c t cung Hình 1.6 C ch epigenetics bao g m hi n t ng methyl hóa DNA, bi n đ i histone vƠ micro - RNA Hình 1.7 Q trình methyl hóa DNA Hình 1.8 C ch methyl hóa DNA chuy n hóa - cystosine (5C) thƠnh methylcytosine (m5C) Hình 1.λ C ch bi n đ i epgenetics gơy im l ng gen Hình 1.10 Mơ hình methyl hóa DNA Hình 1.11 Mơ hình methyl hóa DNA Hình 1.12 trình t l p l i nh gen p16INK4 NST s λ thu c b gen ng i Hình 1.13 Quá trình phiên mƣ gen p16INK4 Hình 1.14 C ch hình thƠnh kh i u Hình 1.15 X lỦ sodium bisulfite vƠ s chuy n đ i cytosine thƠnh uracil PH N V T LI U VÀ PH NG PHÁP Hình 2.1 Chu trình nhi t bi n đ i bisulfite c a EZ DNA METHYLATION GOLDTM kit Hình 2.2 Chu k gradient nhi t cho ph n ng PCR Hình 2.3 Chu k nhi t cho ph n ng PCR PH N K T QU VÀ TH O LU N Hình 3.1 T n s methyl hóa trung bình có tr ng s c a gen p16INK4 m u ung th ph i, vú, c t cung vƠ đ i tr c trƠng Hình 3.2 T n s methyl hóa trung bình có tr ng s c a gen p16INK4 ung th vú vƠ phơn chia theo giai đo n b nh Hình 3.3 T n s methyl hóa trung bình có tr ng s c a gen p16INK4 ung th c t cung vƠ phơn chia theo giai đo n b nh Hình 3.4 nh v gen p16INK4 NST s λ Hình 3.5 S đ hình nh vùng đ o CpG trình t gen kh o sát Hình 3.6 V trí vùng promoter, 5’UTR vƠ exon trình t kh o sát Hình 3.7 Trình t gen p16INK4 vƠ s phơn b v trí phiên mƣ trình t Hình 3.8 V trí trình t m i methyl vùng trình t promoter thu c gen p16INK4 Hình 3.λ K t qu ph n ng gradient nhi t đ kho ng 51 oC - 62 oC Hình 3.10 K t qu n di s n ph m MSP Hình 3.11.K t qu gi i trình t s n ph m MSP DANH M C CH VI T T T 5C V trí carbon s BSP Bisulfite sequencing PCR C Cytosine CDK Cyclin Depedent Kinase CKIs Cyclin Kinase Inhibitors CpG Cystosine phosphate Guanine DNMT DNA methyltranfarase EDTA Ethylenediaminetetraacetic acid G Guanidine Kb Kilo base pair M5C Nhóm methyl (CH3) g n vƠo C s MSP Methylation specific PCR NCBI National Center for Biotechnology Information NST Nhi m s c th OD Optical Density PCR Polymerase Chain Reaction Pha G Pha GAP Pha M Mitosis Pha S Pha Synthesis pRB Product of retinoblastoma RT - PCR Real time PCR SAM S - adenosyl - L - methionine SDS Sodium dodecyl sulfat TBBT T bƠo bình th TBUT T bƠo ung th Tm Nhi t đ nóng ch y ng Ta Nhi t đ lai UTCTC Ung th c t cung UTDTT Ung th đ i tr c trƠng UTP Ung th ph i UTR Untranlated region UTV Ung th vú M CL C L ic m n DANH M C CỄC B NG BI U DANH M C HÌNH V VÀ S DANH M C CH TV N VI T T T PH N T NG QUAN TÀI LI U 1.1 TÌNH HÌNH NGHIểN C U TRONG VÀ NGOÀI N C 1.2 T NG QUAN V UNG TH 1.2.1 Khái ni m ung th 1.2.2 Tình tr ng ung th th gi i vƠ Vi t Nam 1.2.3 T ng quan ung th vú 1.2.4 T ng quan ung th c t cung 10 1.3 EPIGENETICS 13 1.3.1 nh ngh a 13 1.3.2 Hi n t 1.3.3 ng epigenetics 13 o CpG 14 1.3.4 Chu k t bƠo vƠ gen p16INK4 15 1.3.5 Methyl hóa DNA 16 1.3.6 T ng quan v gen p16INK4 20 1.3.7 Con đ ng d n đ n s u hòa b t th ng gen p16INK4 b nh ung th 22 1.3.8 Các ph ng pháp phát hi n s methyl hóa 24 PH N V T LI U VÀ PH 2.1 TH I GIAN VÀ 2.2 V T LI U VÀ PH NG PHỄP A I M NGHIểN C U 26 NG PHỄP NGHIểN C U 26 2.2.1.V t li u 26 2.2.2 Ph ng pháp 26 PH N K T QU VÀ TH O LU N 3.1 K T QU KHAI THỄC D LI U 35 3.1.1 Khai thác d li u m t s lo i ung th 35 3.1.2 Khai thác d li u ung th vú vƠ ung th c t cung 40 3.2 KH O SỄT IN SILICO 46 3.2.1 Xác đ nh c u trúc gen, v trí gen p16INK4 46 3.2.2 Kh o sát v trí vƠ c u trúc đ o CpG thu c vùng promoter gen p16INK4 46 3.2.3 ánh giá m i 49 3.3 K T QU TH C NGHI M 52 3.3.1 K t qu tách chi t DNA 52 3.3.2 K t qu ph n ng MSP m u mô đúc paraffin 53 3.3.3 Gi i trình t s n ph m MSP 55 3.3.4 Mô t đ c m m u b nh ph m ung th vú theo ch tiêu lơm sƠng vƠ c n lơm sƠng 57 3.3.5 Xác đ nh m i t ng quan gi a s methyl hóa gen p16INK4 vƠ b nh ung th 58 Khóa lu n t t nghi p B ng 3.15 M i t ng quan gi a t l methyl hóa t i vùng promoter gen p16INK4 v i y u t lơm sƠng b nh nhơn ung th vú M u b nh Nhóm tu i Methyl p16INK4 Không methyl p16INK4 N1 (50) (16,7%) (50,0%) Giá tr p* 0,5241 Giai đo n b nh I (50%) (0%) II (50%) (33,3%) III (0%) (33,3%) IV (0%) (33,3%) Giá tr p* 0,2330 p* > 0,05,  Th ng kê b ng ph n m m Medcal (B , 2014) Ghi chúμ m u mô vú c a b nh nhơn tu i 50 l n l tu i nh h n 40, t 40 - 50 vƠ cao h n t lƠ N1, N2, N3 M u mô vú giai đo n I, II, III, IV i v i y u t ắnhóm tu i” c a b nh nhơn đ c nghiên c uμ Nhóm b nh nhơn N1 (có đ tu i < 40) chi m t l ph n tr m cao nh t nhóm tu i kho ng 50,0% , u nƠy ch ng t nguy c ung th vú tr c giai đo n ti n mƣn kinh Phơn tích th ng kê, cho th y y u t nhóm tu i vƠ m c đ methyl hóa gen p16INK4 khơng có s khác bi t có Ủ ngh a giá tr p - value b ng 0,5241 cao h n 0,05 (đ c trình bƠy B ng 3.14, B ng ph l c 2) i v i y u t ắgiai đo n” ung th μ s li u đ c trình bƠi B ng 3.14, B ng ph l c 3, nh n th y giai đo n ung th vú, m c đ methyl hóa giai đo n I, II có ph n tr m cao nh t chi m kho ng > 50,0% hay nói cách khác methyl hóa đ c bi u hi n s m trình sinh u vƠ trình b nh hóa c a ung th vú Tuy nhiên, giá tr p - value đ t 0,2330 cao h n 0,05 nên th ng kê khơng có Ủ ngh a Lý Th Tuy t Ng c Trang 59 Khóa lu n t t nghi p 3.3.5 Kh o sát m c đ liên quan (t su t chênh - Odd ratio - OR vƠ nguy c t ng đ i - relative risk - RR) gi a tính ch t methyl hóa v i ung th T su t chênh (Odd ratio) vƠ y u t nguy c t giá hay đo l ng đ i (ralative Risk) dùng đ đánh ng m c đ m nh y u c a m i liên quan m c đ methyl hóa v i lo i m u ung th vú (m u lƠnh vƠ m u ung th ) K t qu th ng kê cho th y, t su t chênh (OR = 2,6667) vƠ y u t nguy c (RR = 2,0000) có giá tr p - value l n l t lƠ 0,4968; 0,5087 v i p > 0,05 khơng có s khác bi t th ng kê Nguyên nhơn lƠ s m u nghiên c u h n h p, n u t ng s l ng m u ung th vú có kh n ng th hi n đ cs t ng quan v m c đ methyl hóa v i lo i m u (m u lƠnh vƠ m u ung th ) B ng 3.16 M c đ liên quan gi a tính ch t methyl hóa t i vùng promoter gen p16INK4 v i ung th vú Lo i m u p16INK4 Giá tr p* Không methyl Methyl T ng M u lƠnh (66,67%) (33,33%) (30%) M u ung th vú (42,86%) (57,17%) (70%) (40,0%) (60%) 10 (100%) p* = 0,6726 T su t chênh (OR) 2,6667 0,4968 T s nguy c (RR) 2,0000 0,5087 p* > 0,05,  Th ng kê b ng ph n m m Medcal (B , 2014) Lý Th Tuy t Ng c Trang 60 Khóa lu n t t nghi p 4.1 K T LU N Thu th p thông tin, xác đ nh v trí vƠ mơ t c u trúc vùng promoter, đ o CpG vƠ exon thu c gen p16INK4a Th ng kê t n s methyl hóa gen p16INK4 m u ung th vú, ung th c t cung, ung th ph i, ung th đ i tr c trƠng l n l 83,02% t ng s 24 cơng trình nghiên c u đ t lƠ 52,5%, 68,28%, 3λ,6λ%, c công b th gi i Kh o sát vƠ đánh giá t n s methyl hóa gen p16INK4 10 m u mơ vú đ paraffin Vi t Nam T n s methyl hóa t bƠo ung th , m u lƠnh l n l 57,14%, 33,33% đ ng th i xác đ nh m i t promoter c đúc t lƠ ng gi a t l methyl hóa t i vùng gen p16INK4 v i y u t lơm sƠng b nh nhơn ung th vú đ tu i nh h n 40 (50%) hay giai đo n I (50%) vƠ II (50%) T s OR (t su t chênh Odd ratio - OR) đ t 2,6667 vƠ t s RR (nguy c t ng đ i - relative risk) đ t 2,0000 4.2 NGH Kh o sát th c nghi m gen p16INK4 nhi u b nh ung th , t ng c kh o sát tình tr ng methyl hóa m u ung th ng c m u Vi t Nam Ti p t c ti n hƠnh th c nghi m kh o sát tính ch t methyl hóa k t h p v i ghi v phơn lo i mô h c đ đánh giá kh n ng s d ng tính ch t methyl hóa nƠy nh m t d u ch ng sinh h c (Bio - marker) h u hi u cho b nh ung th vú Vi t Nam Lý Th Tuy t Ng c Trang 61 Khóa lu n t t nghi p TÀI LI U THAM KH O Ti ng Vi t [1] Lê Th Trúc Linh, H B o Khuyên, Lê Huy n Ễi Thúy (2010), Xơy d ng quy trình Methylation specific PCR nh m kh o sát m c đ methyl hóa t i đ o CpG thu c vùng promoter hai gen Trail - R3, Trail - R4 b nh nhơn b ung th c t cung, T p chí Cơng ngh sinh h c, 8(3B), tr 1091 ậ 1096 Ti ng Anh [2] AS B (1999), Blood , 5(94), pp 1773 - 1781 [3] Baker D.J (2008), Opposing roles for p16INK4 and p19Arf in senescence and ageing caused by BubR1 insufficiency, Nat Cell Biol, 10(7), pp 825 - 836 [4] Bean G.R (2007), Morphologically normal - appearing mammary epithelial cells obtained from high - risk women exhibit methylation silencing of INK4a/ARF, Clin Cancer Res, 13(22), pp 6834 - 6841 [5] Bearzatto A (2002), Hypermethylation p16INK4 detected by Fluorescent Methylation - Specific PCR in plasmas from non - small cell lung cancer, Clin Cancer Res, 3782 (8), pp 3782 - 3787 [6] Carestiato F N (2013), An upward trend in DNA p16INK4 methylation pattern and high risk HPV infection according to the severity of the cervicial lesion, Rev Inst Med Trop Sao Paulo, 55(5), pp 329 - 334 [7] Choi B Y (2005), The tumor suppressor p16INK4 prevents cell transformation through inhibition of c - Jun phosphorylation and AP activity, Nature Structural and Molecular Biology Cell Biol., 12, pp 699 - 707 Lý Th Tuy t Ng c Trang 62 Khóa lu n t t nghi p [8] Coleman K.G (1997), Identification of CDK4 sequences involved in cyclin D1 and p16 binding, J Biol Chem, 272(30), pp 18869 18874 [9] Cutts F.T (2007), Humanpapillo virus and HPV vaccines: a review, Bulletin of the World Health Organization [10] Das P.M (2004), DNA methylation and cancer, Journal of Clinical Oncology, 22(22), pp 4632 - 4642 [11] Deep J S (2012), Aberrant methylation in promoters of GSTP1, p16, p14 and RASSF1A genes in smokers of North India, International Scholarly Research Network ISRN Pulmonology, ID 247631, pp - [12] Delpu Y (2013), DNA methylation and cancer diagnosis, Int J Molecular Sciences, 14(7), pp 15029 - 15058 [13] Herman J.G (1996), Methylation - specific PCR: A novel PCR assay for methylation status of CpG islands, Medical Sciences, 93, pp 9821 - 9826 [14] Hibi K (2001), Mitochondrial DNA alteration in esophageal cancer, International Journal of Cancer, 92(3), pp 319 - 321 [15] Lee J.J (2012), Tumor suppressor gene in primary breast cancer tissue and their quantitative p16INK4 hypermethylation in plasma by Real - Time PCR, The Korean Journal of Pathology, 46, pp 554 - 561 [16] Hu X.C (2003), Tumor - derived aberrant methylation in plasma of invasive ductal breast cancer patients: clinical implications, Oncol Rep., 10(6),pp 1811 - 1815 [17] Jha A K (2012), p16INK4 and p15INK4 gene promoter methylation in cervical cancer patients, Oncology Letters, 3, pp 1331 - 1335 [18] Janzen V (2006), Stem - cell ageing modified by the cyclin dependent kinase inhibitor p16INK4, Nature, 443, pp 421 - 426 [19] Jarmalaite S (2003), Aberrant p16 promoter methylation in smokers and former smokers with nonsmall cell lung cancer, Lý Th Tuy t Ng c Trang 63 Khóa lu n t t nghi p Iternational Journal of Cancer,106(6), pp 913 - 918 [20] Jemal A (2011), Global cancer statistics, CA Cancer J Clin, 61(2), pp 69 - 90 [21] Jennifer B (2009), Promoter methylation and the detection of breast cancer, Cancer causes control, 20(9), pp 1539 - 1550 [22] Jie G (2007), Relationship between expression and methylation status of p16INK4 and the proliferative activity odifferent areas' tumour cells in human colorectal cancer, Int J Clin Pract, 61(9), pp 1523 1529 [23] Jing F (2007), Hypermethylation of tumor suppressor genes BRCA1,p16 and 14 - - 3sigma in serum of sporadic breast cancer patients, Onkologie, 30(1 - 2), pp 14 - 19 [24] Kamb A (1994), A cell cycle regulator potentially involved in genesis of many tumor types, Science, 264(5157), pp 436 - 440 [25] Kim N.B (2005), Methylation and expression of p16INK4 tumor suppressor gene in primary colorectal cancer tissues, Internationa Journal of Oncology, 2, pp 1217 - 1226 [26] Jane B.R (2011), The cell cycle, Campell biology, Chapter 12 [27] Kim W.Y (2006), The regulation of INK4/ARF in cancer and aging, Cell, 127(2), pp 265 - 275 [28] Kim, Y.T and M Zhao (2005), Aberrant cell cycle regulation in cervical carcinoma, Yonsei Medical, 46(5), pp 597 - 613 [29] Krishnamurthy J (2004), INK4/ Arf expression is a Bio - marker of aging, J Clin Invest, 114, pp 1299 - 1307 [30] Laird, P.W (2003), The power and the promise of DNA methylation markers, Nature Publishing Group, [31] Latest world cancer statistics Global cancer burden rises to 14.1 million new cases in 2012: Marked increase in breast cancers must be addressed, (2013), International Agency for Research on Cancer Lý Th Tuy t Ng c Trang 64 Khóa lu n t t nghi p [32] Le T.T.L., Ho T.B.P., Ton N.T.K., Doan T.P.T, Le H.A.T (2011), Appraisal of potential methylation Bio - markers: BRCA1, p16INK4, cyclin D2, GSTP1, RASSF1A in breast cancer early detection, Journal of Science and Technology, 49(1A), pp 329 - 337 [33] Li J (2002), Novel insights into the INK4 - CDK4/6 - Rb pathway: counter action of gankyrin against INK4 proteins regulates the CDK4 mediated phosphorylation of Rb, Biochemistry, 41, pp 3977 - 3983 [34] Li J (2011), The Regulatory Mechanisms of Tumor Suppressor p16INK4 and Relevance to Cancer, Biochemistry, 25(50), pp 5566 5582 [35] Liggett W.H (1998), Role of the p16 Tumor Suppressor Gene in Cancer, Jounnal of Clinical Oncology, (16)3, pp 1197 - 1206 [36] Lo P K (2008), Epigenomics and breast cancer, Pharmacogenomics, (9)12, pp 1879 - 1902 [37] LoF - Ohlin Z.M (2011), Hypermethylation of promoter regions of the APC1A and p16INK4genes in relation to prognosis and tumor characteristics in cervical cancer, International Journal of Oncology, 39, pp 683 - 688 [38] Lu Q (2012), DNA methylation changes in cervicial cancer, Cancer Epignetics Method in Molecular Biology, 863, pp 155 - 176 [39] Malhotra P (2010), Aberrant promoter methylation of p16 in colorectal adenocarcinoma in North Indian patients, World J Gastrointest Oncol, 2(7), pp 295 - 303 [40] Man J.H (2010), Gankyrin plays an essential role in Ras - induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells, J Clin Invest, 120, pp 2829 - 2841 [41] Marcus W K (2013), Epigenetic changes in patients with multiple sclerosis, Nature Reviews Neurology ,9, pp 35 - 43 [42] Manel E (2007), Epigenetic gene silencing in cancer: the DNA Lý Th Tuy t Ng c Trang 65 Khóa lu n t t nghi p hypermethylome, Human Molecular Genetics, 16(1), pp 50 - 59 [43] Matouk Charles C (2008), Epigenetic Regulation of Vascular Endothelial Gene Expression, Circulation Research, 102(8), pp 873 887 [44] Mohammed A (2009), Status of p16INK4 and E - Cadheringene promoter methylation in Moroccan patients with cervical carcinoma, Oncology Research, 18, pp - 100 [45] Molofsky AV (2006), Increasing p16INK4 expression decreases forebrain progenitors and neurogenesis during aging, Nature, 443(7110), pp 448 - 452 [46] Nakayama G (2007), p16 Methylation in Serum as a potential marker for the malignancy of colorectal carcinoma, Anticancer Reasearch, 27 (5A), pp 3367 - 3370 [47] Nephew K.P (2002), Epigenetic gene silencing in cancer initiation and progression, Cancer Letters, 190(2), pp 125 - 133 [48] Nguyen H.L (2010), Cost of treatment for breast cancer in central Vietnam, Glob Health Action, 6: 18872 [49] Ono A (2011), Epigenetic aberrant hypermethylation of DNA in endometrial cancer: application as a Bio - marker, Journal of Cancer Therapy, 2, pp 610 - 615 [50] Lamy A (2002), Aberant methylation of the CDKN2A/p16 gene promoter regionon prevasive bronchial leison: aprospectives study in high - SK patients without invasive cancer, Int J Cancer, 100(2), pp 189 - 193 [51] Ota N (2006), Prognostic significance of hypermethylation p16INK4 in non - small cell lung cancer is evident by quantitative DNA methylation analysis, Anticancer Research, 26, pp 3729 - 3732 [52] Peter B., World cancer Report 2008 [online], WHO press, The International Agency for Research Cancer (IARC) Lý Th Tuy t Ng c Trang 66 Khóa lu n t t nghi p [53] Portela A (2010), Epigenetic modifications and human disease, Nature biotechnology, 28(10), pp 1057 - 1067 [54] Russo A.A (1998), Structural basis for inhibition of the cyclin dependent kinase Cdk6 by the tumour suppressor p16INK4, J Nature, 395(6699), pp 237 - 243 [55] Serizawa H (1998), Cyclin - dependent kinase inhibitor p16INK4 inhibits phosphorylation of RNA polymerase II by general transcription factor TFIIH, J Biol Chem, 273(10), pp 5427 - 5430 [56] Sharma G (2007), Promoter hypermethylation of p16INK4, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients, Life Sci, 80(20), pp 1873 - 1881 [57] Sharma S (2010), Epigenetics in cancer, Carcinogenesis, 31(1), pp 27 - 36 [58] Sherr CJ (1999), CDK inhibitors: positive and negative regulators of G1 - phase progression, Genes Dev., 13(12), pp 1501 - 1512 [59] Shima K (2011), Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: cohort study and literature review, Int J Cance, 128(5), pp 1080 1094 [60] Shin E A (2009), p16INK4 methylation and the correlation to immunohistochemical expression in cervical neoplasia, Journal of Women’s Medicine, 2(1) [61] Smith J.R (1996), Replicative senescence: implications for in vivo aging and tumor suppression, Science, 273(5271), pp 63 - 67 [62] Spathis A (2011), Promoter Methylation of p16INK4, hMLH1, And MGMT In Liquid - Based cervical cytology samples compared with clinicopathological findings and HPV presence, Infectious Diseases in Obstetrics and Gynecology, ID 927861, pp - Lý Th Tuy t Ng c Trang 67 Khóa lu n t t nghi p [63] Szalmas A (2009), Epigenetic alterations in cervical carcinogenesis, Semin Cancer Biol, 19(3), pp 144 - 52 [64] Szyf M., (2008), The role of DNA hypermethylation and demethylation in cancer and cancer therapy, Current Oncology, 15(2), pp 72 - 75 [65] Tsao H (1998), Novel mutations in the p16/CDKN2A binding region of the cyclin - dependent kinase - gene, Cancer Res, 58(1), pp 109 - 113 [66] Vallian S (2009), Methylation status of p16INK4 tumor suppressor gene in Iranian patients with sporadic breast cancer, Journal of Cancer Research and Clinical Oncology, 135(8), pp 991 - 996 [67] Veganzones - de - Castro S (2012), p16 gene methylation in colorectal cancer patients with long - term follow - up, Reveso Ennferm Dig, 104(3), pp: 111 - 117 [68] Vinci D A (2005), p16INK4 promoter methylation and protein expression in breast fibroadenoma, International Journal of Cancer, 114, pp 414 - 421 [69] Voyatzi S (2010), Promoter methylation of p16INK4, RASSF1A, and RAR2 genes in tumor DNA from patients with breast cancer (BC) in correlation with clinical recurrence, Journal finical Oncology, 28(15), 1551 [70] Walkley C.R (2006), Rb is dispensable for self - renewal and multilineage differentiation of adult hematopoietic stem cells, Proc Natl Acad Sci U S A, 103(24), pp 9057 - 9062 [71] Yang H.J (2004), Detection of hypermethylated genes in tumor and plasma of cervical cancer patients, Gynecologic Oncology, 93, pp 435 - 440 [72] Yang L (2006), Aberrant promoter methylation of p16 and MGMT genes in lung tumors from smoking and never - smoking lung cancer Lý Th Tuy t Ng c Trang 68 Khóa lu n t t nghi p patients, Neoplasia, 8(1), pp 46 - 51 [73] Yoruker E.E (2012), Promoter and histone methylation and p16 gene expression in colon cancer, Experimental and therapeutic medicine ,4(5), pp 865 - 870 [74] Zhao Y F (2010), Methylation and expression of gene p16INK4 and RB in breast carcinoma, NCBI, 39(6), pp 377 - 381 [75] Frederique M (2001), Selective association of the methyl - CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia , 98(9), pp 4990 - 4995 [76] Yakov C (2005), Melanoma genetics and the development of rational therapeutics, The Journal of Clinical Investigation, 115: pp 813 ậ 824 [77] Zou H (2002), Detection of aberrant p16 methylation in the serum of colorectal cancer patients, Clinical Cancer Research, 36(7), pp 188 - 191 Internet [78] http://globocan.iarc.fr/Pages/Map.aspx, Globocan 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012 [79] http://screening.iarc.fr/atlasclassifwho.php?lang=1,WHO histological classification of tumours of the uterine cervix, Classification uterine cervicix [80] http://www.who.int/cancer/en/, Definition cancer [81] http://ungthu.net.vn/tinh - hinh - mac - benh - ung - thu - tren - gioi - va - o - viet - nam, (MED - ADI) [82] http://www.benhvienthuduc.vn/detail?id=184, Ths.Bs.Nguy n Thu H ng, ch n đoán xác đ nh ung th vú BMR (Bach Mai Radiology) [83] http://www.cancer.gov/cancertopics/pdq/treatment/breast/Patient/pa ge5, Treatment Option Overview, Treatment Breast cancer Lý Th Tuy t Ng c Trang 69 Khóa lu n t t nghi p [84] http://www.cancer.gov/cancertopics/factsheet/detection/probability - breast - cancer, Detection breast cancer [85] http://www.cancer.gov/cancertopics/pdq/treatment/breast/Patient/pa ge2, Stages of Breast Cancer, National Cancer Institute, Stage of breast cancer [86] http://www.cancer.gov/cancertopics/pdq/treatment/breast/healthpro fessional/page, Treatment breast cancer [87] http://www.cancer.gov/cancertopics/pdq/treatment/cervical/Patient/ page2, Stage cervicial cancer [88] http://ghr.nlm.nih.gov/gene/CDKN2A, Where is the CDKN2A gene located? [89] http://www.hgu.mrc.ac.uk/people/r.meehan_researchb.html, Epigenetic Mechanisms in Development and Disease [90] http://kadinalani.com/rahim - agzi - kanseri - serviks/, Cervicial cancer [91] http://www.cancer.org/cancer/cervicalcancer/detailedguide/cervical - cancer - treating - general - information, Treatment cervicial cancer [92] http://www.dieutri.vn/daicuongungthu/14 - - 2013/S3597/Dai cuong - chan - doan - benh - ung - thu.htm, đ i c ng ch n đoán b nh ung th Lý Th Tuy t Ng c Trang 70 PH L C B ng ph c l c M c đ promoter liên quan gi a tính ch t methyl hóa t i vùng gen p16INK4 v i ung th vú K t lu n methyl T ng K t lu n ung th Ểm D ng LƠnh (30%) Ung th (70%) Total (40,0%) (60%) 10 (100%) Chi - squared 0,179 DF 1,0 Significance level p = 0,6726 B ng ph l c M c đ liên quan tính ch t methyl hóa t i vùng promoter gen p16INK4 v i nhóm tu i/ đ tu i c a b nh nhơn ung th vú K t lu n methyl Phơn nhóm T l methyl tu i Ểm N1 (50) (16,7%) (50,0%) (30%) (60%) (40%) 10 (100%) Chi - squared 1,319 DF Significance p = 0,5241 D ng B ng ph l c M c đ liên quan tính ch t methyl hóa t i vùng promoter gen p16INK4 v i giai đo n b nh b nh nhơn ung th vú Giai đo n K t lu n methyl (N,%) I 0% II 33,3% III 33,3,% IV 33,3% K t lu n methyl T l methyl Giai đo n Ểm D I (50%) (0%) (28,6%) II (50%) (33,3%) (42,9%) III (0%) (33,3%) (14,3%) IV (0%) (33,3%) (14,3%) (57,1%) (42,9%) (100%) Chi - squared 4,278 DF Significance p = 0,2330 B ng ph l c Thông tin b nh nhơn đ ng c s d ng nghiên c u: 10 m u ung th vú M u H tên b nh nhơn Lo i Giai K t qu m u đo n methyl NG TH H 44 M Lể TH KIM A 49 UM TR N NG C T 37 UM TR N TH THANH L 52 M TR N TH N 65 UM T 48 UM TR Tu i TR NG TH MỌ UT NGUY N TH NG C H 52 M HU NH NG C N 32 N UM NGUY N TH H 27 N UM 10 NGUY N T 22 N M Ghi chú: N: m u lƠnh, Mô UTμ mô ung th , UM: Unmethyl, M: Methyl