Khảo sát sự hiện diện của các gen ebna 1 và lmp q của epsrein bar virus trên bệnh ung thư vòm họng ở người việt nam nghiên cứu khoa học

TRƯỜNG ĐẠI HỌC MỞ THÀNH PHỐ HỒ CHÍ MINH BÁO CÁO TỔNG KẾT ĐỀ TÀI NGHIÊN CỨU KHOA HỌC CỦA SINH VIÊN THAM GIA XÉT GIẢI THƯỞNG CẤP TRƯỜNG Tên đề tài: KHẢO SÁT SỰ HIỆN DIỆN CỦA CÁC GEN EBNA-1 VÀ LMP-1 CỦA EPSREIN-BARR VIRUS TRÊN BỆNH UNG THƯ VỊM HỌNG Ở NGƯỜI VIỆT NAM KHOA CƠNG NGHỆ SINH HỌC CHUYÊN NGÀNH VI SINH-SINH HỌC PHÂN TỬ TP.HCM, tháng 02 năm 2016 TRƯỜNG ĐẠI HỌC MỞ THÀNH PHỐ HỒ CHÍ MINH BÁO CÁO TỔNG KẾT ĐỀ TÀI NGHIÊN CỨU KHOA HỌC CỦA SINH VIÊN THAM GIA XÉT GIẢI THƯỞNG CẤP TRƯỜNG Tên đề tài: KHẢO SÁT SỰ HIỆN DIỆN CỦA CÁC GEN EBNA-1 VÀ LMP-1 CỦA EPSREIN-BARR VIRUS TRÊN BỆNH UNG THƯ VÒM HỌNG Ở NGƯỜI VIỆT NAM KHOA CÔNG NGHỆ SINH HỌC CHUYÊN NGÀNH VI SINH-SINH HỌC PHÂN TỬ Sinh viên thực 1: Ngô Đông Kha Nữ Năm 04/04 Lớp DH12SH01 Sinh viên thực 2: Hồ Tá Giáp Nam Năm 04/04 Lớp DH12SH01 Sinh viên thực 3: Thiều Hồng Huệ Nữ Năm 04/04 Lớp DH12SH02 Sinh viên thực 4: Nguyễn Thị Hương Nữ Năm 04/04 Lớp DH12SH01 Sinh viên thực 5: Nguyễn Hải Châu Nam Năm 04/04 Lớp DH12SH03 Giảng viên hướng dẫn: ThS Lao Đức Thuận TP.HCM, tháng 02 năm 2016 MỤC LỤC DANH MỤC VIẾT TẮT ix ĐẶT VẤN ĐỀ PHẦN I 1.TỔNG QUAN VỀ UNG THƢ 1.1.Khái niệm ung thƣ 1.2.Tình hình ung thƣ giới Việt Nam 2.TỔNG QUAN VỀ UNG THƢ VÒM HỌNG 2.1.Sơ lƣợc ung thƣ vòm họng 2.2.Tình hình ung thƣ vòm họng giới Việt Nam 2.3.Tác nhân gây bệnh ung thƣ vòm họng 3.EPSTEIN-BARR VIRUS (EBV) 3.1.Tổng quan Epstein-Barr virus 3.2.Cơ chế xâm nhiễm EBV 3.3.Cấu trúc gen 3.3.1.Gen EBNA-1 (Epstein-Barr Nuclear Antigen-1) 3.3.2.Gen EBNA-2 (Epstein-Barr Nuclear Antigen-2) 3.3.3.Gen LMP-1 (Lantent membrane protein-1) 3.3.4.Gen LMP-2(Lantent membrane protein-1) 11 3.3.5.Gen BALF5 11 4.PHƢƠNG PHÁP POLYMERASE CHAIN REACTION 11 PHẦN II 13 1.VẬT LIỆU 14 i 1.1.Các phần mềm trang web đƣợc sử dụng 14 1.2.Mẫu thí nghiệm 14 1.3.Dụng cụ - thiết bị - hóa chất 15 1.3.1.Dụng cụ 15 1.3.2.Thiết bị 15 1.3.3.Hóa chất 15 1.4.Tiến trình nghiên cứu thực nghiệm 16 2.PHƢƠNG PHÁP NGHIÊN CỨU 16 2.1.Khai thác sở liệu 16 2.2.Khảo sát in silico 17 2.3.Quy trình thực nghiệm 17 2.3.1.Tách chiết DNA theo phƣơng pháp Phenol/Chloroform 17 2.3.2.Kiểm tra độ tinh sản phẩm DNA tách chiết 18 2.3.3.Khuếch đại trình tự gen mong muốn 19 2.3.4.Giải trình tự so sánh với sở liệu 19 PHẦN III 20 1.KẾT QUẢ KHẢO SÁT CƠ SỞ DỮ LIỆU 21 1.1.Kết khảo sát in silico 21 1.2.Kết khảo sát mồi 28 1.2.1.Kết kiểm tra thông số vật lý cặp mồi 28 1.2.2.Kết kiểm tra độ đặc hiệu cặp mồi 30 1.3.Chu trình nhiệt tham khảo 33 2.KẾT QUẢ THỰC NGHIỆM 34 ii 2.1.Kết tinh DNA 34 2.2.Kết tối ƣu hóa quy trình 35 2.2.1.Kết tối ƣu hóa quy trình phát gen LMP-1 35 2.2.2.Kết tối ƣu hóa quy trình phát gen LMP-2 37 2.2.3.Kết tối ƣu hóa quy trình phát gen EBNA-1 38 2.2.4.Kết tối ƣu hóa quy trình phát gen EBNA-2 39 2.2.5.Kết tối ƣu hóa quy trình phát gen BALF5 41 2.3.Kết khuếch đại trình tự gen 42 2.3.1.Kết khuếch đại gen EBNA-1 42 2.3.2.Kết khuếch đại gen EBNA-2 44 2.3.3.Kết khuếch đại gen LMP-1 45 2.3.4.Kết khuếch đại gen LMP-2 47 2.3.5.Kết khuếch đại gen BALF5 49 3.KẾT QUẢ PHÂN TÍCH THỐNG KÊ 52 4.KẾT LUẬN 57 4.1.Kết khảo sát in silico 57 4.2.Kết thực nghiệm 58 4.3 Cơng trình nghiên cứu đƣợc công 57 4.4 Các hội nghị khoa học tham 57 PHẦN IV 60 1.THẢO LUẬN 59 2.ĐỀ NGHỊ 60 TÀI LIỆU THAM KHẢO 61 iii PHỤ LỤC 70 iv DANH MỤC HÌNH ẢNH Hình 1.1 Giải phẫu vòm họng Hình 1.2 Tỷ lệ mắc phải tử vong loại ung thƣ Việt Nam Hình 1.3 Bản đồ hệ gen EBV dạng episome Hình 1.4 Cấu trúc protein EBNA1 Hình 1.5 Cấu trúc protein EBNA2 Hình 1.6 Cấu trúc protein LMP1 10 Hình 1.7 Nguyên tắc phƣơng pháp PCR 12 Hình 2.1 Tiến trình nghiên cứu nghiệm 16 Hình 3.1 Tần số phát gen LMP-1, LMP-2, EBNA-1, EBNA-2 25 Hình 3.2 Biểu đồ trung bình có trọng số loại mẫu sinh thiết mẫu máu 27 Hình 3.3 Kết Annhyb cặp mồi EBNA1-F/EBNA1-R 31 Hình 3.4 Kết Annhyb cặp mồi EBNA2-F/EBNA2-R 31 Hình 3.5: Kết Annhyb cặp mồi LMP1-F/ LMP1-R 32 Hình 3.6 Kết Annhyb cặp mồi LMP2-F/ LMP2-R 32 Hình 3.7 Kết Annhyb cặp mồi BALF5-F/BALF5-R 33 Hình 3.8 Chu trình nhiệt chung gen 33 Bảng 3.9 Thông tin nhiệt độ bắt cặp thời gian kéo dài gen 34 Hình 3.9 Kết PCR lần 1, gen LMP-1 35 Hình 3.10 Kết PCR lần 2, gen LMP-1 36 Hình 3.11 Kết PCR lần 3, gen LMP-1 36 Hình 3.12 Kết PCR lần 1, gen LMP-2 37 Hình 3.13 Kết PCR lần 2, gen LMP-2 37 v Hình 3.14 Kết PCR lần 3, gen LMP-2 38 Hình 3.15 Kết PCR tối ƣu gen EBNA-1 38 Hình 3.16 Kết PCR lần 1, gen EBNA-2 39 Hình 3.17 Kết PCR lần 2, gen EBNA-2 40 Hình 3.18 Kết PCR lần 3, gen EBNA-2 40 Hình 3.19 Kết PCR lần 4, gen EBNA-2 41 Hình 3.20 Kết PCR tối ƣu gen BALF5 41 Hình 3.21 Kết khảo sát diện gen EBNA-1 mẫu bệnh mẫu lành 42 Hình 3.22 Kết giải trình tự gen EBNA-1-R 43 Hình 3.23 Kết Blast trình tự giải trình tự gen EBNA-1 43 Hình 3.24 Kết khảo sát diện gen EBNA-2 mẫu bệnh mẫu lành 44 Hình 3.25 Kết giải trình tự gen EBNA-2-F 45 Hình 3.26 Kết Blast trình tự giải trình tự gen EBNA-2 45 Hình 3.27 Kết khảo sát diện gen LMP1 mẫu bệnh mẫu lành 46 Hình 3.28 Kết giải trình tự gen LMP-1-F 46 Hình 3.30 Kết khảo sát diện gen LMP-2 mẫu bệnh mẫu lành 48 Hình 3.31 Kết giải trình tự gen LMP-2-F 48 Hình 3.32 Kết Blast trình tự giải trình tự gen LMP-2 49 Hình 3.33 Kết khảo sát diện gen BALF5 mẫu bệnh mẫu lành 49 Hình 3.34 Kết giải trình tự gen BALF5-R 50 vi Hình 3.35 Kết khuếch đại gen LMP-1, LMP-2 với chứng nội 51 Hình 3.36 Kết khuếch đại gen EBNA-1, EBNA-2 với chứng nội 51 vii DANH MỤC BẢNG BIỂU Bảng 3.1 Các cơng trình tần số phát gen LMP-1 21 Bảng 3.2 Các cơng trình tần số phát gen LMP-2 22 Bảng 3.3 Các công trình tần số phát gen EBNA-1 23 Bảng 3.4 Các cơng trình tần số phát gen EBNA-2 24 Bảng 3.5 Kết trung bình trọng số gen 24 Bảng 3.6 Tỉ lệ phƣơng pháp đƣợc sử dụng để phát gen EBV 26 Bảng 3.7 Bảng trung bình có trọng số mấu mô sinh thiết mẫu máu 27 Bảng 3.8 Thông số vật lý cặp mồi 28 Bảng 3.9 Bảng nồng độ DNA đƣợc tách chiết từ mẫu mơ sinh thiết vịm họng 34 Bảng 3.10 Kết phân tích thống kê gen EBNA-1, EBNA-2, LMP-1, LMP-2 BALF5 52 Bảng 3.11 Kết phân tích thống kê kết hợp hai gen EBNA-1 EBNA-2 54 Bảng 3.12 Kết phân tích thống kê kết hợp hai gen LMP-1 LMP-2 55 Bảng 3.13 Kết phân tích thống kê phát đa gen 56 Bảng 3.14 Giá trị PI (PCR index) 57 viii Evaluation of EBNA-1 (Epstein-Barr virus nuclear antigen-1) gene prevalence in nasopharyngeal carcinoma in Vietnamese patients Lao Duc Thuan1,2, Nguyen Hoang Anh Tuan1, Ngo Dong Kha2, Thieu Hong Hue2, Ho Ta Giap2, Nguyen Hai Chau2, Nguyen Huu Dung3, Le Huyen Ai Thuy2,* University of Science, Vietnam National University Ho Chi Minh City, Vietnam Ho Chi Minh City Open University, Vietnam Cho Ray Hospital, Ho Chi Minh City, Vietnam *Author for Correspondence: thuy.lha@ou.edu.vn Abstract This study examined the presence of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) based on the detection of EBNA-1 (Epstein-Barr virus nuclear antigen-1) by polymerase chain reaction (PCR), in Vietnamese population Firstly, we systematically analyzed the mean of percentage weighted of the presence of EBNA-1 in previous relevant studies Experimentally, 31 nasopharyngeal cancer biopsies and 20 healthy samples were enrolled in current to evaluate the frequency of candidate genes, as the results, the frequency of EBNA-1 was 77.42% Whereas, none of any cases of healthy samples were found to positive to target gene The p value < 0.05 (EBNA-1: p = 0.0001) showed that it was significant correlation between the presence of those candidate genes and nasopharyngeal cancer Moreover, a high odd ratio (OR) and relative risk (RR) of candidate gene, (OR = 68.16, RR = 2.41) were calculated Therefore, the detection of EBNA-1, which performed by PCR, could serve as a good supplement to early diagnosis and prognosis of NPC in Vietnamese population Keywords: EBNA-1, Nasopharyngeal carcinoma, Vietnamese population Introduction Nasopharyngeal carcinoma (NPC) is a highly invasive and malignant tumor of the nasopharynx, the uppermost region of the pharynx NPC has a striking and geographic and ethnic distribution, which encountered in Asian region, gravitating toward Southeast Asia, especially in China and Vietnam (da Costa et al, 2015; Pathmanathan et al, 1995) According to data provide by GLOBOCAN 2012, in the world, the total of number of NPC was 86,691 cases (Agestandardized rate – ASR = 1.2/100,000), while the number of death was 50,831 (ASR = 0.7/100,000) Among these cases, Vietnam was one of countries contributed to the most to these indices, because the total number was 4,931 cases (ASR = 5.4/100,000) and deaths was 2,885 cases (ASR = 3.3/100,000) (GLOBOCAN, 2012) The etiology of NPC is strongly suggested to be involved to several genetic susceptibility and environmental factors Notably, within ambiguous symptoms, such as hearing loss, nosebleeds, headache, trouble opening the mouth, etc., thus, most patients present with the stage III or IV cancer when diagnosed (Epstein, Jones, 1993; Hao et al, 2004), thus, it could be a challenge in treatment Therefore, to achieve favorable treatment and increasing of patient’s survival, early diagnosis and prognosis are necessary to be appropriate managed To date, three major etiological factors, including viral infection, genetic and environment factors, are defined (Frappier, 2012; Lung et al, 2014; Lo et al, 2004) Many studies have proved that NPC is strongly linked to Epstein-Barr virus (EBV) infection, which is a gamma herpes virus discovered in 1964 by Epstein and Barr, taxonomically called Human herpes virus (Frappier, 2012; Lung et al, 2014; Epstein et al, 1964; Chan, Wong, 2014) EBV infection was considered to be an early event, pre-requisite step in nasopharyngeal tumorigenesis and may contribute to its pathogenesis (Chan, Wong, 2014; Yip et al, 2010) Since then, viral infection has been received much attention and represented a prospective biomarker for prognosis and diagnosis of NPC For the past decade, owing to the increase in EBV detection, numerous molecular studies were carried out on NPC, which mainly focused on polymerase chain reaction (PCR), in situ hybridization, real-time PCR methods to detect the presence of EBV in various sources of NPC Moreover, many previous studies indicated the prevalence of EBV attributed to NPC were reported variations in different ages, sex, geographic regions (Chang, Adami, 2006; Khan, Hashim, 2014) For instance, in the high incidence regions, such as East Asia, South Asia, South East Asia and North Africa and Middle East, etc., within the prevalence were up to 100% (Chang, Adami, 2006; Khan, Hashim, 2014), nevertheless, the overall EBV sero-prevalence in samples from North and South China were 80.78% and 79.38%, respectively (Xiong et al, 2014) EBV consists a double-stranded 184-kbp-long DNA enclosed in a protein capsid and that encode more than 85 genes Evidences showed that multiple copies of EBV genome are presented nasopharyngeal carcinoma and EBV DNA is also found in the pre-invasive state of NPC (Pathmanathan et al, 1995; Yap et al, 2007) The EBV genomic organization that code for EBNAs (Epstein-Barr virus nuclear antigens) including EBNA-1, 2, 3A, 3B, 3C and LP; LMP-1, 2A and 2B (latent membrane protein); two small EBERs (non-coding nuclear RNAs) (Hammerschmidt, Sugden, 2013; Young et al, 1988) Individually, EBNA-1 plays an important role of EBV infection As function, EBNA-1 is essential for EBV immortalization of cell and responsible for EBV DNA episome replication, segregation and persistence of viral genome EBNA-1 coded-protein is only protein which expressed in both latent and lytic modes of infection (Sivachandran et al, 2012; Hammerschmidt, Sugden, 2013) Additionally, EBNA-1 is necessary to maintain EBV genome in circular episome with multicopies in the infected cell (Thompson, Kurzrock, 2004) The study of evaluation prevalence of EBV, by detection of EBNA-1, was still no research carried out in Vietnamese population Whereas, only research was carried out in development the EBV genetic variation of LMP1 (Nguyen-Van D et al, 2008) and MMSP (multiplex methylation PCR) (Zhang et al, 2012) for Vietnamese patients with nasopharyngeal carcinoma Therein, the aim of the present study was to systematically evaluate the mean of EBNA-1 based on previous studies and to analyze the presence of those genes in Vietnamese nasopharyngeal carcinoma tissues by PCR Materials and methods Data mining, the identification of presence of EBNA-1 in previous relevant studies The following keywords: Epstein-Barr virus, nasopharyngeal carcinoma, EBNA-1, etc., were applied in identification of detection of EBV genomic DNA for early diagnosis and prognosis of nasopharyngeal cancer Moreover, the following databases were search: PubMed, ScienceDirect, etc The selection period lasted until the end of 2015 Consequently, the data of EBNA-1 frequencies were extracted and used to calculate of means of their presences Samples collection, DNA isolation From June, 2015 to December, 2015, thirsty one nasopharyngeal tumor biopsies (Male: 74.19% and female: 25.81%) were retrieved from Cho Ray Hospital, Vietnam All of those nasopharyngeal biopsies were submitted to histopathological department to confirm NPC Additionally, twenty nasopharyngeal swab samples were collected from healthy volunteers were used as negative controls DNA was extracted from clinical sample by means of an enzyme digestion using 700 μl lysis buffer (NaCl 5M, Tris-HCl 1M, EDTA 0.5M, SDS 10% and Proteinase K mg/ml) The samples were incubated at 56oC overnight Then, DNA obtained and purified by Phenol/Chloroform extraction and ethanol precipitation The quality and purity of DNA extraction was measured by the proportion of A260/A280 Then, the DNA solution was stored at EDTA 0.5M, -20oC for further used Detection of EBNA-1 Viral DNA detection was carried out by PCR method for EBNA-1 gene amplification The forward and reverse primer sequences were: 5’-GTCATCATCATCCGGGTCTC-3’ and 5’TTCGGGTTGGAACCTCCTTG-3’, respectively (Telenti et al, 1990) Amplification of human beta-actin gene was used as internal control for marker of the presence of intact genomic DNA The forward and reverse primer sequence for beta-actin amplification were: 5’ATCATGTTTGAGACCTTCAACAC-3’ and 5’- CATCTCTTGCTCGAAGTCCAG-3’, respectively For PCR assay, the amplification was done in a total volume of 15 μl, containing μg DNA template PCR reaction was subjected to initial at 95oC for minutes, followed by 35 cycles at 95oC for 30 seconds, 63oC for 30 seconds, 72oC for 30 seconds, and finally 72oC for 10 minutes Each PCR product was directly loaded onto a 2.0% agarose gel, stained with Ethidium bromide, and directly visualized under UV illumination Then, PCR product was sequenced to confirm target gene amplification Statistical analysis The statistical analysis was done by using the Medcalc® software The frequency of EBNA-1 was calculated and the Chi-squared test was used to compare the frequencies of categorical variables between groups The differences of frequency of EBNA-1 among groups were considered statistically significant for p ≤ 0.05 Statistical comparisons were carried out between each gene and match-pairs tested by κ-value, sensitivity, specificity, positive and negative predictive values, likelihood ratio and receiver-operating characteristic Results Data mining, presence of EBNA-1 in previous studies The criteria for selection related research articles in database were the selection period lasted until 2015 within EBNA-1 detection on various samples, such as nasopharyngeal carcinoma tissues, nasopharyngeal swab, etc., by different methods as PCR, RT-PCR, immunohistochemistry, etc., as the result, total of 16 relevant studies were found As the results, the detection of target gene in nasopharyngeal cancer cells from various sources such as biopsy tissue, serum, nasopharyngeal swab, peripheral blood, etc and carried out on various countries, such as India, Malaysia, Iran, etc However, the predominant kind of sample was biopsy tissue For target genes detection, among several methods used, such as PCR, RT-PCR, nested-PCR, etc., PCR was the most common method for detection of EBNA-1 The frequency of EBNA-1 was ranged from 65.0% to 100% The estimated frequencies of EBNA-1 variants in the samples from NPC patients were necessary to be calculated, counting for 93.50% Additionally, the mean of detection of EBNA-1 in non-cancer samples, including various types as tissues, blood, swab or paraffin-embedded tissues, were 13.8%, respectively, shown in Fig Figure The frequency of EBNA-1 in Nasopharyngeal cancer and healthy specimens in previous studies The center of the circle indicated the previous reported frequencies and the size of circle indicated the size of studies (n = number of samples), number of circle indicated amount of previous studies The frequencies of EBNA-1 in nasopharyngeal cancer specimens and healthy samples Total samples were enrolled in PCR for detection of EBNA-1 prevalence The frequency of EBNA-1 was evaluated by PCR assay including nasopharyngeal cancer biopsy specimens and healthy specimens As well as κ-value, sensitivity, specificity, positive and negative predictive values, likelihood ratio and receiver-operating characteristic were shown in Table As the results, of the 31 NPC tumors, the frequency of EBNA-1 detection was 77.42% (24 of 31 cases) Concerning to healthy specimens, total of healthy specimens were negative to EBNA-1 detection Table Validity data for individual gene detection in nasopharyngeal cancer specimens and healthy samples EBNA-1 Nasopharyngeal P (n(%)) 24 (77.42) specimens N (n(%)) (22.58) Healthy P (n(%)) (0.00) specimens N (n(%)) 20 (100.00) κ 0.729 AUC 0.887 Se 77.42% Sp 100.00% Pp 100.00% Np 74.07% LR- 0.23 p-value < 0.0001 Note: P: positive; N: negative; κ: Kappa value; Se: Sensitivity; Sp: Specificity; Pp: Positive Predictive Value; Np: Negative predictive value; LR-: Negative Likelihood Ratio; AUC: Area under the ROC curve The PCR products were observed by electrophoresis in distinctly different sizes and easily identified, shown in Fig The EBNA-1, forward and reverse primer yielded a PCR product of 269 bp For internal control, the beta-actin PCR was 319 bp Furthermore, PCR product was confirmed by DNA sequencing The signal of peaks in PCR product sequencing were good for reading nucleotide (Fig 3) According to Blast results, EBNA-1 gene’s sequence was similar to Human Herpesvirus (Epstein-Barr virus) EBNA-1 sequence was similar to KP735248, within Total score = 462, Ident = 100% and E-value = 1e-126 Figure Agarose gel electrophoresis showing the present of EBNA-1 and beta-actin in representative samples (1)(2): NPC biopsy samples; (3)(4) Healthy samples; NC: Negative control; PC: positive control MW: molecular weight ladder 100 bp Figure Sequencing profile of EBNA-1 of represent nasopharyngeal samples Chi-squared test was used to access the correlation between candidate genes and nasopharyngeal carcinoma was calculated by Medcalc® software, it indicated that, in our study, the presence of EBNA-1 was strongly correlated with nasopharyngeal carcinoma (EBNA-1: p = 0.0001) Additionally, the risk of nasopharyngeal cancer was also calculated via odd ratio (OR) and relative risk (RR) by analysis of detection of EBNA-1 in samples Results indicated that, in the case of EBNA-1, OR = 68.16 (95% CI = 3.58 - 1314.13, p = 0.0050) and the RR = 2.41 (95% CI = 1.37 – 4.27, p = 0.0022) Therefore, it could be induced that the presence of EBNA-1 was significant feature of nasopharyngeal carcinoma, in Vietnamese population Discussion Due to unclear symptoms, nasopharyngeal cancer often presents in the last stage when first diagnosis Therefore, there is a challenge for finding a simple and non-invasive methods access to NPC In current study, the data mining provided the evidence that presence of EBNA-1 was a striking characteristics of genomic EBV contributed in nasopharyngeal cancer In our data mining, the range of frequency of EBNA-1 were ranged from 65.0% to 100% These results were in accordance to previous statement that prevalence of the presence of EBNA-1 was various in different countries (da Costa et al, 2015; Pathmanathan et al, 1995) As shown in figure 1, the mean of presence of EBNA-1 gene was 93.50% Whereas, total healthy samples were found of 13.8% Therefore, it indicated that the presence of EBNA-1 could be considered as the potential molecular biomarker in early diagnosis of NPC In Vietnam, there was still no research related to evaluate prevalence of EBV, only research was carried out in evaluation of LMP1 (Nguyen-Van D et al, 2008) and MMSP (multiplex methylation PCR) (Zhang et al, 2012) for Vietnamese patients with nasopharyngeal carcinoma According to Zhang et al (2012), an ideal cancer screening and detection test should be non-invasive to be performed, however, in our study, the biopsy tissues were enrolled, because biopsy samples is highly accurate since these samples were obtained from nasopharyngeal tumors In our study, in the nasopharyngeal cancer biopsy set, the frequency of EBNA-1 was 77.42%, which were lower than the mean of those genes in our data mining In the healthy control, it was according to previous studies that lower than nasopharyngeal carcinoma, especially no positive case was found Based on figure 2, the PCR product was easily distinguished within the different length, among them, the human beta-actin and positive control for EBNA-1 gave indication on the quality of the extracted DNA and specific of primer sets used According to table 2, a value p < 0.05 (EBNA-1: p = 0.0001) showed a strong correlation between the presence of each EBNA-1 with nasopharyngeal carcinoma, inferred the presence of EBV, were significant associated to nasopharyngeal cancer development The OR and RR were 68.16 (p = 0.0050) and 2.41 (p = 0.0022) for EBNA-1 It meant that the detection of candidate gene was strongly correlated with nasopharyngeal cancer via the OR and RR with the significant statistic Based on the OR, the odds for positive EBNA-1 in nasopharyngeal cancer was 68.16 times higher than in the case without EBNA-1 detection, which meant that without EBV infected More, the positive of EBNA-1 was 2.41 times higher than negative in nasopharyngeal cancer Based on those results, the detection of EBNA-1 which indicated the infection of EBV, could be served as the potential biomarker for detection of EBV presence and predictor for nasopharyngeal cancer development Moreover, specificity and positive predictive value, which reach to 100% and other values, including the highest sensitivity, κ-value, area under the receiver-operating characteristic curve in nasopharyngeal biopsy samples, making it suitable and specific gene for diagnosis of NPC Conclusion During the data mining, total of studies was enrolled to evaluate mean of presence of candidate gene, which was a striking geographic and ethnic distribution of NPC cases, within the mean of EBNA-1 was 93.50% In our study, EBNA-1 detection in nasopharyngeal biopsy samples obtained in 77.42% Notably, none of heathy control were positive Moreover, the p value showed the strong correlation between the detection of EBNA-1 and NPC Additionally, the OR and RR were calculated, counting for 68.16 (p = 0.0050) and 2.41 (p = 0.0022), respectively Therefore, the candidate gene detection in tissue obtained from nasopharyngeal biopsy suggested that PCR assay done in candidate genes on the patients’ samples, which was easily, rapid for diagnosing NPC, in Vietnamese population In future studies, non-invasive samples will be continuously carried out to find out the non-invasive method in early diagnosis and prognosis for nasopharyngeal cancer in Vietnamese population Acknowledgment This research was conducted on the support of the Ho Chi Minh City Open University References Chan, J Y., Wong, S T (2014) The role of plasma Epstein-Barr virus DNA in the management of recurrent nasopharyngeal carcinoma, Laryngoscope, 124(1), 126-130 Chang, E T., Adami, H O (2006) The enigmatic epidemiology of nasopharyngeal carcinoma, Cancer 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nasopharyngeal carcinoma, PLoS One., 7, e45908 Khảo sát diện gen EBNA-1 (Epstein-Barr virus nuclear antigens-1) ung thư vòm họng người Việt Nam Lao Đức Thuận1,2, Nguyễn Hồng Anh Tuấn1, Ngơ Đơng Kha2, Thiều Hồng Huệ2, Hồ Tá Giáp2, Nguyễn Hải Châu2, Nguyễn Hữu Dũng3, Lê Huyền Ái Thúy2,* Trường Đại Học Khoa Học Tự Nhiên, Đại Học Quốc Gia Thành phố Hồ Chí Minh Trường Đại Học Mở Thành phố Hồ Chí Minh Bệnh viện Chợ Rẫy Thành phố Hồ Chí Minh *email: thuy.lha@ou.edu.vn Tóm tắt Trong nghiên cứu này, diện EBV (Epstein-Barr virus) ung thư vịm họng đánh giá thơng qua việc phát gen EBNA-1 (Epstein-Barr virus nuclear antigen-1) phương pháp PCR (Polymerase chain reaction) Đầu tiên, tiến hành thống kê tần số trung bình trọng số gen EBNA-1 thơng qua cơng trình nghiên cứu giới Về thực nghiệm, tiến hành khảo sát diện gen EBNA-1 31 mẫu sinh thiết ung thư vòm họng 20 mẫu dịch phết vòm họng thu nhận từ người lành, kết cho thấy tần số diện gen EBNA-1 đạt 77.42% Trong đó, tất mẫu lành tính không phát gen EBNA-1 Đồng thời, giá trị p < 0.05 (EBNA-1: p < 0.0001) cho thấy có tương quan diện gen EBNA-1 với ung thư vòm họng Giá trị OR RR tính tốn 68.16 2.41 Từ kết trên, nhận thấy việc phát gen EBNA-1, phương pháp PCR, dấu chứng sinh học đặc trưng ứng dụng việc chẩn đốn sớm ung thư vịm họng người Việt Nam Từ khóa: EBNA-1, Ung thư vịm họng, Người Việt Nam ... THÔNG TIN KẾT QUẢ NGHIÊN CỨU CỦA ĐỀ TÀI Thông tin chung: - Tên đề tài: KHẢO SÁT SỰ HIỆN DIỆN CỦA CÁC GEN EBNA- 1 VÀ LMP -1 CỦA EPSREIN- BARR VIRUS TRÊN BỆNH UNG THƢ VÒM HỌNG Ở NGƢỜI VIỆT NAM - Sinh... ĐẠI HỌC MỞ THÀNH PHỐ HỒ CHÍ MINH BÁO CÁO TỔNG KẾT ĐỀ TÀI NGHIÊN CỨU KHOA HỌC CỦA SINH VIÊN THAM GIA XÉT GIẢI THƯỞNG CẤP TRƯỜNG Tên đề tài: KHẢO SÁT SỰ HIỆN DIỆN CỦA CÁC GEN EBNA- 1 VÀ LMP -1 CỦA EPSREIN- BARR... gen EBNA- 1 từ 65 -10 0% mẫu bệnh từ 15 ,8-88,4% mẫu lành Gen EBNA- 2 từ 66-96% mẫu bệnh Gen LMP -1 LMP- 2 lần lƣợt 6,7 -10 0% 45-57,6% mẫu bệnh Trong số 31 mẫu ung thƣ nghiên cứu, tần số phát gen EBNA- 1,