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MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENSE VIETNAM MILITARY MEDICAL UNIVERSITY DANG CHIEU DUONG RESEARCH ON EXPRESSION OF MICRORNA-21, MICRORNA-122 PLASMA IN HEPATITIS B VIRUS-RELATED HEPATOCELLULAR CARCINOMA PATIENTS Major: Internal Medicine Code: 9720107 SUMMARY OF MEDICAL DISSERTATION HANOI- 2020 THIS STUDY HAS BEEN COMPLETED AT VIETNAM MILITARY MEDICAL UNIVERSITY Research adviser: 1. Prof. Ph.D. Nguyen Linh Toan 2. Ph.D. Ngo Tat Trung Reviewer 1: Prof. Ph.D. Vu Van Khien Reviewer 2: Prof. Ph.D. Phan Van Chi Reviewer 3: Prof. Ph.D. Bui Vu Huy The dissertation will be defended in front of Oral Examination Committee at Vietnam Military Medical University at: ... hour, ... date ... month ... 2020. The dissertation can be found at: - National Library - Library at Vietname Military Medical University 1 INTRODUCTION Hepatocellular carcinoma (HCC) represents more than 90% of primary liver cancers, with the 6th highest incidence and the 4th highest death rate among the common cancer types in the world. In Vietnam, HCC ranks first in terms of morbidity and mortality, over 80% is associated with infection with the hepatitis B virus. A good screening strategy will help to make early diagnosis of HCC, which is often the combination of abdominal ultrasound and AFP. Besides classic AFP, a number of other biomarkers have been studied and applied such as AFP-L3, DCP ... but the desired effects have not been achieved yet. MicroRNAs (miRNAs) are small endogenous RNA that do not encode proteins and play a key role in regulating gene expression in the post-transcription phase. Many studies in the world have demonstrated that miR-21, miR-122 plasma are derived from liver tumor cells and are valuable in the diagnosis, prognosis, and evaluation of treatment response to HCC. In Vietnam, there have not been many studies evaluating the expression of plasma miRNA in HCC patients infected with hepatitis B virus. Therefore, we conducted the study: “Research on expression of microRNA-21, microRNA-122 plasma in hepatitis B virus-related hepatocellular carcinoma patients "with the two following objectives. 1. Evaluate plasma microRNA-21 and microRNA-122 expression levels in hepatitis B virus-related hepatocellular carcinoma patients. 2. Analyze the relationship between microRNA-21, microRNA-122 plasma with some clinical, subclinical and stage 2 indicators in hepatitis B virus-related hepatocellular carcinoma patients. CONTENT OF THE DISSERTATION This dissertation consists of 137 pages, of which: statements of the problem – 2 pages, literature review – 35 pages, study respondents and methods – 20 pages, research results - 45 pages, discussion - 33 pages, conclusion - 1 page, recommendation - 1 page. In the disseratation, there are 36 tables, 35 charts, 10 images, 120 references (15 Vietnamese documents, 105 English documents). NOVELTY OF THE DISSERTATION This is the first study in Vietnam to study the expression of miR-21, miR-122 plasma in hepatitis B virus-related hepatocellular carcinoma patients. The results of the study showed that miR-21 and miR-122 expression increases in plasma of HCC group compared to CHB and HC groups, even in cases of early stage of HCC and HCC with AFP ≤ 20ng / ml. MiR-21 and miR-122 have higher sensitivity and specificity than AFP in the diagnosis of HCC with CHB. In the case of early stage HCC, HCC with AFP ≤ 20ng / ml, miR-21, miR-122 still have high accuracy when differentiating HCC from CHB (AUC = 0.848; 0.979); (AUC = 0.806; 0.915), HCC from HC (AUC = 0.894; 0.993); (AUC = 0.935; 0.991) respectively. The effectiveness of HCC diagnosis with CHB group increases when combining miR-21, miR-122 with AFP (AUC = 0,868; 0,914). 3 No relationship between miR-21, miR-122 plasma has been found with ages, genders, some clinical, subclinical symptoms and the stage of disease in HCC patients with HBV infection. There is poor correlation between miR-21 and miR-122 plasma with HBV DNA. CHAPTER 1. OVERVIEW 1.1. MicroRNA MicroRNAs (miRNAs) are small endogenous RNA that does not encode proteins and play an important role in regulating gene expression. MiRNA prevents code translation by promoting degradation of the target mRNA. The miRNAs were found to be involved in histone modification and gene methylation promoters. The first MiRNA was discovered in 1993 by Victor Ambors and his colleagues when studying the development of the C. elegans nematode. By 2014, 2588 human miRNAs were identified. MiRNA plays an important role in the physiological and cellular pathologies, such as regulating proliferation, differentiation or apoptosis. In recent years, a link between miRNAs and chronic liver disease has been recognized, especially miR-21 and miR-122 in HCC. Due to its specific tumor properties, circulation and sustainability in body fluids, miR-21, miR-122 are also found to be stable in plasma stored at room temperature or negative temperature. Therefore, this is the scientific basis for the quantitative application of miR-21, miR-122 in plasma with the tendency as useful biomarkers for the diagnosis and prognosis of cancer cells. 4 1.2. MicroRNA-21 MicroRNA-21 (miR-21) is a short RNA segment consisting of 22 nucleotides, the human miR-21 gene is located on chromosome 17 at position 17q23.1, in a transmembrane protein coding gene 49 (TMEM49) also known as 49 vacuolar membrane protein 49. Mature miR-21 molecule is made up of 22 nucleotides, which is one of the first miRNA described to be associated with cancer. High concentrations of miR-21 are found in the plasma of patients with melanoma, which makes it a potential biomarker for cancer. MiR-21 has been linked to several cancers such as esophageal cancer, stomach cancer, and colorectal cancer but the most common is HCC. Recent studies have shown that miR-21 has a higher plasma expression in patients with HCC and has a higher sensitivity and specificity than AFP in diagnosing HCC with chronic liver cancer without cancer. Furthermore, miR-21 has been shown to be associated with vascular proliferation, invasion and monitoring of response to treatment. 1.3. MicroRNA-122 MicroRNA-122 (miR-122) was originally identified in mouse liver tissue, accounting for 72% of miRNA analyzed in the liver. In humans, miR-122 is encoded by a single gene in chromosome 18 at position 18q21.31. MiR-122 is regulated by Rev Erba alpha, involved in gene expression by adjusting the expression of many target mRNA molecules. MiR-122 was found to be highly specific for liver tissue. In the case of HCC related to HBV, miR-122 inhibits viral replication by targeting NDRG3 (N-myc downstreamregulated gene 3), a member of the N-myc gene family. 5 Research has shown that both miR-122 and NDRG3 in treatment are feasible for HCC related to HBV. MiR-122 may represent the primary biomarker in diagnosis and has the potential for a combination of HBV-related treatment of HBV. The expression of miR-122 was found to be sharply reduced in the liver tumor tissue itself, while it was found to increase in the plasma of HCC patients. This is probably because miR-122 is transferred from tumor tissue into the bloodstream. Many studies in the world have shown that miR-122 has high specific sensitivity in HCC diagnosis. Moreover, miR-122 has been found to be valuable in monitoring response to some HCC treatments. CHAPTER 2. SUBJECTS AND METHODS 2.1. SUBJECTS Group of diseases: Including 101 patients with HCC infected with HBV inpatient treatment at 108 Military Central Hospital . Control group: Including 46 patients with chronic hepatitis B (CHB) inpatient treatment at 108 Military Central Hospital and 103 healthy control (HC). Research period: from March 2014 to March 2017. 2.1.1. Inclusion criteria 2.1.1.1. Group disease - Patients with HCC were diagnosed following the criteria issued by the Ministry of Health of Vietnam in 2012: + There is evidence of anatomy. + Typical image on CT scan taken with contrast injection or CHT with contrast agent + AFP> 400 ng / ml. 6 + Typical image of HCC on CT scan of abdominal abdomen with contrast or CHT with contrast inhibitors + AFP higher than normal (less than 400ng / ml) + hepatitis B, C virus infection - Patients with HCC have positive HBsAg test. 2.1.1.2. Control group * Patients with chronic hepatitis B: We selected patients with chronic hepatitis B diagnosed under the guidance of the Ministry of Health of Vietnam in 2014. - HBsAg (+) > 6 months or HBsAg (+) and Anti HBc IgG (+). - AST, ALT increase in installments or over 6 months. - There is evidence of progressive histopathological damage identified by a liver biopsy. * Healthy control: are voluntary blood donors without any medical history, who test negative for HBV, HCV, HIV (including rapid test and NAT technique: nucleic acid test). 2.1.2. Exclusion criteria - Patients with HCC have positive anti-HCV test. - Patients with treated HCC: surgery, Transcatheter arterial chemoembolization, Radiofrequency ablation, Percutaneous Ethanol Injection or treatment with sorafenib. - Patients with co-morbid conditions: Severe heart failure, respiratory failure, gastrointestinal bleeding and other cancers. - Patients with severe coagulation disorders: PLT <50 G / L; PT <50%. - People who do not agree to participate in the research. 7 2.2. METHODS 2.2.1. Research design Cross-sectional descriptive study, with comparative control. 2.2.2. Study sample size The sample size is identified by convenient sampling method. 2.2.3. Research facilities - 9700 PCR machine and Realtime PCR 7500 machine of Applied Biosystems in USA, installed at Department of Molecular Biology Department, 108 Military Central Hospital . 2.2.4. Quantitative miR-21, miR-122 The miR-21, miR-122 were quantified relatively to the internal standard by Realtime PCR. Take 5ml of intravenous blood in the morning when the patient has not got breakfast into the tube with Ethylenediaminetetra acid anticoagulant (EDTA), bring immediately to the Department of Molecular Biology, 108 Military Central Hospital . Centrifuge 3000 rpm for 5 minutes, separate the plasma and stored in minus 800c refrigerator. When collecting enough samples, quantify miR-21 and miR-122. 2.2.5. Research indicators - Age, gender, clinical and subclinical symptoms, characteristics of liver tumors, stage of liver cancer according to BCLC. - Expression of miR-21, miR-122 plasma in the study subjects. 2.2.6. Statistical analysis - Data were processed by medical statistical methods, using SPSS 21.0 software. 8 - Calculate percentage, average value, standard deviation, median. Make comparisons to find differences between observations, determine the diagnostic value of biomarkers using the ROC curve, calculate the area under the curve (AUC), the sensitivity, the specificity. Analyze relationships using logistic regression, calculating "OR". Analyze correlation, calculate the correlation coefficient "r" 2.2.7. Research ethics The study obeys the ethics of medical research. CHAPTER 3. RESULTS The study was conducted at 108 Military Central Hospital from March 2014 to March 2017. Included 101 patients with HCC, 46 patients with CHB, 103 healthy people eligible for selection. 3.1. Research group characteristics 3.1.1. Clinical and laboratory characteristics of research subjects - Average age of patients with HCC (55,6 ± 12,34), patients at least 23 years old, the largest patients are 92 years old. Male account for the majority (93.1%), female (6.9%), male / female ratio ~ 13/1. - Common functional symptoms of liver cancer are fatigue (63.4%), anorexia (61.4%), pain in the lower right flank (61.4%), weight loss (47.5%). - Systemic symptoms are frequently seen as vascular disease (14.85%), lipstick hands (11.88%), jaundice (10.89%), less common symptoms such as fever, edema, subcutaneous hemorrhage have the same rate (1.98%).