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: Prof Ph.D Nguyen Linh Toan 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 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 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 Evaluate plasma microRNA-21 and microRNA-122 expression levels in hepatitis B virus-related hepatocellular carcinoma patients Analyze the relationship between microRNA-21, microRNA-122 plasma with some clinical, subclinical and stage 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 – pages, literature review – 35 pages, study respondents and methods – 20 pages, research results - 45 pages, discussion - 33 pages, conclusion - page, recommendation - 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) 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 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 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 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 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 + 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 (+) > months or HBsAg (+) and Anti HBc IgG (+) - AST, ALT increase in installments or over 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 0,05 > 0,05 > 0,05 < 0,05 Comment: No correlation between miR-21 and platelet counts, prothrombin ratio, AST, ALT, total bilirubin, total protein, Abumin and AFP levels in patients with HCC (| r | 0.05) There was a weak correlation between miR-21 plasma and HBV DNA (r = 0.502; p 0,05 > 0,05 > 0,05 > 0,05 > 0,05 > 0,05 > 0,05 > 0,05 < 0,05 Comment: No correlation between miR-122 and platelet counts, prothrombin ratio, AST, ALT, total bilirubin, total protein, Abumin and AFP levels in patients with HCC (| r | 0.05) There was a weak correlation between miR-122 plasma and HBV DNA (r = 0,369; p 0.05) Table 3.36 Relationship between miR-122 and liver function according to Child - Pugh classification, tumor tumor characteristics and disease stage according to BCLC miR-122 Indicator OR p Child – Pugh 1,316 0,665 Tumor number 1,031 0,941 Tumor size 0,489 0,112 Vascular invasion 0,731 0,511 Tumor differentiation 1,219 0,575 BCLC 0,730 0,272 (95% CI) 0,379 - 4,565 0,457 - 2,328 0,202 - 1,182 0,286 - 1,864 0,609 - 2,441 0,416 - 1,280 Comment: There is no relation between miR-122 plasma and liver function according to Child - Pugh classification, tumor number, tumor size, vascular invasion, tumor differentiation, and stage of cancer by BCLC classification (p> 0.05) Chapter DISCUSSIONS 4.1 Research group characteristics HCC can appear in patients at any age, but there are differences in incidence in different age groups In our study, the age range of HCC patients prevention is quite wide, the youngest is 23 years old, the oldest is 92 years old, the average age (55.6 ± 12.34), 22 the male account for the majority (93, 1%), female (6.9%), male / female ratio ~ 13/1 HCC usually progresses silently Once the symptoms are visible, they are at an advanced stage, the tumor is large in size or with impaired hepatic function The results of our study showed that common mechanical symptoms are fatigue (63.4%), anorexia (61.4%), right upper quadrant pain (61.4%), weight loss ( 47.5%) The most physical symptom is hepatomegaly (21.78%) The average AFP concentration in HCC patients (339.29 ± 574.02) ng / ml, of which 35.6% of HCC patients had serum AFP at ≤ 20ng / ml In our study, the majority of cases have large size liver tumors, the largest average size of the tumor (7.78 ± 3.41) cm Patients with liver tumors of less than 5cm size only account for (28.71%) Patients had one tumor (64.36%), multiple liver tumors were found in 35.64% of which 2-3 tumors (13.86%), over tumors (21.78%) Our results are similar to the findings of some domestic and foreign authors, the majority of HCC patients have a liver tumor Vascular invasion is a sign of late stage of cancer, showing the spread and destruction of the cancer organization into nearby blood vessels such as portal vein, liver vein and lower vena cava The pattern of vascular invasion is shown by the image of venous thrombosis, the proportion of portal venous thrombosis in our study accounts for 22.77% Cellular differentiation is an indispensable factor when it comes to any type of cancer In HCC, differentiation is considered to be an important prognostic factor In our study, patients with HCC had moderate cell differentiation (45.55%), high differentiation (24.75%), and low-grade (9.90%) Unspecified (19.80%), these are 23 cases of bright cell type HCC and fine needle aspiration FNA is applied Our rate of differentiation of cancer cell group is similar to that of Thai Doan Ky, Nguyen Tien Thinh and Le Van Truong In this study, we used the BCLC classification system to assess the stage of disease of patients with cancer The research results showed that patients with HCC mediated stage B accounted for 49.51%, the progression of BCLC C (34.65%), the early stage BCLC A (15.84%), there was no late stage BCLC D HCC patient 4.2 Expression of miR-21, miR-122 in HCC patients In our research findings, miR-21, miR-122 showed higher plasma expression in HCC group than in chronic hepatitis B patients and healthy people with p