Summary of Medical dissertation: Research on expression of microrna-21, microrna-122 plasma in hepatitis B virus-related hepatocellular carcinoma patients

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.

DANG CHIEU DUONG

RESEARCH ON EXPRESSION OF MICRORNA-21,

MICRORNA-122 PLASMA IN HEPATITIS B

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,

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

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)

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

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 downstream-regulated 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 2 SUBJECTS AND METHODS

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 (+) > 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

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

- 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"

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%)

- The most common physical symptom in hepatocellular carcinoma is hepatomegaly (21.78%), less common symptoms such

as splenomegaly (6.93%), portal vena cava (3.96%), No patients had ascites

- Hepatic enzyme activity of AST, ALT, total bilirubin and HBV load in HCC group are lower than in CHB group (p <0.05) AFP concentration was higher in the HCC group than in the CH group (p <0.05)

- Patients with HCC had AFP ≤ 20ng / ml, accounting for 35.6%

3.1.2 Liver tumor characteristics

- Patients with HCC have one tumor (64.36%), 2-3 tumors (13.86%), over 3 tumors (21.78%)

- The largest average size of the tumors (7.78 ± 3.41) cm Patients with liver tumors of less than 5cm size (28.71%)

- Patients with HCC have portal vein thrombosis (22.77%)

- Patients with HCC have moderate cell differentiation in the majority (45.55%), high differentiation (24.75%), low differentiation (9.90%)

- Classification of stage Barcelona Clinic Liver Cancer (BCLC): A (15.84%), B (49.51%), C (34.65%)

3.2 Expression of miR-21, miR-122 in the study subjects

3.2.1 Expression of miR-21, miR-122 in patients with HCC, CH and HC

Table 3.13 Expression of miR-21 plasma in patients with HCC,

Comments: The value of miR-21 plasma fluctuated in a large

range among the study groups MiR-21 in the HCC group had greater manifestations than the CHB and HC groups

Table 3.14 Expression of miR-122 plasma in patients with HCC,

Comments: The miR-122 plasma values fluctuated in a wide

range among the study groups MiR-122 of the HCC group had

greater expressions than the CHB and HC groups

Figure 3.5 ROC curves of miR-21, miR-122 in patients with HCC

and CHB

Comments: In the diagnosis between patients with HCC and

CHB, miR-21 has sensitivity (68.3%), specificity (78.3%); miR-122 has a sensitivity (79.2%) and specificity (89.1%) The specificity of miR-122 is higher than the specificity of miR-21 in the diagnosis between the HCC and CHB groups

Figure 3.7 ROC curves of AFP, miR-21, miR-122 in patients with

HCC and CHB

Comment: The charge under the curve of miR-21 is greater

than the charge under the curve of AFP but not statistically significant (p> 0.05) The area under the curve of miR-122 is larger than the area under the curve of AFP and miR-21 is significant with (p <0.05)

- When combining miR-21 with AFP, the charge under the curve increased significantly compared with miR-21 and AFP alone (p <0.05)

- When combining miR-122 and AFP, the charge under the curve was higher than that of AFP (p <0.05), but not significantly increased compared to miR-122 (p> 0.05)

- The area under the curve of miR-21 in combination with AFP is smaller than the area under the curve of miR-122 in combination with AFP (p <0.05) In combination with miR-21, miR-

122 with AFP showed an increase in charge under the curve compared with miR-21 in combination with AFP (p <0.05)

Figure 3.12 ROC curves of miR-21, miR-122 in patients with HCC

and HC

Comment: In the diagnosis between patients with HCC and

HC, miR-21 had sensitivity (81.2%), specificity (94.2%); miR-122 has a sensitivity (94.1%) and specificity (99.0%)

3.2.2 Expression of miR-21, miR-122 in patients with HCC with AFP ≤ 20ng / ml

- Expression of miR-21, miR-122 plasma in patients with HCC with AFP ≤ 20ng / ml is higher than in patients with CHB and

Sensitivity (%)

Specificity (%)

miR-122 (2-∆Ct) 0,915 55,33 80,6 89,1

Comments: In the diagnosis between patients with HCC with

AFP ≤ 20ng / ml and CH: miR-21 has sensitivity (75%), specificity (78.3%); miR-122 has sensitivity (80.6%), specificity (89.1%) Table 3.25 Sensitivity, specificity of miR-21, miR-122 in the diagnosis of HCC with AFP ≤ 20ng / ml and HC

HCC (n = 36)

HC (n = 103) AUC

Cut point

Sensitivity (%)

Specificity (%)

Comments: In the diagnosis between patients with HCC with

AFP ≤ 20ng / ml and HC: miR-21 has sensitivity (83.3%) specificity (98.1%); miR-122 has a sensitivity (94.4%) and specificity (99%)