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

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 downstream- regulated 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%). 9 - 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%). 10 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, CHB and HC miR-21 (2-ΔCt) Median SDX  Value range HCC (n=101) 10,78 194,05 ± 564,16 0,10 – 3743,05 CHB (n=46) 2,02 3,79 ± 7,46 0,12 – 50,91 HC (n=103) 0,73 0,83 ± 0,55 0,10 – 2,42 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, CHB and HC miR-122 (2-ΔCt) Median SDX  Value range HCC (n=101) 436,54 3158,58 ± 7707,36 0,14 – 47975,16 CHB (n=46) 3,61 31,63 ± 84,27 0,002 – 508,46 HC (n=103) 0,10 1,07 ± 2,09 0,00002 – 13,17 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. 11 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 12 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 13 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 HC (p <0.001). Table 3.23. Sensitivity, specificity of miR-21, miR-122 in the diagnosis of HCC with AFP ≤ 20ng / ml and CHB HCC (n = 36) CH (n = 46) AUC Cut point Sensitivity (%) Specificity (%) miR-21 (2-∆Ct) 0,806 3,62 75,0 78,3 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 (%) miR-21 (2-∆Ct) 0,935 2,17 83,3 98,1 miR-122 (2-∆Ct) 0,991 8,3 94,4 99,0 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%). 14 3.2.3. Expression of miR-21, miR-122 in patients with early stage HCC - Expression of miR-21, miR-122 increased in plasma of early stage HCC plasma (BCLC: A) compared with CH and HC group (p <0.001). Table 3.27. Area under the curve of AFP, miR-21, miR-122 in early stage HCC and CH diagnosis Indicator Area under the curve (AUC) P AFP (1) 0,617 p1,2 < 0,05 p1,3 < 0,05 p2,3 < 0,05 miR-21 (2) 0,848 miR-122 (3) 0,979 Comments: miR-21, miR-122 are highly valuable for early diagnosis of early stage HCC and CHB. The area under the curve of miR-21, miR-122 is larger than the area under the curve of AFP in early stage HCC and CHB diagnosis (p <0.05). Table 3.28. Area under the curve of miR-21, miR-122 in early stage diagnosis of HCC and HC Indicator Area under the curve (AUC) p miR-21 0,894 p < 0,05 miR-122 0,993 Comments: miR-21, miR-122 have high value in early stage diagnosis of HCC and HC. The area under the curve of miR-122 is larger than the area under the curve of miR-21 in early stage diagnosis and HC (p <0.05). 15 3.3. The relationship between miR-21 and miR-122 with some clinical, subclinical and disease indicators in patients with HCC. Table 3.29. Correlation between miR-21 and miR-122 with age Age miR-21 miR-122 R P r p HCC 0,157 > 0,05 0,163 > 0,05 Comment: No correlation between miR-21 and miR-122 plasma has been found with age in patients with HCC (| r | 0.05). Table 3.30. Relationship between miR-21, miR-122 and gender Gender miR-21 miR-122 OR P (95% CI) OR p (95% CI) HCC 0,383 0,266 0,071 - 2,075 1,391 0,676 0,295 - 6,559 Comment: No relationship between miR-21, miR-122 plasma and gender was found in the patients with HCC (p> 0.05). 16 Table 3.33. Correlation between miR-21 and some laboratory indicators in patients with HCC miR-21 Indicator Correlation coefficients (r) p Platelet (G/L) - 0,139 > 0,05 Prothrombin (%) 0,102 > 0,05 AST (U/L) - 0,037 > 0,05 ALT (U/L) 0,012 > 0,05 Blirubin TP (µmol/l) - 0,107 > 0,05 Protein TP (g/l) - 0,128 > 0,05 Albumin (g/l) 0,047 > 0,05 AFP (ng/ml) - 0,008 > 0,05 HBV-DNA{Log(copy/ml)} 0,502 < 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). 17 Table 3.34. Correlation between miR-122 and some laboratory indicators in patients with HCC miR-122 Indicator Correlation coefficients (r) p Platelet (G/L) - 0,051 > 0,05 Prothrombin (%) 0,186 > 0,05 AST (U/L) - 0,097 > 0,05 ALT (U/L) - 0,037 > 0,05 Blirubin TP (µmol/l) - 0,131 > 0,05 Protein TP (g/l) - 0,076 > 0,05 Albumin (g/l) 0,159 > 0,05 AFP (ng/ml) 0,004 > 0,05 HBV-DNA {Log(copy/ml)} 0,369 < 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 3.35. Relationship between miR-21 and liver function according to Child - Pugh classification, liver tumor characteristics and disease stage according to BCLC Indicator miR-21 OR p (95% CI) Child – Pugh 1,316 0,665 0,379 - 4,565 Tumor number 0,868 0,733 0,384 - 1,961 Tumor size 0,883 0,777 0,373 - 2,092 Vascular invasion 1,148 0,771 0,453 - 2,913 Tumor differentiation 1,710 0,141 0,837 - 3,492 BCLC 1,092 0,756 0,627 - 1,902 18 Comment: There is no relation between miR-21 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). Table 3.36. Relationship between miR-122 and liver function according to Child - Pugh classification, tumor tumor characteristics and disease stage according to BCLC Indicator miR-122 OR p (95% CI) Child – Pugh 1,316 0,665 0,379 - 4,565 Tumor number 1,031 0,941 0,457 - 2,328 Tumor size 0,489 0,112 0,202 - 1,182 Vascular invasion 0,731 0,511 0,286 - 1,864 Tumor differentiation 1,219 0,575 0,609 - 2,441 BCLC 0,730 0,272 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 4. 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), the male account for the majority (93, 1%), female (6.9%), male / female ratio ~ 13/1. 19 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 3 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 cases of bright cell type HCC and fine needle aspiration FNA is applied. Our rate of differentiation of cancer cell 20 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 <0.001. High expression of miR-21 in hepatocellular carcinoma has been reported for many years. In a study at Ohio University in the US in 2007, author Meng F. et al. reported an increased expression of miR-21 in liver cancer tissue compared with healthy liver tissue in humans. Research by Tomimaru Y. et al. (2012) in Japan showed that miR-21 expression was higher in plasma of HCC patients compared with CHB patients and healthy control with p <0.0001. In a study in China in May 2017, author Guo X. and his colleagues studied serum miR-21 expression in 453 respondents including 175 patients with HCC, 64 patients with chronic hepatitis B, 78 cirrhosis patients and 136 healthy volunteers. Results showed that serum miR-21 was significantly higher in HCC group than in chronic hepatitis B group, cirrhosis and healthy people (p <0.0001). Kutay H. (2006) from Ohio State University Medical School studied the changes of miRNA in mouse liver tissue experimentally induced by HCC. Results showed that miR-122 was strongly expressed in healthy liver tissue, while miR- 122 was severely reduced in cancerous liver tissue. While the 21 expression of cancerous liver tissue was reduced, miR-122 was found to be increased in plasma in patients with HCC compared with those without HCC. The study involved 152 respondents, including 70 HCC patients infected with HBV, 48 patients with chronic hepatitis B without cancer and 34 healthy volunteers by author Qi P. et al. (2011). Results showed that expression of miR-122 plasma was statistically significantly increased in patients with HCC compared with healthy subjects (p <0.001) and chronic hepatitis B (p <0.05). Similar to our study, the study on HCC patients in India in 2019, Bharali, D and colleagues found that miR-21 and miR-122 expression increased significantly in the HCC patient group compared with cirrhotic group, chronic hepatitis and healthy people with p <0.05. Investigate the values of miR-21, miR-122 in distinguishing HCC diagnosis from CHB and HC. Our research results show that miR-21, miR-122 have high specific sensitivity. In particular, the area under the curve, the specific sensitivity of miR-122 is higher than the charge under the curve, the specific sensitivity of miR-21 in diagnosis between HCC patients and CHB patients and HC. Moreover, when comparing the diagnostic value of miR-21, miR-122 with AFP through the area under the curve it shows that the area under the curve of miR-21, miR-122 is larger than the area under the curve of AFP. Our results are similar to those of some authors in the world that miR-21, miR-122 have higher specific sensitivity than AFP in HCC diagnosis for control groups. When combining miR-21 with AFP, the charge under the curve increased significantly compared with miR-21 and AFP alone p <0.05. Or when combining 22 miR-122 with AFP, the charge under the curve is higher than that of single AFP p <0.05. This result shows that the combination of miR- 21, miR-122 with AFP increases the diagnostic efficacy of AFP and therefore can complement AFP in HCC diagnosis. Moreover, our research results showed that the expression of miR-21, miR-122 increased in plasma in the group of HCC patients with AFP ≤ 20ng / ml compared to the group of CHB patients and healthy (p < 0.001). In the diagnosis among HCC group, there was AFP ≤ 20ng / ml with CHB, the area under the curve, the sensitivity, specificity of miR-21, miR-122 were at (0.806; 75.0%; 78.3%). and (0.915; 80.6%; 89.1%) respectively. Similarly, between HCC group, AFP ≤ 20ng / ml and healthy group, area under curve, sensitivity, specificity of miR-21, miR-122 (0.935; 83.3%; 98.1% ) and (0.991; 94.4%; 99.0%) respectively. In both cases, miR-122 had a higher specific sensitivity than miR-21 (p <0.05). We investigated the diagnostic value of miR-21, miR-122 in early HCC patients group (BCLC A) with control groups including CHB and HC. The results showed that miR-21, miR-122 were highly effective in diagnosing HCC with CHB (AUC = 0.848; 0.979), HCC with HC (AUC = 0.894; 0.993). Especially miR-21, miR-122 had higher value than AFP in diagnosis between early stage HCC group and CH g