Nghiên cứu mức độ biểu hiện và giá trị chẩn đoán, tiên lượng của một số microRNA ở bệnh nhân nhiễm khuẩn huyết TT TIENG ANH

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE 108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES - TRAN THI LIEN EVALUATE THE USED OF CIRCULATING MIRCRONA IN SEPSIS DIAGNOSIS AND SEPSIS PROGNOSIS Speciality: Infectiousdisieases and Tropical diseases Code: 62720153 ABSTRACT OF MEDICAL PHD THESIS Hanoi – 2021 THE THESIS WAS DONE IN: 108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES Supervisor: 1.PhD Vu Viet Sang 2.PhD Ngo Tat Trung Reviewer: This thesis will be presented at Institute Council at: 108 Institute of Clinical Medical and Pharmaceutical Sciences Month Day Year The thesis can be found at: National Library of Vietnam Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences Central Institute for Medical Science Infomation and Tecnology INTRODUCTION Sepsis is one of the leading causes of death in the world and the most common cause of death in the intensive care units (ICU) Early diagnosis and timely and rational initiation of antibiotic treatment are important for improving mortality rate Blood culture is the gold standard for diagnosis of sepsis, but it takes a long time, and the rate of positive is not high Biological markers are used to support the diagnosis and prognosis of sepsis, such as procalcitonin, Interleukin (IL-6), CRP, lactate are limited MicroRNAs (MiRNAs) are endogenous RNAs which are not involved in protein synthesis but control gene regulation in the posttranscription phase MiRNA is present at various stages in the pathogenesis of sepsis such as early inflammatory response, antiinflammatory response, excessive inflammatory response, immunosuppression, programmed cell death Alter expression levels of miRNAs may be used as a biological marker in the diagnosis and prognosis of sepsis But these results have not been consistent From above reasons, we proceed to the thesis with the following two objects: Investigating the expression level of miRNAs: miRNA- 146-3p, miRNA-147b, miRNA-155, miRNA-223 in patients with sepsis, dengue fever and healthy people Identifing the role of miRNAs: miRNA-146-3p, miRNA- 147b, miRNA-155, miRNA-223 in the diagnosisand prognosis of septic patients * New contributions of the thesis The thesis provides more research results on new biomarkers that can be used in the diagnosis and prognosis of sepsis The study results showed that miRNAs: miRNA-146-3p, miRNA-147b, miRNA155, miRNA-223 were valuable in diagnosing sepsis and septic shock, but had little predictive value in mortality in patients with sepsis and septic shock Patients with bacteremia miRNA is a new biomarker being studied in many diseases, including sepsis and was first studied in Vietnam as a biomarker of sepsis • Thesis structure includes 108 pages Question pages; Chapter 1.Document overview 29 pages; Chapter Research subjects and methods: 20 pages; Chapter Research results: 25 pages; Chapter Discussion: 30 pages; Conclusion 02 pages; Recommend 01 page The thesis has 32 tables, 14 charts, 02 diagrams, 06 drawings; 183 references including 05 Vietnamese references and 178 English refrences Chapter OVERVIEW 1.1 Background of sepsis 1.1.1 Definition of sepsis According to Sepsis-3, current sepsis is defined as a serious organ dysfunction which is life-threatening due to the host's uncontrolled response to infection 1.1.2 Etiology, primary foci of infection and risk factors 1.1.3 Pathogenesis of sepsis 1.1.4 Role of biomarkers in sepsis 1.2 Overview of miRNA 1.2.1 The basics of miRNA Micro RNA was first discovered in 1993 in the Caenorhabditis elegans nematode MiRNAs are short RNAs of about 19 –24 nucleotides that are not involved in protein synthesis Of which, nearly 70% of the miRNAs involved in the regulation of transcription process make circulating RNAs (RNAs), 30% remaining have not been fully functionalized 1.2.2 Mechanism of action of miRNA in humans MiRNAs play an important role in networks that regulate and control complex biological processes involving cells, thereby controlling innate and adaptive immune responses 1.2.3 Biological properties of miRNA 1.2.4 The techniques quantification of miRNA 1.2.5 The role of miRNA in the diagnosis and prognosis of sepsis MiRNAs can act as bio-available markers and may help distinguish the different stages of sepsis Analysis results of some miRNAs - miRNA-143, miRNA-145, miRNA-146a, miRNA-150, miRNA-155, miRNA-182 and miRNA-584 - were found in peripheral blood mononuclear cells (PBMCs) of septic patients Gangli has investigated that microRNA-147b produced in macrophages activates multiple TLRs and plays a role in controlling the negative responses of signals related to bell-receptors (TLRs) TLRs are primary receptors that allow inflammatory cells to recognize invading bacterial pathogens Wang et al used Solexa sequencing and identified six miRNAs to predict septic patients including miRNA-146a, miRNA223 Huang showed that two miRNAs - miRNA-146a, miRNA-223 could be biological markers in diagnosis of sepsis Increased MiRNA-223 correlated with increase in TNFα level and severity of disease Using miRNA as circulating biomarker for sepsis is still primitive However, at this time, some miRNAs have been initially confirmed to be used as biomarkers in the diagnosis and prognosis of sepsis, so further research is needed and many more studies should be done to increase the sensitivity and specificity of this method Chapter SUBJECTS AND METHODS OF RESEARCH 2.1 Subjects of research The research was conducted on 125 patients with sepsis at 02 hospitals: 108 Military Central Hospital and Vietnamese-Czech Friendship Hospital of Hai Phong for years (from December 2014 to December 2017) 2.1.1 Criteria for selecting research patients 2.1.1.1 Criteria for selecting patients with sepsis - Patients more than 18 years old, diagnosed with sepsis according to the international guidelines on the management of respiratory infections and septic shock (2016) - Patients agreed to participate in research 2.1.1.2 Criteria for selecting a control group We selected the control group according to the ratio of ≈ diseases - person being healthy and person with other infectious diseases (dengue patients) - Including 71 healthy people who voluntarily participated in blood donation or came to periodically health check-up at 108 Military Central Hospital - 69 patients were diagnosed with dengue according to WHO standards (2011) 2.1.2 Exclusion criteria - Patients were under 18 years old - Pregnant women - Patients were infected with HIV/AIDS, HBV, HCV - The patient did not agree to participate in the study 2.3 Methods of the research 2.3.1 Research design Cross-sectional descriptive studies, with comparison of control cases 2.3.2 Methods of the research 2.3.2.1 Proceed of sampling, handling and preserving samples ● With sepsis patients Patients with qualified sepsis would provide 04 ml of blood contained in EDTA K2 tube for use as multi-primer PCR test and plasma retention to quantify miRNA relative ● With the control group - healthy people and patients with dengue Healthy people and patients with dengue eligible for the study would provide an additional 02 ml of blood contained in EDTA K2 tube to quantify the relative expression level of plasma miRNA The blood samples in EDTA K2 tube were centrifuged 5000 rounds for 15 minutes at room temperature, then aspirated the upper phase plasma to Epfendor tube, recorded the patient information and stored in the refrigerator at minus 20 degrees Celsius 2.3.2.2 Select miRNA as internal standard From the results of screening and testing phase, we selected miRNA-16 as internal standard 2.3.2.3 Process for quantifying miRNA - Reactive ingredients: Master mix SYBR luminar: 5µl, Primer mix – FR: 1µl/ miRNA (miRNA-16, miRNA- 146-3p, miRNA- 147b, miRNA- 150, miRNA-155, miRNA-223), cDNA: µl - 45 SYBR thermal cycles: 50 ° C- minutes, 95 ° C- 10 minutes, 95 ° C- 15 seconds, 58 ° C-min, 95 ° C- 15 seconds, 60 ° C1 minute, 95 ° C-15 seconds Each sample was repeated times and averaged results for the study The miRNA expression level was quantified relative on the realtime PCR Agilent machine system - Primer: We used a pair of forwards and reverse primers specifically designed by the Department of Molecular Biology of 108 Military Central Hospital for miRNAs: miRNA-16, miRNA146-3p, miRNA-147b, miRNA-155, miRNA-223 - Extracting RNA, store at minor 20 ° C until being used to synthesize cDNA Synthesizing CDNA, using cDNA general commercial crates of Ferenzymtas company Using a self-designed cDNA synthetic Primer set consisting of 16 primers (cDNA Stemloop primer) ● PCR operating condition: 25 C 10 minutes 60 minutes minutes 42 C 70 C - After synthesis, cDNA was diluted in free RNA, DNAwater to get a total volume of 100µl Using 5µl for each realtime PCR reaction QRT-PCR reaction: Using Sybr Green and self-designed primers to operatereal-time PCR the miRNA-16, miRNA-146-3p, miRNA147b, miRNA-155, miRNA-223 ● Real-time PCRoperating condition: 500C - minutes Repeat cycle 950C - 10 minutes Repeat cycle 95 C - 15 seconds 580C - minute Repeat 45 cycles - All reactions were performed on Agilent machines - Internal standard: MiRNA-16 had been selected as the internal standard - Quantitative miRNA assay was performed at the Department of Molecular Biology of 108 Military Central Hospital ● Analysing the result: - Using available software on Agilentmachine to determine threshold cycles of miRNAs and relative expression level of miRNAs based on calculation formula - Results were analyzed based on the ratio between miRNAs candidates and internal standard according to Livac's formula The formula for the relative expression of miRNA MiRNA= 2-∆Ct (∆Ct = CtmiRNA NC - CtmiRNA-16) In which: - Ct miRNA NC was threshold cycle - Ct miRNA-16: threshold cycle of the internal standard miRNA 2.4 Research content 2.4.1 Research indicators on general characteristics of septic patients General clinical features General clinical features: age, sex, entry, number of impaired Subclinical indicators 11 Figure 2.2 Research diagrams 12 Chapter RESEARCH RESULTS 3.1 Characteristics of septic patients Table 3.1 General characteristics of septic patients Characteristics of septic patients Age (X±SD) (year) (min – max) Sex (male) Ratio n=125 (%) 57,6 ± 17,5 (18-87) 90 Length of hospital stay (days) 72,6 12 (5 - 19) Rate of mechanical ventilationpatients 37 29,6 Percentage of dialysis patients 27 21,6 SOFA score 6,1±4,1 Rate of septic shock 50 40 Mortality rate 45 36,0 76 68,8 Rate of positive blood cultures Rate of the incidence of chronic diseases Location of first infection Unknown 18 14,4 Skin, mucous membranes 17 13,6 Respiratory 22 17,6 Digist 27 21,6 Urinary 18 14,4 Nerve central 23 18,4 Conclusions: The average age of the septic group was 57.6 years old, male accounted for 72.6% The overall rates of septic shock and death of the study group were 40% and 36.0% respectively 3.2 Relative expression level of miRNAs in plasma of septic patients 13 Table 3.7.Expression levels of candidate circulating miRNAs in septic patient compared with healthy controls miRNA miRNA-1463p miRNA-147b miRNA-155 miRNA-223 Groups Median(IQR) Sepsis patients Healthyl control (n=125) (n=71) 0,0002 0,02 (0,00007-0,001) (0,0007-0,63) 2,12 (0,07-50,07) 0,0173 (0,0059-0041) 0,023 (0,002-0,24) 0,0055 (0,0008-0,06) 0,0025 (0,00076-0,0072) 0,0008 (0,0004-0,0014) p