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 SE

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

The thesis can be found at:

1 National Library of Vietnam

2 Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences

3 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 6 (IL-6), CRP, lactate are limited

MicroRNAs (MiRNAs) are endogenous RNAs which are not involved in protein synthesis but control gene regulation in the post-transcription phase MiRNA is present at various stages in the pathogenesis of sepsis such as early inflammatory response, anti-inflammatory 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:

1 Investigating the expression level of 4 miRNAs: 146-3p, miRNA-147b, miRNA-155, miRNA-223 in patients with sepsis, dengue fever and healthy people

miRNA-2 Identifing the role of 4 miRNAs: 146-3p, 147b, miRNA-155, miRNA-223 in the diagnosisand prognosis of septic patients

miRNA-* 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 4 miRNAs: miRNA-146-3p, miRNA-147b, miRNA-

155, 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 2 pages; Chapter 1.Document overview 29 pages; Chapter

2 Research subjects and methods: 20 pages; Chapter 3 Research results: 25 pages; Chapter 4 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 1

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, miRNA-

223 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 2 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 3 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 ≈ 2 diseases - 1 person being healthy and 1 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: 5 µl

- 45 SYBR thermal cycles: 50 ° C- 2 minutes, 95 ° C- 10 minutes, 95 ° C- 15 seconds, 58 ° C-min, 95 ° C- 15 seconds, 60 ° C-

1 minute, 95 ° C-15 seconds

Each sample was repeated 2 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 5 miRNAs: 16, 146-3p, miRNA-147b, miRNA-155, miRNA-223

miRNA 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 4 primer)

QRT-PCR reaction: Using Sybr Green and self-designed primers to operatereal-time PCR the miRNA-16, miRNA-146-3p, miRNA-147b, miRNA-155, miRNA-223

● Real-time PCRoperating condition:

950C - 10 minutes Repeat 1 cycle

950C - 15 seconds

Repeat 45 cycles

580C - 1 minute

- 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

● Hematological changes

● Biochemical changes in blood

● Evaluation of biomarkers

- Procalcitonin: Procalcitonin concentration in plasma (ng/ml)

was measured by immunofluorescence method (LUMItest PCT of Brahms Diagnostica company, Germany) The threshold of detection was 0.05 ng/ml, the coefficient of variation at low and high concentrations was 12% and 5%, respectively

- MiRNA:Evaluation of the relative expression level of

miRNAs: miRNA-146-3p, miRNA-147b, miRNA-155, miRNA-223 The assay was carried out by the method of real-time quantitative gene sequencing on Agilent machine system (Section 2)

2.5 Machines, biologicals and techniques used in research

2.5.1 Clinical examination

Daily clinical examination was performed by clinicians at research sites in collaboration with researchers, detecting symptoms and recording in sample research records

2.5.2 Basic hematology and biochemical tests

Basic hematology and biochemical tests were performed according to standard procedure of laboratory testing in hospitals Criteria for evaluating some test indicators were based on Vietnamese biological constants[116]

2.7

2.8

Ethical Council of the 108

Research Institute, and the Vietnamese

Hai Phong

Data processing

- Data were managed

- Data were processed using SPSS software version 22.0

managed with Excel 2010 software

Data were processed using SPSS software version 22.0

The research protocol was approved by the Scientific and Ethical Council of the 108 Clinical Medicine and Pharmacy Research Institute, and the Vietnamese

Excel 2010 software

Data were processed using SPSS software version 22.0

The research protocol was approved by the Scientific and

Clinical Medicine and Pharmacy Research Institute, and the Vietnamese-Czech Friendship Hospital of

Data were processed using SPSS software version 22.0

The research protocol was approved by the Scientific and

Clinical Medicine and Pharmacy Czech Friendship Hospital of

Figure 2.2 Research diagrams Figure 2.2 Research diagrams Figure 2.2 Research diagrams

Chapter 3 RESEARCH RESULTS 3.1 Characteristics of septic patients

Table 3.1 General characteristics of septic patients

Characteristics of septic patients n=125 Ratio

(%)

Age (X±SD) (year) (min – max) 57,6 ± 17,5 (18-87)

Length of hospital stay (days) 12 (5 - 19)

Rate of mechanical ventilationpatients 37 29,6

Rate of positive blood cultures

Rate of the incidence of chronic diseases 76 68,8

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 4 miRNAs in plasma of septic patients

Table 3.7.Expression levels of candidate circulating miRNAs

in septic patient compared with healthy controls

miRNA

Groups

Median(IQR)

p Sepsis patients

(n=125)

Healthyl control (n=71)

Table 3.8.Expression levels of candidate circulating miRNAs in

septic patient compared with DHF patients

miRNA Groups (Median (IQR))

p Sepsis (n=125) DHF (n=69)

3.2.3 Relative expression level of 4 miRNAs with organ dysfunction

3.2.3.1 Relative expression level of miRNAsin septic patients with respiratory dysfunction

Bảng 3.13 The expression level ofmiRNAs in septic patients with

(0,0308-miRNA-155 0,042 (0,0046-0,751) 0,0089 (0,0013-0,136)

miRNA-223 0,0113

(0,00134-0,475)

0,0043 0,0415)

(0,0005-Comments:The expression level ofmiRNAs in septic patients with respiratory dysfunction groups is higher than other groups

3.2.4.2 The expression level of miRNAs in septic patients with cardiovascular dysfunction

Bảng 3.14 The expression level of miRNAs in septic patients with

Comments: The relative expression level of miRNA-155 in septic patients with cardiovascular impairment was significantly higher than in the group without cardiovascular dysfunction

3.2.4.3.The relative expression level of miRNA-155 in septic patients withacute kydney injury

Table 3.15 The relative expression level of miRNAs in septic patients withacute kydney injury

Comments: The expression level of miRNA-147b and miRNA-155 in septic patients withAKI groups were higher than other groups (p<0,05)

3.2.5 The expession level of miRNA in septic shocks patients compare with septic patients

Bảng 3.16 The expession level of miRNA in septic shocks patients

compare with septic patients

miRNA

Septic shock

Trungvị (Khoảngtứphânvị) P Have (n=50) Not have (n=75)

3.2.8 The relative expession level of miRNAs in according to

treatment result sin septic patients

Table 3.19.The expression levels of miRNAs according to treatment results