Tóm tắt: Nghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết Dengue

VIETNAM MILITARY MEDICAL UNIVERSITYNGUYEN MINH NAM RESEARCH ON THE EXPRESSION LEVELS ANDPROGNOSTIC VALUE OF SOME LONG NON-CODING RNAs LncRNAs IN DENGUE HEMORRHAGIC FEVER PATIENTS SpecialNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết DengueNghiên cứu mức độ biểu hiện và giá trị tiên lượng của một số long non-coding RNA (LncRNA) ở người bệnh sốt xuất huyết Dengue

VIETNAM MILITARY MEDICAL UNIVERSITY

NGUYEN MINH NAM

RESEARCH ON THE EXPRESSION LEVELS ANDPROGNOSTIC VALUE OF SOME LONG NON-CODING RNAs

(LncRNAs) IN DENGUE HEMORRHAGIC FEVER PATIENTS

Specialty: Infectious and tropical diseases

Code: 9 72 01 09

THE SUMMARY OF THE MEDICAL DOTORAL THESIS

HANOI – 2024

AT VIETNAM MILITARY MEDICAL UNIVERSITY

Scientific Supervisors:

1 Assoc Prof Dr Hoang Van Tong

2 Assoc Prof Dr Do Tuan Anh

Reviewer 1: Prof Dr Bui Vu Huy

Reviewer 2: Assoc Prof Dr Phan Quoc Hoan

Reviewer 3: Assoc Prof Dr Hoang Vu Hung

The doctoral thesis was defended at the University’s Committee

of Grading at Vietnam Military Medical University at … on ……20…

Searching for dissertation at:

1 National Library

2 Vietnam Military Medical University’s library

Dengue hemorrhagic fever (DHF) is endemic in over 100countries, with approximately 70% of cases occurring in Asia.Around 500,000 severe cases of DHF are reported each year, with

a mortality rate of about 10% However, this rate can be reduced

to less than 1% if patients are diagnosed early and treatedpromptly Long non-coding RNAs (LncRNAs) RNA molecules that

do not code for proteins and are longer than 200 nucleotides.Many lncRNAs are involve in antiviral responses and play a role invarious interactions between viruses and hosts Research on cellmodels has shown that in DENV-infected cells, lncRNAs such asEPB41L4A-AS1, IFI6lnc1, IFI6lnc2, HID1-AS1, ILK 4, OAS1 lnc102exhibit increased expression compared to uninfected cells.Therefore, studying the expression of these lncRNAs can providefurther insights into their mechanisms and potential applications

in the early prognostication of severe dengue risk in patients.Therefore, we conducted the study with two objectives:

1 To determine the expression levels of specific plasma lncRNAs in patients with dengue hemorrhagic fever according to various disease severity.

2 To analyze the relationship between the expression levels of these plasma lncRNAs and clinical, paraclinical characteristics as well as prognostic value in patients with dengue hemorrhagic fever.

NEW CONTRIBUTION OF THE THESIS

1 The expression levels of the lncRNAs EPB41L4A-AS1,IFI6lnc1, IFI6lnc2, HID1-AS1, Lnc-ILK-4, and OAS1 lnc102 have

been identified in patients with Dengue hemorrhagic fever.

2 Research findings indicate that the expression levels oflncRNAs (EPB41L4A-AS1, IFI6lnc1, IFI6lnc2, HID1-AS1, Lnc-ILK-

4, OAS1 lnc102) are associated with hemorrhagic status, and variouslaboratory tests (including blood creatinine and liver enzymes AST,

ALT) in patients with Dengue hemorrhagic fever These lncRNAs demonstrate both diagnostic and prognostic value in Dengue

hemorrhagic fever

STRUCTURE OF THE THESIS

The thesis comprises 123 pages (excluding references andappendices), and is divided into 4 chapters It includes 32 tables, 24figures, 12 references in Vienamese and 140 references in English.The content is organized as follows: 2 pages introduction, 33 pages forthe literature overview, 25 pages for subjects and methods, 31 pages forresults, 29 pages for the discussion, 2 pages for the conclusion, 1 pagefor the recommendations

CHAPTER 1 - LITERATURE OVERVIEW

1.1.Epidemiology of Dengue hemorrhagic fever

1.5 Clinical and paraclinical characteristics of Dengue fever

Dengue fever has a variety of clinical manifestations,

ranging swiftly from mild to severe levels The onset of the disease

is typically abrupt and progresses through three distinct stages:the febrile phase, the critical phase, and the recovery phase

1.6 Overview of lncRNA

Long non-coding RNA (lncRNA) refers to a group of RNAmolecules that do not encode proteins (non-coding RNA) and arelonger than 200 nucleotides They share structural similaritieswith mRNA but lack an open reading frame

1.7 Overview of lncRNA and studies on lncRNA in Dengue

hemorrhagic fever

1.7.1 LncRNA in research

LncRNA EPB41L4A-AS1 (EPB41L4A antisense RNA 1) islocated on chromosome 5q22.2, ), has a length of 1,194 bp Studieshave shown that EPB41L4A-AS1 is closely associated with theregulation of cellular metabolism LncRNA-IFI6 (IFI6lnc1) (alsoknown as lncRNA RP11–288L9.4; lnc-FAM76A-1:1) is located onchromosome 1 (p36, 11) with a length of 489 bp IFI6lnc2, alsoknown as LINC02574 (Long Intergenic Non-Protein Coding RNA2574), is located on chromosome 1 (length 441 bp) HID1-AS1located on chromosome 17 (length 830 bp) Lnc-ILK4 is located on

chromosome 11 (380 bp) OAS1 lnc102 (also known as 1:1) is located on chromosome 12 (length 575 bp).

lnc-RASAL1-CHAPTER 2- SUBJECTS AND METHODS OF THE STUDY

2.1 Subjects of the study

2.1.1 Patient group: The study included 377 patients diagnosed

and treated for Dengue hemorrhagic fever at Military Hospital

103, categorized as follows: 150 patients diagnosed with Denguehemorrhagic fever (DHF); 159 patients diagnosed with Denguehemorrhagic fever with warning signs (DWS); 68 patientsdiagnosed with severe Dengue hemorrhagic fever (SD) Among the

377 patients, 59 were monitored for the levels of lncRNAs on day

2, day 5 and day 8 of the disease

2.1.1.1 Selection criteria for patient group

- Age ≥ 16

- Patients diagnosed with Dengue hemorrhagic fever

* Diagnostic criteria for Dengue hemorrhagic fever were based onthe 2019 guidelines of the Ministry of Health and the 2009 WorldHealth Organization recommendations

+ Positive DENV NS1 antigen test and/or positive DENV IgMantibody test

2.1.1.2 Exclusion criteria for patient group

- Pregnant women; patients with co-infected with other viralpathogens (eg: HCV, HBV, SARS-CoV-2, influenza etc ); patientsundergoing treatment for other chronic diseases such as: cancer,autoimmune diseases, or those using immunosuppressants,corticosteroids, or on dialysis

- Patients with diseases that may cause bleeding such as:thrombocytopenic purpura, hemophilia, cirrhosis were alsoexcluded

2.1.2 Control group: This group consists of 128 volunteers who

meet the criteria

- Age ≥ 16; No clinical symptoms of Dengue hemorrhagic fever

- No history of diseases such as: hepatitis, cancer, autoimmunediseases, kidney failure, or diseases that may cause bleeding (e.gthrombocytopenic purpura, hemophilia, cirrhosis ).

- Fundamental hematology and biochemistry indicators areobserved to be within the normal range

2.5 Procedure for quantifying plasma lncRNA concentrations

2.5.1 Generation of standard concentration cDNA samples

Table 2.2 Information of the recombinant plasmid samples

Concentratio

n (Mean, ng/µl)

102, 103, 104, 105, 106 copies/µl were obtained to construct a

standard curve for the real-time quantitative PCR method

2.5.2 Construction of standard curve

Based on the average Ct results obtained from five runs withstandard samples at concentrations of 5, 101, 102, 103, 104, 105, 106 copies/µl, the study established a standard curve for 6 lncRNAs Thestandard curves for all lncRNAs demonstrated a correlationcoefficient R2 >0.99 and an amplification coefficient >90%

2.5.3 Quantification of plasma lncRNA concentrations

2.5.3.1 Plasma RNA isolation using the abGenix automated RNA extraction system

2.5.3.2 Synthesis of cDNA from total RNA samples

2.5.3.3 Conduct Real-Time PCR reaction to determine Ct value of the lncRNAs

2.5.3.4 Plasma lncRNA concentrations

The concentration of lncRNA in plasma (unit is copies/ml plasma)was calculated by the formula:

- VPCR is the volume of extracted cDNA used for each PCR reaction

- Vext is the volume of plasma used for extraction (ml)

2.6 Statistical analysis

Data collection was performed using Microsoft Excel 2016.Data processing was conducted using SPSS 22.0, adhering tobiomedical statistical algorithms

CHAPTER 3- RESULTS 3.1 General characteristics of the study subjects

3.2 Expression of plasma lncRNAs in the study subjects

Table 3.7 Concentrations of lncRNAs EPB41L4A-AS1, IFI6lnc1, IFI6lnc2, HID1-AS1, Lnc-ILK-4 in study groups

during the critical phase.

0.75 (0.33-1.45)

3.95 (1.64-7.04)

14.75 (6.71-26.94) p* p(1,2)<0.001; p(2,3)<0.001; p(3,4)<0.001; p(1,4)<0.001 IFI6ln

c1

Median

(Quartiles)

0.11 (0.05-0.33)

0.40 (0.16-0.84)

1.60 (0.80-3.40)

10.50 (4.53-22.53) p* p(1,2)<0.001; p(2,3)<0.001; p(3,4)<0.001; p(1,4)<0.001

0.46 (0.20-0.29)

4.22 (2.28-7.49)

7.88 (4.13-15.78) p* p(1,2)<0.001; p(2,3)<0.001; p(3,4)<0.001; p(1,4)<0.001

0.23 (0.09-0.44)

1.31 (0.72-2.65)

2.98 (1.51-7.35) p* p(1,2)<0.001; p(2,3)<0.001; p(3,4)<0.001; p(1,4)<0.001

0.31 (0.13-0.66)

2.28 (1.23-3.77)

5.37 (2.65-10.56) p* p(1,2)<0.001; p(2,3)<0.001; p(3,4)<0.001; p(1,4)<0.001

* Mann Whitney test, CG: Control group

Table 3.7 shows that the lncRNAs EPB41L4A-AS1, IFI6lnc1,IFI6lnc2, HID1-AS1, Lnc-ILK-4 were expressed in all studysubjects (both control and patient groups) The concentrations ofthe these lncRNAs increased gradually with the severity of thedisease The differences between the groups were statisticallysignificant with p<0.001

Table 3.8 OAS1-lnc102 expression in study groups during

the critical phase LncRNA (10 5 copies/ml) CG (1)

(n=128)

DHF (2) (n=150)

DWS (3) (n=159)

SD (4) (n=68)

0.04 (0.02 -0.09)

0.16 (0.08-0.31)

0.50 (0.23-0.86)

p p(1,2)<0.001* p(2,3)<0.001* p(3,4)<0.001* p(1,4)<0.001*

* Mann Whitney test,  Chi-Square test, CG: Control group

Table 3.8 shows that the proportion of samples with expressinglncRNA OAS1-lnc102 in the control group was lower than in thepatient groups (the difference was statistically significant withp<0.01) The median concentration of lncRNA OAS1 increasedgradually with the severity of the disease The lowestconcentration was in the control group; the highest was in the SDgroup The differences between the groups were all statisticallysignificant with p<0.001

Figure 3.1 Concentrations of lncRNAs in the follow-up group

Figure 3.1 shows that the concentrations of 6 lncRNAs in DHFpatients at different testing points were all higher than those in thecontrol group, the difference was significant with p<0.01

The concentration of lncRNA EPB41L4A-AS1 at time point T1 wassignificantly higher than at time point T0, with p < 0.05

The concentrations of lncRNAs IFI6lnc1, IFI6lnc2, HID1-AS1, ILK-4, OAS1 lnc102 at time points T0, T1, T2 corresponding to thefever phase, the critical phase, and the recovery phase in DHFpatients had no difference (p>0.05)

Lnc-3.3 Relationship between the expression of plasma lncRNAs and some clinical symptoms, laboratory tests and severity of

Dengue hemorrhagic fever in the critical phase

3.3.1 Relationship between lncRNAs and DENV diagnostic testing

Table 3.11 Concentration of lncRNAs in the group of patients

positive and negative for DENV-IgG antibodies.

LncRNA (10 5 copies/ml)

Anti DENV -IgG p*

Positive (n=228) Negative(n=128) EPB41L4A-

(n=356) Median (Quartiles)

1.82 (0.68-5.65)

0.65 (0.22-1.85) p<0.01IFI6lnc2

(n=356) Median (Quartiles)

4.01 (1.34-8.06)

0.67 (0.27-3.21) p<0.01HID1-AS1

(n=356) Median (Quartiles)

1.30 (0.48-2.83)

0.35 (0.15-1.09) p<0.01Lnc-ILK-4

(n=356) Median (Quartiles)

2.32 (0.77-4.42)

0.43 (0.15-1.82) p<0.01

* Mann Whitney test

The results in Table 3.11 show that the median concentrations of lncRNAs EPB41L4A-AS1, IFI6lnc1, IFI6lnc2, HID1-AS1, Lnc-ILK-4, OAS1 lnc102 in DHF patients with positive DENV-IgG test were all higher than the median concentrations of the these lncRNAs in the group with negative DENV IgG test (the difference is statistically significant with p<0.01)

3.3.2 Relationship between lncRNA concentrations and some clinical and hematological symptoms in patients with dengue hemorrhagic fever

Table 3.14 Concentrations of lncRNAs in patient groups according to platelet levels in the critical phase LncRNA (10 5 copies/ml)

Platelet levels p**

<50G/L (n=186)

50≤PL≤100G/

L (n=57)

>100G/L (n=134) EPB41L4A -

AS1 (n=377)

Median (Quartiles)

5.51 (2.72-11.05)

3.13 (0.90-7.88)

0.67 (0.31-1.42)

p<0.0 1 IFI6lnc1

(n=377)

Median (Quartiles)

2.41 (1.06-8.70)

1.48 (0.40-4.01)

0.40 (0.16-0.84)

p<0.0 1 IFI6lnc2

(n=377)

Median (Quartiles)

5.24 (2.87-8.72)

2.99 (0.54-6.61)

0.46 (0.19-0.98)

p<0.0 1 HID1-AS1

(n=377)

Median (Quartiles)

1.69 (1.0-3.75)

0.96 (0.24-2.1)

0.24 (0.08-0.44)

p<0.0 1 Lnc-ILK-4

(n=377)

Median (Quartiles)

2.99 (1.76-5.42)

1.72 (0.32-3.34)

0.30 (0.12-0.64)

p<0.0 1

0.20 (0.10-0.47)

0.12 (0.03-0.27)

0.04 (0.02-0.10)

p<0.0 1

** Kruskal-wallis, PL: Platelet

The results in Table 3.14 show that in patients with Dengue hemorrhagic fever, as the platelet levels decrease, the median concentrations of the lncRNAs increase

Table 3.17 Concentrations of lncRNAs in patients with and without mucosal hemorrhage

LncRNA (10 5 copies/ml) Mucosal hemorrhage p*

Yes (n=148) No (n=229) EPB41L4A -AS1

(n=377)

Median (Quartiles)

5.58 (2.73-10.87)

1.26 (0.50-3.84) p<0.01IFI6lnc1 (n=377) (Quartiles)Median 2.20

(1.02-8.03)

0.70 (0.28-2.11) p<0.01IFI6lnc2 (n=377) (Quartiles)Median 4.67

(2.65-8.17)

0.99 (0.32-3.97) p<0.01HID1-AS1

(n=377)

Median (Quartiles)

1.58 (0.92-3.01)

0.42 (0.16-1.36) p<0.01Lnc-ILK-4

(n=377)

Median (Quartiles) (1.73-4.93)2.74 (0.19-2.24)0.56 p<0.01

(Quartiles)

0.21 (0.09-0.46)

0.07 (0.03-0.19)

* Mann Whitney test

The results in Table 3.17 indicate that in Dengue hemorrhagic fever patients with bleeding symptoms, the median concentration oflncRNAs was higher than in the group of patients without bleedingsymptoms The difference between the two groups was statisticallysignificant with p<0.01

Table 3.19 Concentrations of lncRNAs in patient groups

according to ALT enzyme levels LncRNA (10 5 copies/ml)

<80U/L (n=233) 80≤ALT≤400U/L (n=99) >400U/L(n=42) EPB41L4A

3.46 (1.41-7.04)

14.8 (7.79-27.44)

p<0.0 1

1.66 (0.84-4.57)

12.0 (4.64-21.77)

p<0.0 1

3.89 (1.32-7.29)

8.93 (5.93-18.84)

p<0.0 1

1.32 (0.52-2.74)

3.10 (1.53-7.59)

p<0.0 1

2.08 (0.94-3.53)

6.21 (3.14-11.14)

p<0.0 1

0.16 (0.08- 0.34)

0.50 (0.26- 0.80)

p<0.0 1

** Kruskal-Wallis

The results in Table 3.19 show that in dengue fever patients,

as the ALT levels increased, the median concentrations of thelncRNAs also increased The difference between the groups wasstatistically significant, with p < 0.01

3.3.4 Diagnostic and prognostic value of lncRNAs in Dengue hemorrhagic fever during the critical phase

Table 3.21 Discriminatory value of lncRNAs between severe and severe Dengue hemorrhagic fever groups LncRNA AUC 95% CI p Sensitivity (%) Specificity (%) (10 5 Cut off copies/ml)

0.828 0.779-0.876 p<0.001 73.5 77.3 5.11HID1-AS1

0.827 0.778-0.875 p<0.001 76.5 74.8 1.49Lnc-ILK-4

0.861 0.818-0.905 p<0.001 92.6 66.7 1.90OAS1-

Table 3.22 Univariate regression analysis of factors related to severity in patients with Dengue hemorrhagic fever

Gender -0.136 0.873 0.516-1.478 >0.05 Subcutaneous hemorrhage 1.039 2.825 1.652-4.831 <0.01 Mucosal hemorrhage 1.206 3.342 1.935-5.770 <0.01