Application of lncRNA MIR3945HG in the preparation of a diagnostic kit for detecting sepsis

Abstract

The present invention relates to an application of lncRNA MIR3945HG in preparing a diagnostic kit for detecting sepsis. The kit can be used for early diagnosis of sepsis and has the advantages of good sensitivity and strong specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to an application of lncRNA MIR3945HG in preparing a diagnostic kit for detecting sepsis. Background Art

[0002] According to the latest definitions from the Society of Critical Care Medicine (SCCM) and the European Society of Intensive Care Medicine (ESICM), sepsis is a life-threatening organ dysfunction caused by a dysregulated host immune response to infection. It can further develop into septic shock and multiple organ dysfunction syndrome (MODS). It is a common, severe complication and leading cause of death in critically ill patients with infections, burns / trauma, shock, and other conditions. It poses a serious threat to human health, with high mortality and treatment costs. Despite significant advances in anti-infective therapy and organ function support technologies in recent years, there is still no effective treatment for sepsis, and the mortality rate remains as high as 30%-70%. Multicenter epidemiological surveys show that the incidence of sepsis in intensive care units (ICUs) in my country exceeds 8%, with a mortality rate as high as 45%. Since the International Sepsis Alliance launched the Surviving Sepsis Campaign (SSC) in 2001, issued the Barcelona Declaration in 2002, and established five goals for the first World Sepsis Day, the International Sepsis Alliance and the World Health Organization (WHO) have been committed to jointly promoting public awareness of sepsis and developing new sepsis management guidelines in 2016. In 2017, WHO members published an article titled "Confirming Sepsis as a Global Health Priority—A WHO Resolution" in The New England Journal of Medicine. In 2018, the SSC Executive Committee reaffirmed the Surviving Sepsis Campaign and updated the SSC guidelines. Controlling sepsis has become a major issue that demands urgent attention in the medical community.

[0003] Sepsis has a complex pathogenesis, involving multiple tissues and organs throughout the body. It is a severe disease with a high mortality rate. Currently, the main bottlenecks are difficulty in diagnosis, delayed response time, and difficulty in treatment. According to sepsis treatment guidelines, time is a crucial factor in rescuing patients with sepsis, making early prediction and diagnosis of sepsis particularly important. Early diagnosis is a prerequisite for the earliest possible initiation of treatment. The diagnosis of sepsis is primarily based on clinical manifestations and laboratory tests, but the clinical presentation of sepsis is diverse and lacks a specific clinical presentation. Currently, pathogen culture is the most important indicator for diagnosing sepsis. However, it is time-consuming, does not reflect efficacy, and culture methods vary across regions, resulting in low positive rates, false negatives, and even the inability to rule out contamination, hindering early diagnosis and treatment. Consequently, numerous studies have focused on identifying biomarkers of sepsis for diagnosis and prognostic assessment. However, the reliability of currently commonly used nonspecific infection biomarkers in clinical practice (PCT, CRP, WBC, etc.) is limited. CRP and WBC levels increase in conditions such as common local infections, trauma, inflammation, and certain tumors, resulting in low specificity. PCT also has low specificity and poor agreement with pathogen culture results. PCT increases to varying degrees after surgery, cardiogenic shock, acute graft-versus-host disease, bone marrow suppression, and end-stage renal disease. In particular, PCT undergoes physiological changes within 48 hours of birth, limiting its use in early-onset neonatal sepsis and preventing rapid diagnosis of sepsis in the critical early stages. Finding biomarkers with both high sensitivity and specificity for use in the diagnosis, prognosis, and treatment monitoring of sepsis is of great clinical significance. Summary of the Invention

[0004] The present invention aims to provide a use of lncRNA MIR3945HG in the preparation of a diagnostic kit for detecting sepsis, wherein the kit can be used for diagnosing early sepsis and has the advantages of good sensitivity and strong specificity.

[0005] The technical solution of the present invention is:

[0006] Application of peripheral blood leukocyte lncRNA MIR3945HG in the preparation of a diagnostic kit for detecting sepsis.

[0007] The encoding gene ID of the lncRNA MIR3945HG is ENSG00000251230.

[0008] The transcript ID of the lncRNA MIR3945HG is ENST00000511703, and its sequence is shown in SEQ ID NO.5.

[0009] When the fluorescent dye method is used, the kit includes: lncRNA MIR3945HG expression detection specific primers, SYBR Green fluorescent dye, dNTP and Taq enzyme.

[0010] When the fluorescent probe method is used, the kit includes: lncRNAMIR3945HG expression detection specific primers, TaqMan fluorescent probes, dNTPs and Taq enzyme.

[0011] The sequences of the lncRNAMIR3945HG expression detection specific primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] The kit of the present invention provides a new sepsis diagnostic indicator, which can be used for the diagnosis of early sepsis and has good sensitivity and strong specificity.

[0013] The applicant's experiments showed that the expression level of peripheral blood leukocyte lncRNA MIR3945HG in clinical sepsis patients was significantly higher than that in healthy volunteers. ROC analysis found that the peripheral blood leukocyte lncRNA MIR3945HG of the subjects can be used as a new biomarker for the diagnosis of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Graph showing the detection results of lncRNA MIR3945HG in peripheral blood leukocytes of sepsis patients and healthy volunteers in Example 1 of the present invention;

[0015] Figure 2 Schematic diagram showing the ROC curve analysis of the diagnostic effect of lncRNA MIR3945HG in sepsis in Example 1 of the present invention;

[0016] Figure 3 Graph showing the detection results of lncRNA MIR3945HG in peripheral blood leukocytes of sepsis patients and healthy volunteers in Example 2 of the present invention;

[0017] Figure 4 Schematic diagram showing the ROC curve analysis of the diagnostic effect of lncRNA MIR3945HG in sepsis in Example 2 of the present invention. DETAILED DESCRIPTION

[0018] The following will describe the embodiments of the present invention through specific examples in conjunction with the accompanying drawings. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0019] Experimental methods without specific conditions specified in the examples are generally carried out under conventional conditions or conditions recommended by the manufacturers.

[0020] Based on the applicant's previous research and screening of a large number of biomarkers related to the development and progression of sepsis, the present invention found that the expression level of long non-coding RNA MIR3945HG (lncRNA MIR3945HG) is closely related to the development and progression of sepsis, and can be used as a good biomarker for the diagnosis of sepsis.

[0021] First, a sepsis lncRNA omics dataset (GSE134347) from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo) was analyzed. The expression levels of the peripheral blood lncRNA MIR3945 were differentially analyzed between 156 sepsis patients and 83 healthy volunteers. The researchers found that the expression levels of the lncRNA MIR3945HG in the peripheral blood of sepsis patients were significantly higher than those in healthy volunteers. Further diagnostic test analysis showed that the AUC for the diagnosis of sepsis using the peripheral blood lncRNA MIR3945HG was 0.954 (95% CI = 0.930-0.979), with a specificity and sensitivity of 90.4% and 89.1%, respectively. This suggests that the expression level of the lncRNA MIR3945HG in the peripheral blood of subjects can serve as an important biomarker for the diagnosis of sepsis with high sensitivity and specificity.

[0022] Furthermore, quantitative PCR (SYBR Green fluorescent dye method) was used to measure the expression of lncRNA MIR3945HG in peripheral blood leukocytes from 40 patients with sepsis and 20 healthy volunteers. The researchers found that the expression of lncRNA MIR3945HG in peripheral blood leukocytes was significantly elevated in patients with sepsis compared with healthy volunteers. Diagnostic test analysis showed that the AUC for the diagnosis of sepsis by lncRNA MIR3945HG in peripheral blood leukocytes was 0.875 (95% CI = 0.758-0.992), with a specificity and sensitivity of 80.0% and 82.1%, respectively. This confirms the value of peripheral blood lncRNA MIR3945HG expression as a diagnostic biomarker for sepsis.

[0023] Example 1

[0024] Reanalysis and ROC analysis of lncRNA MIR3945HG expression in the GEO database

[0025] The lncRNA omics dataset of sepsis patients (GSE134347, sequencing platform: GPL17586[HTA-2_0]Affymetrix Human TranscriptomeArray 2.0[transcript(gene)version)) was downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo). The signal value of lncRNA MIR3945HG (transcript ID: ENST00000511703, probe ID: TC04002881.hg.1) was extracted. The independent sample T test statistical method was first used to compare the expression signal values ​​of lncRNA MIR3945 in the peripheral blood of 156 sepsis patients and 83 healthy volunteers. The results showed that lncRNA MIR3945 in the peripheral blood leukocytes of sepsis patients was significantly higher than that in the peripheral blood leukocytes of sepsis patients. The expression level of MIR3945HG was 81.66±36.96, which was significantly higher than that of healthy volunteers (43.05±6.42). The difference in expression was statistically significant (P=1.75×10 -26 ) (See Figure 1 ). Further analysis using the receiver operating characteristic (ROC) diagnostic test evaluation method showed that the area under the curve (AUC) of peripheral blood lncRNA MIR3945HG for diagnosing sepsis was 0.954 (95% CI = 0.930-0.979), with a specificity and sensitivity of 90.4% and 89.1%, respectively (see Figure 2 ). This indicates that the expression of lncRNA MIR3945HG in the peripheral blood of subjects can be used as an important biomarker for the diagnosis of sepsis with high sensitivity and specificity.

[0026] Example 2

[0027] Validation of the predictive value of peripheral blood lncRNA MIR3945HG for the diagnosis of sepsis

[0028] Peripheral blood samples were collected from 40 clinical sepsis patients on admission and 21 healthy volunteers. Leukocytes were separated and total RNA was extracted for reverse transcription. Quantitative PCR primers specific for lncRNA MIR3945HG expression were used. The primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The housekeeping gene GAPDH was used as an internal reference. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The expression level of lncRNA MIR3945HG was detected by 2 -△△CT (△CT=CT lncRNA -CT GAPDH , △△CT=△CT 脓毒症 -△CT 健康自愿者) The relative expression difference between sepsis patients and healthy volunteers was calculated using 2 -△CT The expression level of lncRNA MIR3945HG in each sample was calculated, and ROC analysis was used to evaluate the diagnostic value of lncRNA MIR3945HG for sepsis.

[0029] The analysis results showed that the expression level of lncRNA MIR3945HG in peripheral blood leukocytes of patients with sepsis was significantly higher than that of healthy volunteers (difference fold = 2.18 ± 2.02, P = 0.005) ( Figure 3 ), further ROC analysis showed that the AUC of peripheral blood lncRNA MIR3945HG for diagnosing sepsis was 0.875 (95% CI = 0.758-0.992), with specificity and sensitivity of 80.0% and 82.1%, respectively ( Figure 4 ). The clinical value of the peripheral blood leukocyte lncRNA MIR3945HG expression level found in Example 1 as a biomarker for the diagnosis of sepsis was confirmed.

[0030] In summary, the present invention found that the expression level of lncRNA MIR3945HG in the peripheral blood leukocytes of clinical sepsis patients was significantly higher than that in healthy volunteers. Receiver-operating characteristic (ROC) analysis revealed that lncRNA MIR3945HG in the peripheral blood leukocytes of these subjects can serve as a new biomarker for the diagnosis of sepsis. Therefore, the present invention provides lncRNA MIR3945HG as a biomarker for the diagnosis of sepsis. Sequence Listing <110> Chinese People's Liberation Army Special Medical Center <120> Application of lncRNA MIR3945HG in the preparation of a diagnostic kit for detecting sepsis <141> 2022-05-24 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> LncRNA MIR3945HG quantitative PCR primer <400> 1 ctcagtcaca agacggctgc 20 <210> 2 <211> twenty one <212> DNA <213> LncRNA MIR3945HG quantitative PCR primer (Lncrna mir3945hg quantitative PCR primer) <400> 2 tttaggttcc tgagcctgga g <210> 3 <211> 21 <212> DNA <213> Internal reference GAPDH quantitative PCR primer <400> 3 ccaaaagggt catcatctct g <210> 4 <211> 21 <212> DNA <213> Internal reference GAPDH quantitative PCR primer <400> 4 catgagtcct tccacgatac c <210> 5 <211> 729 <212> DNA <213> LncRNA MIR3945HG(LncRNA MIR3945HG) <400> 5 60. aagcctatct ccttttcctg ccttgtgtct ttccagtcac cgctgtgtct aggtgtggcc atatgagtag ttctcaccag tggaatgtag tgaaaacgag atgctgtctc ttagggacta aggaagctat ctttcctctt ccgacatttg ctgactggtc gatgctagat gatcttgaga ccctgtgctg aggactggag aactacagga aaggagcagt atctctgaat 240 tatcgtgtgg aaggtcactt gtctagcccc tgatgactgt gagcctcttc ctcctgcttg 300 ccacctctca gtcacaagac ggctgctgcg actcaggctc atgtcccaat tcaaggcagc 360 aagaaggcca tggagcggca cctgccagca gatgcccctg caggccatct ctccaggctc 420 aggaacctaa agaagaatct acccagatgt ggtggctcac acctgtgatc ccaccacttt 480 gggaggctga ggcaagagga tcacttgagc ccaggagttg gagaccaacg taggcaatac 540 agcaagactc ccatctctac aaaaaaaaa aagaaagaaa gaaagaaaga aaagaaatta 600 gctggctgtg gtgctgcaca tttgtaatac cagctactct agaggctgag gctggaggat 660 cacttgagcc gaggagtgag ctatgaccac accattgcat ttcagcctca gtgacagagc 720 aaggccctg 729

Claims

1. Use of a detection reagent for peripheral blood leukocyte lncRNA MIR3945HG in the preparation of a diagnostic kit for detecting sepsis, the detection reagent comprising specific primers as shown in sequences SEQ ID NO.1 and SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The encoding gene ID of the lncRNA MIR3945HG is ENSG00000251230.

3. The use according to claim 1, characterized in that: The transcript ID of the lncRNA MIR3945HG is ENST00000511703, and its sequence is shown in SEQ ID NO.

5.

4. The use according to claim 1, characterized in that: The kit also includes SYBR Green fluorescent dye, dNTP and Taq enzyme.

5. The use according to claim 1, characterized in that: The kit also includes TaqMan fluorescent probe, dNTP, and Taq enzyme.

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