Application of KLRG1 gene as a marker in preparation of SLE secondary HLH detection preparation

Abstract

The application discloses application of a killer cell lectin-like receptor G1 (KLRG1) gene as a marker in preparation of a systemic lupus erythematosus (SLE) secondary hemophagocytic lymphohistiocytosis (HLH) detection preparation, and the preparation is used for detecting the expression level of the KLRG1 gene in a biological sample. Research finds that the expression level of the KLRG1 gene in SLE secondary HLH patients is significantly lower than that of healthy control groups and SLE patients, and has good diagnostic sensitivity and specificity. The application also discloses a preparation for detecting the expression level of the KLRG1 gene in a biological sample of a patient, the preparation contains a primer pair for detecting the mRNA level of the KLRG1 gene expression, or contains an antibody for detecting the protein level of the KLRG1 gene expression, and only needs to extract peripheral blood mononuclear cells (PBMC) after blood drawing to detect whether SLE patients are secondary to HLH, and the preparation is simple, rapid, and accurate in result.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of disease diagnosis, and particularly relates to application of KLRG1 gene as a marker in preparation of SLE secondary HLH detection preparation. BACKGROUND

[0002] Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening severe complication of systemic lupus erythematosus (SLE), and its diagnosis is based on the HLH-2004 standard proposed by the International Histiocyte Association, and there is a lack of biomarkers for early diagnosis of HLH. HLH specifically includes 8 diagnostic criteria, which are as follows: (1) fever: body temperature > 38.5℃, duration > 7 days; (2) splenomegaly; (3) pancytopenia (involving peripheral blood two or three systems); (4) hypertriglyceridemia and / or hypofibrinogenemia; (5) finding of hemophagocytosis in bone marrow, spleen, liver or lymph node; (6) ferritin elevation > 500 μg / L; (7) NK cell activity reduction or absence; (8) sCD25 elevation; meeting 5 of the above 8 criteria can diagnose HLH. According to the standard for diagnosing HLH, although it has high specificity, due to the large number of standards, and the standards such as fever and pancytopenia overlap with the clinical manifestations of SLE, hemophagocytosis needs invasive operation to be clear, and sCD25 and NK cell activity are not developed in medical units, and need to be detected by external sending companies, resulting in delayed diagnosis of SLE secondary HLH, and high rate of missed diagnosis, leading to poor prognosis. SUMMARY

[0003] The purpose of the present application is to solve the problems of complicated HLH-2004 standard diagnosis, high rate of missed diagnosis and the like in the prior art, and provide application of KLRG1 gene as a marker in preparation of SLE secondary HLH detection preparation, which can quickly and accurately diagnose.

[0004] The technical scheme of the present application is described in detail as follows:

[0005] In a first aspect, the present application provides application of KLRG1 gene as a marker in preparation of SLE secondary HLH detection preparation, and the preparation is used for detecting the expression level of KLRG1 gene in a biological sample.

[0006] KLRG1, killer cell lectin-like receptor G1, the research found that the expression level of KLRG1 in SLE secondary HLH patients was significantly lower than that in HC (healthy control) and SLE group, which had good sensitivity and specificity for the diagnosis of SLE secondary HLH. The expression level of the KLRG1 gene can be the mRNA expression level or the protein expression level.

[0007] Optionally or preferably, the preparation is used for detecting the mRNA expression level of KLRG1 gene in the biological sample.

[0008] Optionally or preferably, the mRNA expression level of KLRG1 gene in the biological sample of SLE secondary HLH patients is significantly lower than that in normal healthy people (HC) and SLE patients.

[0009] Optionally or preferably, the biological sample is peripheral blood mononuclear cells (PBMC).

[0010] In the second aspect, the present application provides a preparation for detecting the expression level of KLRG1 gene in the biological sample of patients, which contains a primer pair for detecting the mRNA level of KLRG1 gene expression, or contains an antibody for detecting the protein level of KLRG1 gene expression.

[0011] Optionally or preferably, the preparation contains a primer pair for detecting the mRNA level of KLRG1 gene expression, and the nucleotide sequence of the primer pair is as follows:

[0012] Sense strand: 5'-AACCCAAGCCCAGAATGACT-3'(SEQ ID NO: 1),

[0013] Antisense strand: 5'-CAGCTGGCACAAGTGGAGTA-3'(SEQ ID NO: 2).

[0014] Optionally or preferably, the preparation further contains a qPCR reaction solution.

[0015] Optionally or preferably, the preparation further contains a primer pair for detecting the expression level of the reference gene GAPDH, and the nucleotide sequence of the primer pair is as follows:

[0016] Sense strand: 5'-TTGCCCTCAACGACCACTTT-3'(SEQ ID NO: 3),

[0017] Antisense strand: 5'-TGGTCCAGGGGTCTTACTCC-3'(SEQ ID NO: 4).

[0018] Optionally or preferably, the preparation further comprises PBMC separation and extraction reagent, total RNA extraction reagent and reverse transcription reagent.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application provides a new diagnostic marker KLRG1 for SLE secondary HLH, which has a significantly lower expression level in SLE secondary HLH patients than in HC and SLE patients, and has good diagnostic sensitivity and specificity.

[0021] The present application provides a preferred biological sample peripheral blood mononuclear cell (PBMC), which has the following advantages: ① The sample is easy to obtain: PBMC is a mixture of mononuclear cells contained in peripheral blood (i.e. blood other than bone marrow), and 2ml of EDTA anticoagulated whole blood can meet the experimental requirements, which can be separated and obtained by percoll non-continuous density gradient centrifugation. ② The sample is closely related to the occurrence and development of the disease: PBMC includes NK cells, T cells and B cells, which are immune cells in the body and are a type of cells commonly used to study the pathogenesis of immune diseases, so paying attention to PBMC is a key link to understand the occurrence and development of SLE secondary HLH. ③ High value for clinical differential diagnosis: the expression level of KLRG1 in PBMC in SLE secondary HLH patients is significantly lower than that in HC and SLE group, which has good sensitivity and specificity for SLE secondary HLH diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The volcano plot drawn after standardization of the differentially expressed mRNA data for screening the differentially expressed mRNA of SLE and SLE secondary HLH groups in the examples;

[0023] Figure 2 The heat map drawn after standardization of the differentially expressed mRNA data for screening the differentially expressed mRNA of SLE and SLE secondary HLH groups in the examples;

[0024] Figure 3 The statistical result graph (left) and ROC curve analysis graph (right) of the mRNA expression level of KLRG1 in PBMC of different groups in the examples. DETAILED DESCRIPTION

[0025] For the person skilled in the art to better understand the present application, the present application will be clearly and completely described below in combination with the embodiments and drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor should fall within the scope of protection of the present application. The instruments and reagents used in the embodiments are obtained from commercial channels if not otherwise specified.

[0026] Example 1 Screening of differentially expressed genes in SLE secondary HLH patients

[0027] There are three groups in the experiment, which are HC (healthy control) group, SLE (systemic lupus erythematosus) group, and SLE secondary HLH (systemic lupus erythematosus secondary hemophagocytosis) group.

[0028] 1. Isolation of human PBMC

[0029] 2 ml of peripheral EDTA anticoagulant whole blood was collected, and Percoll non-continuous density gradient centrifugation method was used to obtain PBMC of healthy people and disease groups, and the specific operation method was as follows:

[0030] 2. Screening of differentially expressed mRNA in HC group, SLE group and SLE secondary HLH group by transcriptome sequencing

[0031] PBMC samples of 3 HC (healthy control), 3 moderate active SLE (systemic lupus erythematosus) patients and 3 SLE secondary HLH patients were sent to Novogene Company for transcriptome sequencing, and differentially expressed mRNA was obtained according to Fold change≥0, P-value<0.05. At the mRNA level, there were 601 differentially expressed genes in the SLE secondary HLH group, of which 365 genes were up-regulated and 236 genes were down-regulated, as shown in Tables 1 and 2. Figure 1 and Figure 2

[0032] 3. Screening of differentially expressed mRNA molecules in SLE secondary HLH patients

[0033] Taking Fold Change>2 and P value<0.05 as the standard for screening of differentially expressed genes, KLRG1 was screened out which showed a progressive downward trend in the expression in HC group, SLE group and SLE secondary HLH group. See Table 1 for details.

[0034] Table 1 mRNA expression of KLRG1 in HC, SLE and SLE-HLH

[0035]

[0036] ​The screening results show that the expression level of KLRG1 in the SLE secondary HLH group is significantly lower than that in the healthy control group and the disease control group (SLE group).

[0037] KLRG1 gene sequence (NM_001329099 1401bp, SEQ ID NO: 5):

[0038]

[0039] The present application simultaneously sets the internal reference gene GAPDH.

[0040] GAPDH gene sequence (NM_001256799 1386bp, SEQ ID NO: 6):

[0041]

[0042] Example 2 Clinical verification of the diagnostic value of mRNA molecule KLRG1 in PBMC in SLE secondary HLH

[0043] HC 14 cases, 50 cases of active SLE patients were collected, among which the SLE patients were divided into SLE group (36 cases) and SLE secondary HLH group (14 cases) according to whether secondary HLH occurred.

[0044] 1. Separation of PBMC

[0045] Take 2ml EDTA anticoagulant whole blood, centrifuge at 3000rpm for 10min, carefully suck the white membrane layer cells, resuspend with 2ml sterile 1xPBS for standby; Prepare 75% and 60% percoll cell separation liquid diluent each 2ml with sterile 1xPBS; Prepare a clean 15ml centrifuge tube, add 75% percoll cell separation liquid, 60% percoll cell separation liquid and resuspended white membrane layer at one time; 2600rpm / min centrifuge for 20min to obtain PBMC layer, mix with 10ml 1xPBS, centrifuge at 1800rpm for 5min, discard the supernatant, add appropriate amount of red blood cell lysis solution, lyse the mixed red blood cells, collect the cell precipitate, which is PBMC.

[0046] 2. Total RNA extraction (total RNA extraction kit of Tiangen company: D419)

[0047] Take 1ml TRIzol (total RNA extraction reagent) to blow the above cell precipitate, stand at room temperature for 10min; add 200μl chloroform, shake vigorously for 1min, stand at room temperature for 3min, centrifuge at 4℃ 12000g for 10min; take 500μl of upper water phase, add 250μl of anhydrous ethanol, mix well and transfer to the RNA extraction column, centrifuge at 4℃ 12000g for 1min; discard the waste liquid, add 500μl of deproteinization liquid in the column, centrifuge at 4℃ 12000g for 1min; discard the waste liquid, add 500μl of washing liquid in the column, stand at room temperature for 2min, centrifuge at 4℃ 12000g for 1min; repeat the above step; discard the supernatant, centrifuge at 4℃ 12000g for 2min; stand at room temperature for 10min until the ethanol completely evaporates; add 30μl of enzyme-free water in the center of the column, stand at room temperature for 2min; centrifuge at 4℃ 12000g for 2min to obtain total RNA, detect the concentration and absorbance A260, A280 and A230 of RNA.

[0048] 3. Reverse transcription (Yisheng company: 11141ES60)

[0049] 1) First remove residual genomic DNA

[0050] Prepare the mix as shown in Table 2 in RNase free centrifuge tube, mix gently by pipetting. Incubate at 42°C for 2 min.

[0051] Table 2 Mix for removing residual genomic DNA

[0052] Component Amount used RNase free ddH2O Up to 30 μL 5x gDNA digester 6 μL Total RNA 4 μg

[0053] 2) Preparation of reverse transcription reaction system

[0054] Add 2x HifairTM II SuperMix plus directly into the reaction tube of Step 1), mix gently by pipetting as shown in Table 3.

[0055] Table 3 Reverse transcription reaction system

[0056]

[0057] 3) Reverse transcription program settings, as shown in Table 4 below:

[0058] Table 4 Reverse transcription standard program

[0059] Temperature Time 25℃ 5 min 42℃ 30 min 85℃ 5 min

[0060] Obtain template cDNA, add 80 μL RNase free ddH2O to each sample for use.

[0061] 4, qPCR (Ying Seng Company: 11184ES08)

[0062] 1) Prepare qPCR mixed solution (see Table 5 below) into a 96-well plate, set 3 replicate wells for each sample, and mix well after sealing and centrifuging.

[0063] Table 5 qPCR mix

[0064]

[0065]

[0066] (Note: The upstream primer has the same nucleotide sequence as the aforementioned sense strand, and the downstream primer has the same nucleotide sequence as the aforementioned antisense strand)

[0067] 2) qPCR program settings

[0068] Table 6 qPCR program

[0069]

[0070] 3) Result calculation

[0071] GAPDH as the internal reference gene, the minimum cycle number used to reach the threshold value set by the instrument as CT value, 2 –ΔΔCT The expression level was calculated.

[0072] 5、Statistical results

[0073] The statistical analysis results show that the mRNA level of KLRG1 in PBMCs is significantly different among the HC group, the SLE group and the SLE secondary HLH group, and the expression level in the SLE secondary HLH group is significantly lower than that in the HC group and the SLE group, see Table 7 below and Figure 3 (left).

[0074] Table 7 Comparison of expression levels of mRNA candidate molecules in PBMCs of three groups

[0075]

[0076] Further ROC curve analysis was performed to detect the performance of KLRG1 for the classification diagnosis of SLE secondary HLH, and the area under the ROC curve (AUC) was 0.927, the specificity was 0.929, and the sensitivity was 0.889, see Table 8 below and Figure 3 (right), which is better than the diagnosis of SLE secondary HLH by the current clinical indicators such as ferritin (Fer), triglyceride (TG) and fibrinogen (FIB).

[0077] Table 8 Diagnostic value of mRNA expression level of KLRG1 in PBMCs in SLE secondary HLH

[0078]

[0079] In summary, the present application has high diagnostic value for diagnosing secondary HLH in SLE patients, with a sensitivity of 0.889 and a specificity of 0.929. It is not only simple and feasible in technology, but also can effectively improve the early diagnosis rate of secondary HLH, provide timely and effective treatment, reduce HLH-related complications and improve the adverse prognosis related to HLH.

[0080] In this paper, specific examples are applied to elaborate the inventive concept in detail. It should be noted that any obvious modification, equivalent replacement or other improvement made by those skilled in the art without departing from the inventive concept should be included in the protection scope of the present application.

Claims

1. Application of KLRG1 gene as a marker in preparation of a preparation for detecting SLE secondary HLH, which is used for detecting the expression level of KLRG1 gene in a biological sample.

2. Use according to claim 1, characterized in that, The preparation is used for detecting the mRNA expression level of KLRG1 gene in a biological sample.

3. Use according to claim 1 or 2, characterized in that, The mRNA expression level in the biological sample of SLE secondary HLH patients is significantly lower than that of normal healthy people and SLE patients.

4. Use according to claim 1 or 2, characterized in that, The biological sample is peripheral blood mononuclear cells (PBMC).

5. Use of a preparation for detecting the level of expression of the KLRG1 gene in a biological sample of a patient in the manufacture of a preparation for detecting SLE secondary HLH, characterized in that, It contains a primer pair for detecting the mRNA level of KLRG1 gene expression, or an antibody for detecting the protein level of KLRG1 gene expression.

6. Use according to claim 5, characterized in that, It contains a primer pair for detecting the mRNA level of KLRG1 gene expression, and the nucleotide sequence of the primer pair is as follows: Positive strand: 5'-AACCCAAGCCCAGAATGACT-3'(SEQ ID NO: 1), Negative strand: 5'-CAGCTGGCACAAGTGGAGTA-3'(SEQ ID NO: 2).

7. Use according to claim 6, characterized in that, It also contains a qPCR reaction solution.

8. Use according to claim 6, characterized in that, It also contains a primer pair for detecting the expression level of the reference gene GAPDH, and the nucleotide sequence of the primer pair is as follows: Positive strand: 5'-TTGCCCTCAACGACCACTTT-3'(SEQ ID NO: 3), Negative strand: 5'-TGGTCCAGGGGTCTTACTCC-3'(SEQ ID NO: 4).

9. Use according to claim 6, characterized in that, It also contains PBMC separation and extraction reagents, total RNA extraction reagents and reverse transcription reagents.

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