Application of klrc3 gene as a marker in preparation of sLE secondary hlh detection preparation

Abstract

The application discloses application of a killer cell lectin-like receptor C3 (KLRC3) 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 KLRC3 gene in a biological sample. Research finds that the KLRC3 gene expression level in SLE secondary HLH patients is significantly lower than that in a healthy control group and SLE patients, and has good diagnostic sensitivity and specificity. The application also discloses a preparation for detecting the KLRC3 gene expression level in a biological sample of a patient, the preparation contains a primer pair for detecting the mRNA level of the KLRC3 gene expression, or contains an antibody for detecting the protein level of the KLRC3 gene expression, and only needs to extract peripheral blood mononuclear cells (PBMC) from blood to detect whether SLE patients are secondary to HLH, and the preparation is simple, rapid and accurate in result.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of disease diagnosis, and particularly relates to application of KLRC3 gene as a marker in preparation of a 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 Organization for Cell Association, which specifically includes eight diagnostic criteria, specifically: (1) fever: body temperature > 38.5 DEG C, duration > 7 days; (2) splenomegaly; (3) pancytopenia (involving peripheral blood two or three systems); (4) hypertriglyceridemia and / or hypofibrinogenemia; (5) bone marrow, spleen, liver or lymph node hemophagocytosis; (6) ferritin elevation > 500 ug / L; (7) NK cell activity reduction or absence: (8) sCD25 elevation; meeting five of the above eight criteria can diagnose HLH. According to the standard for diagnosing HLH, although it has high specificity, due to the multiple standards, and the standards of fever, pancytopenia and the like 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, and thus leading to high mortality of the part of patients. 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 of SLE secondary HLH in the prior art, and provide application of KLRC3 gene as a marker in preparation of a 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 KLRC3 gene as a marker in preparation of a SLE secondary HLH detection preparation, and the preparation is used for detecting the expression level of KLRC3 gene in a biological sample.

[0006] KLRC3, killer cell lectin like receptor subfamily C member 3, the expression level of KLRC3 in SLE patients with secondary HLH is significantly lower than that in HC (healthy control) and SLE group, which has good sensitivity and specificity for diagnosis of SLE secondary HLH. The expression level of the KLRC3 gene can be mRNA expression level or protein expression level.

[0007] Optionally or preferably, the preparation is used for detecting the mRNA expression level of KLRC3 gene in the biological sample.

[0008] Optionally or preferably, the mRNA expression level of KLRC3 gene in the biological sample of SLE patients with secondary HLH 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 KLRC3 gene in the biological sample of patients, which contains a primer pair for detecting the mRNA level of KLRC3 gene expression, or contains an antibody for detecting the protein level of KLRC3 gene expression.

[0011] Optionally or preferably, the preparation contains a primer pair for detecting the mRNA level of KLRC3 gene expression, and the nucleotide sequence of the primer pair is as follows:

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

[0013] Reverse strand: 5'-ATTGCACAGTTACGTTCAGCA-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 internal standard gene GAPDH, and the nucleotide sequence of the primer pair is as follows:

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

[0017] Reverse 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 KLRC3 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 cells (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 anticoagulant whole blood can meet the experimental requirements, which can be separated and obtained by percoll non-continuous density gradient centrifugation method. 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 KLRC3 in PBMC in SLE secondary HLH patients is significantly lower than that in HC and SLE groups, which has good sensitivity and specificity for SLE secondary HLH diagnosis. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0024] Figure 3 The mRNA expression level statistical result graph (left) and ROC curve analysis graph (right) of KLRC3 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 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.

[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 Bioinformatics Technology Co., Ltd. for transcriptome sequencing. According to Fold change≥0, P-value<0.05, differentially expressed mRNA was obtained. 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, KLRC3 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 KLRC3 in HC, SLE and SLE-HLH

[0035]

[0036] ​The screening results show that the expression level of KLRC3 in the SLE secondary HLH group is significantly lower than that in the healthy control group and the disease control group (SLE group).

[0037] KLRC3 gene sequence (NM_002261 1025bp, SEQ ID NO: 5):

[0038]

[0039] The present application simultaneously sets an internal standard 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 KLRC3 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 and extraction of PBMC

[0045] Take 2ml EDTA anticoagulation 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 evaporates completely; 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 DNA, 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 96-well plate, set 3 replicates for each sample, centrifuge after sealing the plate and mix well.

[0063] Table 5 qPCR mix

[0064]

[0065] 2) qPCR program settings

[0066] Table 6 qPCR program

[0067]

[0068]

[0069] 3) Result calculation

[0070] Take GAPDH as the internal reference gene, take the cycle number used to reach the threshold value set by the instrument as the Ct value, and take 2 –ΔΔCT to calculate the expression level.

[0071] 5, statistical results

[0072] Statistical analysis results found that the mRNA level of KLRC3 in PBMC was 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 was significantly lower than that in the HC group and the SLE group, see Table 7 and Figure 1 below. Figure 3 The left graph.

[0073] Table 7 Comparison of mRNA expression level of KLRC3 in PBMC

[0074]

[0075] Further ROC curve analysis was performed to detect the performance of KLRC3 for the classification diagnosis of SLE secondary HLH, and the area under the ROC curve (AUC) was 0.885, the specificity was 0.929, and the sensitivity was 0.806, see Table 8 and Figure 2 below. Figure 3 The right graph, which is better than the current clinical use of ferritin (Fer), triglyceride (TG) and fibrinogen (FIB) and other indicators for the diagnosis of SLE secondary HLH.

[0076] Table 8 Diagnostic value of mRNA molecule KLRC3 in PBMC of SLE patients for SLE secondary HLH

[0077]

[0078] In summary, the present application has high diagnostic value for diagnosing secondary HLH in SLE patients, with a sensitivity of 0.806 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, timely provide powerful treatment, reduce HLH related complications, and improve HLH related adverse prognosis.

[0079] In this paper, specific examples are used to elaborate the inventive concept in detail. The above examples are only used to help understand the core idea of the present application. 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. Use of a preparation for detecting the expression level of KLRC3 gene in the preparation of a product for detecting SLE secondary HLH, wherein the preparation is used for detecting the expression level of KLRC3 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 KLRC3 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. The use according to claim 1, characterized in that, The preparation contains a primer pair for detecting the mRNA level of KLRC3 gene expression, or an antibody for detecting the protein level of KLRC3 gene expression.

6. Use according to claim 5, characterized in that, The preparation contains a primer pair for detecting the mRNA level of KLRC3 gene expression, and the nucleotide sequence of the primer pair is as follows: Forward strand: 5'-GCCAGCATTTTACCTTCCTCA-3' (SEQ ID NO: 1), Reverse strand: 5'-ATTGCACAGTTACGTTCAGCA-3' (SEQ ID NO: 2).

7. Use according to claim 6, characterized in that, The preparation further contains a qPCR reaction solution.

8. Use according to claim 6, characterized in that, The preparation further contains a primer pair for detecting the expression level of internal standard gene GAPDH, and the nucleotide sequence is as follows: Forward strand: 5'-TTGCCCTCAACGACCACTTT-3' (SEQ ID NO: 3), Reverse strand: 5'-TGGTCCAGGGGTCTTACTCC-3' (SEQ ID NO: 4).

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

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