CN116515995B - Serum microRNA marker for detecting hemophagocytic lymphocytohyperplasia and application thereof - Google Patents

Abstract

The invention provides a serum microRNA marker for detecting hemophagocytic lymphocytopenia and application thereof, wherein the serum microRNA marker is selected from hsa-miR-21-5p and/or hsa-miR-106a-5p. The invention belongs to the field of molecular biological detection of hemophagocytic lymphocytosis, and also provides a kit for detecting hemophagocytic lymphocytosis, and the kit and the detection method can be used for carrying out auxiliary diagnosis and detection on the hemophagocytic lymphocytosis noninvasively, rapidly and accurately.

Description

Serum microRNA marker for detecting hemophagocytic lymphocytohyperplasia and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a serum microRNA marker for detecting hemophagocytic lymphocytopenia and application thereof.

Background

Hemophagocytic lymphocytosis (Hemophagocytic Iymphohistiocytosis, HLH) is a fatal clinical syndrome caused by a variety of pathogenic factors such as infection, tumor, immune diseases, etc. The disease is frequently seen in children, but in recent years, the incidence of adult patients is gradually increasing as HLH is becoming more and more known. The incidence rate of the Chinese medicinal composition is about 1.2/100 ten thousand, and the incidence rates of male and female patients are not obviously different. The early mortality rate of HLH is still up to 33% and the survival rate in 3 years is only 55%. In addition, clinical manifestations and laboratory indexes of HLH are not specific, and abnormal indexes similar to HLH clinically can also appear in systemic inflammatory response syndrome, viral infection, tumor and the like, so that misdiagnosis is easy to cause, and the death risk of patients is increased. Therefore, finding new biomarkers for diagnosis and treatment of HLH is necessary, and has important significance for reducing missed diagnosis and misdiagnosis and improving prognosis of patients.

micrornas are a class of non-coding single-stranded small RNA molecules of about 19-24 nt in length, most of which can inhibit translation of a target gene into a protein by complementary binding to the 3' utr region of the target gene, thereby affecting the growth and development of organisms at the cellular, tissue or individual level and involved in a variety of disease processes. The expression profile of miRNA has obvious tissue specificity and specific expression pattern in different diseases. These features make mirnas a new biomarker and therapeutic target for disease diagnosis. It has now been found that there are a variety of miRNAs in HLH that are aberrantly expressed, such as miRNA-205-5p, miR-133 and miR-BART16-1, etc., which occur at different stages of HLH progression, as a molecular biological evidence of HLH occurrence, disease progression and prognosis.

Although few studies reported serum microRNA markers for the detection of hemophagocytic lymphoproliferative disorders, these results were not consistent and did not go through perfect detection performance verification. Therefore, serum microRNA biomarkers which can be finally used for detecting hemophagocytic lymphoproliferative diseases are not shown in theory, and corresponding biomarker combination kits are not shown in the market. Therefore, if the corresponding reagent kit for hemophagocytic lymphocytosis can be developed and applied to the scientific and clinical fields, the screening and diagnosis of the hemophagocytic lymphocytosis and the conversion of scientific research results can be greatly promoted, and the reagent kit can play a great pushing role in diagnosis and treatment of the hemophagocytic lymphocytosis.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a serum microRNA marker for detecting hemophagocytic lymphocytohyperplasia and application thereof. The detection method based on the marker can carry out non-invasive, rapid and accurate auxiliary diagnosis and detection on the hemophagocytic lymphocytosis, and has important application value in screening and diagnosis research of the hemophagocytic lymphocytosis.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the invention provides a serum microRNAhsa-miR-21-5p for detecting hemophagocytic lymphoproliferative disorder, wherein the serum microRNA marker is selected from hsa-miR-21-5p and/or hsa-miR-106a-5p.

Preferably, the serum microRNA markers are a combination of hsa-miR-21-5p and hsa-miR-106a-5p.

The invention detects the expression profile of microRNA in serum of a healthy control group and a patient with hemophagocytic lymphoproliferative disease by using Small RNA-seq in the early stage, screens the microRNA with differential expression, verifies candidate microRNA by using real-time quantitative PCR, analyzes the expression quantity of target microRNA, and obtains 2 differential serum microRNA markers.

microRNA markers hsa-miR-21-5p and hsa-miR-106a-5p which are obtained through screening and related to hemophagocytic lymphocytopenia are differentially expressed in at least one target serum and at least one healthy control serum, so that differential expression in hemophagocytic lymphocytopenia patients and healthy people can be reflected, and high sensitivity and high specificity of diagnosis of hemophagocytic lymphocytopenia are reflected.

In a second aspect, the invention provides a kit for detecting hemophagocytic lymphoproliferative disorder, wherein the kit comprises a reagent for specifically detecting the serum microRNA marker for detecting hemophagocytic lymphoproliferative disorder in the first aspect.

Preferably, the reagent comprises qPCR upstream primers for amplifying serum microRNA markers for detecting hemophagocytic lymphoproliferative disorder according to the first aspect.

Preferably, the qPCR upstream primer for amplifying the marker hsa-miR-21-5p is shown in SEQ ID NO. 4. SEQ ID NO. 4: gccaggcatagcttatcagactg.

Preferably, the qPCR upstream primer of the amplified marker hsa-miR-106a-5p is shown in SEQ ID NO. 5. SEQ ID NO. 5: atcccacaaaagtgcttacagtg.

Preferably, the reagent further comprises a mature precursor sequence of an exogenous reference gene and qPCR upstream primers to amplify the mature precursor sequence.

Preferably, the mature precursor sequence of the exogenous reference gene is shown as SEQ ID NO. 3. SEQ ID NO. 3: ucaccggguguaaaucagcuug.

Preferably, the qPCR upstream primer for amplifying the mature precursor sequence is shown in SEQ ID NO. 6. SEQ ID NO. 6: cgtcgatcaccgggtgtaaa.

The sequences of hsa-miR-21-5p and hsa-miR-106a-5p microRNAs related to the invention are shown in Table 1. The sequence corresponding to hsa-miR-21-5p is SEQ ID NO:1: uagcuuaucagacugauguuga. The sequence corresponding to hsa-miR-106a-5p is SEQ ID NO:2: aaaagugcuuacagugcagguag.

TABLE 1

The qPCR forward primer sequences of the 2 microRNA markers hsa-miR-21-5p and hsa-miR-106a-5p and the exogenous reference gene are shown in Table 2, and the microRNA qPCR primers are designed according to the nucleotide sequences reported by a microRNA database as templates.

The hsa-miR-21-5p and hsa-miR-106a-5p target genes qPCR amplification primers are designed according to the complete microRNA sequence, and the upstream primer is the microRNA mature sequence (U is changed into T).

Furthermore, if the annealing temperature of the forward specific primer designed based on the microRNA sequence is too low, it is necessary to add several bases (mainly G and C) to the 5' -end of the primer.

Further, if the primer annealing temperature is too high, it is necessary to delete several bases at the 5' end.

TABLE 2

Preferably, the reagent further comprises any one or a combination of at least two of Poly-A tailing enzyme, reverse transcriptase, dNTPs, reverse transcription buffer, RNase-free water, magnesium chloride, DNA polymerase, SYBR Green PCR premix.

The kit for auxiliary diagnosis of hemophagocytic lymphocytosis provided by the invention comprises enzymes and reagents commonly used in PCR reaction besides the microRNA markers related to hemophagocytic lymphocytosis. Specifically, the kit also contains Poly-A tailing enzyme, reverse transcriptase, dNTPs, reverse transcription buffer, RNase-free water, qPCR buffer, magnesium chloride, SYBR Green PCR premix and the like. The invention is not particularly limited to other reagents in the kit except for microRNA markers related to hemophagocytic lymphoproliferative disorder, and can be selected according to actual needs by a person skilled in the art.

In a third aspect, the present invention provides a device for detecting haemophagous lymphoproliferative disorders, the device comprising:

sample amplification module: extracting serum miRNA of a sample to be detected, performing tail-adding reverse transcription on target microRNA by using the kit for detecting hemophagocytic lymphocytohyperplasia according to the second aspect, performing real-time fluorescence quantitative reaction, and detecting a fluorescence signal;

and a result analysis module: and (3) calculating the relative content of the target microRNA by adopting a relative quantitative method, comparing the relative content of the target microRNA with the relative content of the target microRNA in normal healthy control serum, and judging whether the sample to be detected is hemophagocytic lymphocytohyperplasia or non-hemophagocytic lymphocytohyperplasia.

Preferably, the reaction conditions for reverse transcription in the sample amplification module are:

reverse transcription: 37 ℃,50-65 min (for example, 50 min, 55 min, 60 min or 65 min, etc.);

enzyme inactivation: 80-85 ℃ (e.g., may be 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, etc.), 5-300s (e.g., may be 5 s, 10 s, 100 s, 150 s, 200 s, 250s, 300s, etc.);

preferably, the reaction conditions of the real-time fluorescent quantitative reaction are as follows:

pre-denaturation: 94-96 ℃ (e.g., 94 ℃, 95 ℃, 96 ℃ or the like) and 9-11 s (e.g., 9 s, 10 s, 11 s or the like);

denaturation annealing: 94-96 ℃ (e.g., 94 ℃, 95 ℃, 96 ℃ or the like), 4-6 s (e.g., 4 s, 5 s, 6 s or the like), 60-64 ℃ (e.g., 60 ℃, 62 ℃, 64 ℃ or the like), 44-46 s (e.g., 44 s, 45 s, 46 s or the like); 38-42 cycles (which may be 38, 39, 40, 41 or 42, for example).

Preferably, the standard for judging whether the sample to be tested is hemophagocytic lymphoproliferative disorder or non-hemophagocytic lymphoproliferative disorder in the result analysis module is:

use 2 ^-△△Ct As the relative expression level of the target genes hsa-miR-21-5p and hsa-miR-106a-5p in serum, the expression level of hsa-miR-21-5p marker>5.1 and expression level of hsa-miR-21-5p marker>1.9, judging positive; otherwise, the result is negative.

In a fourth aspect, the invention provides the serum microRNA marker for detecting hemophagocytic lymphoproliferative disorder in the first aspect and/or the application of the kit for detecting hemophagocytic lymphoproliferative disorder in the second aspect in preparation of products for detecting hemophagocytic lymphoproliferative disorder.

FIG. 4 shows a discovery and verification scheme of the markers, wherein microRNA markers hsa-miR-21-5p and hsa-miR-106a-5p related to hemophagocytic lymphocytopenia are obtained through screening, serum microRNAs of a patient sample and a healthy control sample of the hemophagocytic lymphocytopenia are collected and subjected to real-time fluorescent quantitative detection, the application condition of the markers in noninvasive auxiliary diagnosis of the hemophagocytic lymphocytopenia is verified, and the markers are found to have the sensitivity of 90.92% and the specificity of 87.94% for diagnosis of the hemophagocytic lymphocytopenia, so that the markers have important significance for auxiliary diagnosis of the hemophagocytic lymphocytopenia.

In the invention, the serum microRNA markers hsa-miR-21-5p and hsa-miR-106a-5p related to hemophagocytic lymphocytopenia have high sensitivity and good specificity for auxiliary diagnosis of hemophagocytic lymphocytopenia, and can be used as new serum microRNA markers for noninvasive auxiliary diagnosis of hemophagocytic lymphocytopenia.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has the following beneficial effects:

(1) Compared with the conventional detection method in clinic, the detection method for the peripheral blood serum microRNA marker provided by the invention has the characteristics of high specificity, high sensitivity and no wound.

(2) The invention adopts the Ploy A tailing method, and the qPCR forward primer which is designed independently carries out quantitative analysis on the 2 serum microRNA markers, can complete reverse transcription of various microRNAs only through one reverse transcription reaction, has small required serum sample quantity, and has the advantages of simple and convenient operation, high flux and low cost.

(3) The serum microRNA marker and the detection kit provided by the invention are verified in a patient with hemophagocytic lymphocytosis and a healthy person, and the sensitivity of the marker to diagnosis of hemophagocytic lymphocytosis is 90.92%, the specificity is 87.94%, so that the serum microRNA marker has important significance for auxiliary diagnosis of hemophagocytic lymphocytosis.

Drawings

FIG. 1 is a comparison of hsa-miR-21-5p expression levels in serum of a patient with hemophagocytic lymphoproliferative disorder and a healthy human.

FIG. 2 is a comparison of hsa-miR-106a-5p expression levels in serum of a patient with hemophagocytic lymphoproliferative disorder and a healthy human.

FIG. 3 is a graph of a joint diagnostic ROC of the 2 serum markers hsa-miR-21-5p and hsa-miR-106a-5p.

Fig. 4 is a discovery and validation scheme for the markers.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

In the examples, the present invention uses reverse transcription universal primers and qPCR universal reverse primers, commercially available from Shanghai, inc. of the holy Biotechnology of next generation.

Example 1

The embodiment provides a kit for detecting hematophagous lymphocytosis, which comprises the following components:

(1) qPCR upstream primers for specifically detecting hsa-miR-21-5p and hsa-miR-106a-5 p;

the qPCR upstream primer of the amplified marker hsa-miR-21-5p is shown as SEQ ID NO. 4;

the qPCR upstream primer of the amplified marker hsa-miR-106a-5p is shown in SEQ ID NO. 5.

(2) A mature precursor sequence of an exogenous internal reference gene and qPCR upstream primers to amplify the mature precursor sequence;

the mature precursor sequence of the exogenous internal reference gene is shown as SEQ ID NO. 3;

the qPCR upstream primer for amplifying the mature precursor sequence is shown as SEQ ID NO. 6.

(3) Reagents related to PCR reaction: poly-A tailing enzyme, reverse transcriptase, dNTPs, reverse transcription buffer, RNase-free water, magnesium chloride, DNA polymerase and SYBR Green PCR premix.

Example 2

The present embodiment provides a method for detecting hemophagocytic lymphoproliferative disorder, the method comprising:

1. sample collection

Collecting old blood department and blood department of Xueyu lymphopoiesis hyperplasia cases and healthy physical examination crowd of Xueyu lymphopoiesis hyperplasia cases attached to Xueyu medical university in south Beijing medical science and No. 10 in 2021, screening 87 cases of Xueyu lymphopoiesis hyperplasia patients, and obtaining 44 cases of normal control of peripheral serum specimens of healthy physical examination subjects in the hospital except other diseases such as rheumatism, infection, tumor and the like. The system collects demographic and clinical information of the sample.

2. Serum sample preparation

The study subjects were on an empty stomach in early morning, peripheral venous blood 2 mL was collected by heparin or ethylenediamine tetraacetic acid (EDTA) anticoagulation tube, mixed by shaking gently, and left at room temperature for 20 min, after centrifugation for 10 min at 3000 g, the supernatant was collected, transferred to a 1.5 mL centrifuge tube without RNase, after centrifugation for 10 min at 12000 g, the supernatant was collected, 200. Mu.L was placed in a new 1.5 mL centrifuge tube without RNase, and stored in a refrigerator at-80℃for later use.

3. Serum miRNA extraction

200. Mu.L of Serum was taken from each sample, 20 pmol of an artificially synthesized foreign reference gene sequence (SEQ ID NO. 3) was added and then microRNA enrichment was performed using the QIAGEN extraction Kit miRNeasy Serum/Plasma Kit, see Kit instructions for specific procedures.

4. Reverse transcription reaction

Reverse transcription of the micrornas of interest was performed using tail-biting reverse transcription, as shown in table 3.

TABLE 3 Table 3

The reaction conditions for reverse transcription were 60 minutes at 37 ℃ followed by 5 seconds enzyme inactivation at 85 ℃. At least 100. Mu.L of enzyme-free water was added to each 20. Mu.L of reverse transcription reaction, gently mixed, and transiently separated for subsequent PCR reaction.

5. Real-time fluorescent quantitative reaction

The fluorescent PCR reaction system is 50 mu L, and the required components and the required amounts are shown in Table 4.

TABLE 4 Table 4

The amplification reaction was performed on an ABI 7500 real-time fluorescent quantitative PCR instrument, the reaction program settings are shown in Table 5, and the other settings are all system default values.

TABLE 5

Two duplicate wells were made for each sample and averaged for counting. Melting curves look at primer specificity, with single peaks being specific amplifications. The relative content of hsa-miR-21-5p and hsa-miR-106a-5p in blood-phagocytic lymphoproliferative disorder and normal healthy control serum is detected by adopting a relative quantitative method. Takes artificially synthesized exogenous Cel-miR-39-3p as a reference gene, wherein DeltaCt= (Ct) _{Target gene} -Ct _{Reference gene} ） _{Experimental group} -(Ct _{Target gene} -Ct _{Reference gene} ) _{Control group} Will be 2 ^-△△Ct As the relative expression level of the gene of interest in serum.

6. Expression level analysis

hsa-miR-21-5p and hsa-miR-106a-5p in serum were detected in 87 hemophagocytic lymphoproliferative patients and 44 healthy controls.

Comparison of expression differences of hsa-miR-21-5p and hsa-miR-106a-5p in hemophagocytic lymphoproliferative patients and healthy persons by adopting a delta-Ct method, wherein the differential expression of each miRNA in hemophagocytic lymphoproliferative patients and healthy persons is=2 ^-△△Ct . The SPSS software is used for carrying out result analysis, and the hsa-miR-21-5P and hsa-miR-106a-5P in serum of a patient suffering from hemophagocytic lymphocytopenia are found to be significantly higher than that of a healthy control, and the difference has significant statistical significance (P is less than 0.0001), and the result is shown in figures 1 and 2. Further test analysis, found that when the hsa-miR-21-5p marker expression level>5.1, positive for hsa-miR-106a-5p marker expression level>1.9, judging positive. Further, the positive result is judged when both markers are positive.

Example 3

In this example, 39 patients suffering from hemophagocytic lymphoproliferative disorder and 17 normal control samples were examined according to the method described in example 2, and the effect of the kit of the present invention was further verified.

The detection effect of the serum markers hsa-miR-21-5p on the hemophagocytic lymphoproliferative disease is evaluated by analyzing the expression levels of hsa-miR-21-5p and hsa-miR-106a-5p in serum of 39 hemophagocytic lymphoproliferative disease patients and 17 healthy controls, and analysis shows that the curve area of the serum markers hsa-miR-21-5p is 0.87, the sensitivity is 84.08% and the specificity is 79.91%. The curve area of the serum marker hsa-miR-106a-5p is 0.86, the sensitivity is 94.14%, and the specificity is 64.51%, as shown in Table 6. Further, the combination of the combined markers formed by 2 microRNAs shows that the AUC reaches 0.94, the sensitivity is 90.92%, the specificity is 87.94%, and the AUC is larger than that of a single miRNA, and the result shows that the combined detection combination of 2 miRNAs is helpful for improving the effect of diagnosing the hemophagocytic lymphocytohyperplasia, as shown in figure 3.

TABLE 6

In summary, 2 serum markers related to the hemophagocytic lymphocytosis are screened and found, a kit for diagnosing the hemophagocytic lymphocytosis is prepared based on the markers, a method for diagnosing the hemophagocytic lymphocytosis based on the markers is developed, and the method is verified (as shown in figure 4), and the result shows that the expression level of the 2 serum markers hsa-miR-21-5p and hsa-miR-106a-5p in serum of the hemophagocytic lymphocytosis is obviously up-regulated, and the combined detection of 2 microRNAs has better sensitivity and specificity for diagnosing the hemophagocytic lymphocytosis, is beneficial to diagnosis of the hemophagocytic lymphocytosis, and has potential clinical application value.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims (7)

1. The application of a kit for detecting hemophagocytic lymphocytosis in preparing products for detecting hemophagocytic lymphocytosis is characterized in that the kit comprises a reagent for specifically detecting serum microRNA markers;

the serum microRNA marker is a combination of hsa-miR-21-5p and hsa-miR-106a-5 p;

the reagent comprises qPCR upstream primers for amplifying the serum microRNA markers;

the qPCR upstream primer of the amplified marker hsa-miR-21-5p is shown as SEQ ID NO. 4;

the qPCR upstream primer of the amplified marker hsa-miR-106a-5p is shown in SEQ ID NO. 5.

2. The use according to claim 1, wherein the reagent further comprises a mature precursor sequence of an exogenous reference gene and qPCR upstream primers to amplify the mature precursor sequence;

the mature precursor sequence of the exogenous internal reference gene is shown as SEQ ID NO. 3;

the qPCR upstream primer for amplifying the mature precursor sequence is shown as SEQ ID NO. 6.

3. The use of claim 2, wherein the reagent further comprises any one or a combination of at least two of Poly-a tailing enzyme, reverse transcriptase, dNTPs, reverse transcription buffer, rnase-free water, magnesium chloride, DNA polymerase, SYBR Green PCR premix.

4. Use of a system for detecting hemophagocytic lymphocytosis in the manufacture of a product for detecting hemophagocytic lymphocytosis, said system comprising:

sample amplification module: extracting serum miRNA of a sample to be detected, performing tail-end reverse transcription on target microRNA by using the kit for detecting hemophagocytic lymphocytohyperplasia according to any one of claims 1-3, and then performing real-time fluorescence quantitative reaction to detect a fluorescence signal;

and a result analysis module: and (3) calculating the relative content of the target microRNA by adopting a relative quantitative method, comparing the relative content of the target microRNA with the relative content of the target microRNA in normal healthy control serum, and judging whether the sample to be detected is hemophagocytic lymphocytohyperplasia or non-hemophagocytic lymphocytohyperplasia.

5. The use of claim 4, wherein the reaction conditions for reverse transcription in the sample amplification module are:

reverse transcription: 37 ℃ for 50-65 min;

enzyme inactivation: 80-85 ℃ for 5-300s;

the reaction conditions of the real-time fluorescence quantitative reaction are as follows:

pre-denaturation: 94-96 ℃,9-11 sec;

denaturation annealing: 94-96 ℃,4-6 sec,60-64 ℃ and 44-46 sec;38-42 cycles.

6. The use according to claim 5, wherein the standard for determining whether the sample is a lymphoproliferative disorder with hematophagous cells or a lymphoproliferative disorder with non-hematophagous cells in the result analysis module is:

use 2 ^-△△Ct As the relative expression level of the target genes hsa-miR-21-5p and hsa-miR-106a-5p in serum, the expression level of hsa-miR-21-5p marker>5.1 and expression level of hsa-miR-21-5p marker>1.9, judging positive; otherwise, the result is negative.

7. The application of the reagent for specifically detecting the serum microRNA markers in the preparation of products for detecting hemophagocytic lymphocytohyperplasia is characterized in that the serum microRNA markers are a combination of hsa-miR-21-5p and hsa-miR-106a-5p.