Application of combined miRNA in preparation of endometriosis diagnostic agent and diagnostic kit

Abstract

The invention relates to application of a combined miRNA expression quantity detection agent in preparation of an endometriosis diagnostic agent and an endometriosis diagnostic kit. The expression quantity of the combined miRNA in the serum exosome is used as a diagnostic marker of endometriosis, the sensitivity is 0.846, the specificity is 0.898, the false positive rate is 0.102, and the ROC curve AUC value is 0.921. Therefore, both the sensitivity and the specificity of the combined miRNA are very high, and the combined miRNA has a relatively good diagnostic value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of endometriosis diagnosis, and particularly relates to application of a combined miRNA expression detection agent in preparation of an endometriosis diagnosis agent, and an endometriosis diagnosis kit. BACKGROUND

[0002] Endometriosis (endometriosis for short) is a common gynecological benign disease, which has the characteristics of invasion, metastasis and recurrence similar to malignant tumors, and the incidence rate is as high as 10%-15% in women of childbearing age, and is showing a rising trend year by year. Endometriosis not only causes patients to have symptoms such as progressive and aggravated dysmenorrhea, chronic pelvic pain, and dyspareunia, but also seriously affects the reproductive function. According to statistics, about 30%-50% of endometriosis patients are combined with infertility, which brings great distress to the physical and mental health and quality of life of patients, and also causes heavy social and medical burden.

[0003] At present, the diagnosis of endometriosis faces many challenges. In the traditional diagnosis method, although the ultrasonic examination is a non-invasive method, the detection rate for small lesions or deep infiltrating endometriosis is low, and misdiagnosis and missed diagnosis are easy to occur; the specificity and sensitivity of serum tumor markers such as CA125 are insufficient, and it is difficult to be used as a diagnosis basis. Laparoscopy combined with pathological biopsy is the current "gold standard" for the diagnosis of endometriosis, but this method is a invasive operation, which has problems such as anesthesia risk, surgical complications, and high cost, and cannot be used as a routine screening method, and it is also difficult to meet the needs of patients for non-invasive diagnosis.

[0004] Therefore, finding a non-invasive, efficient and specific endometriosis diagnosis method has become a key problem to be solved in the current gynecological medical field.

[0005] Exosomes, as a kind of vesicular structure with a diameter of about 30-150 nm secreted by cells, widely exist in body fluids such as blood, urine and ascites, can carry nucleic acids, proteins, lipids and other bioactive substances of the source cells, and play an important role in cell-to-cell information transmission and disease development. Because of the good biological stability of exosomes, and the contents of which can reflect the pathological and physiological state of the source cells, it is considered to be a very potential disease diagnosis biomarker carrier.

[0006] MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of about 20-24 nucleotides, which can participate in cell proliferation, apoptosis, invasion and other biological processes by regulating the expression of target genes. Studies have found that the expression profile of miRNA will change significantly under disease conditions, and it is stable in body fluid exosomes. In recent years, more and more studies have shown that the expression level of exosome miRNA in the body fluid (such as serum, ascites) of endometriosis patients is significantly different from that of healthy people, and some specific exosome miRNAs can not only accurately distinguish endometriosis patients from healthy controls, but also reflect the severity and stage of the disease.

[0007] At present, although relevant studies have found many potential exosome miRNA markers for endometriosis, most of the research sample sizes are small, and there is a lack of unified detection standard and diagnostic threshold, so an endometriosis non-invasive diagnosis model based on exosome miRNA has not yet been formed for clinical promotion. SUMMARY

[0008] To solve the above problems, the application provides an application of a miRNA expression detection agent in the preparation of an endometriosis diagnosis agent, wherein the miRNA is a combination of miR-126-3p, miR-122-5p, miR-10b-5p, miR-15b-5p, miR-20b-5p, miR-328-3p and let-7g-5p.

[0009] In one specific embodiment, the miRNA expression detection agent is a Q-PCR detection agent.

[0010] The application also provides an endometriosis diagnosis kit, which comprises a miRNA expression detection agent, wherein the miRNA is a combination of miR-126-3p, miR-122-5p, miR-10b-5p, miR-15b-5p, miR-20b-5p, miR-328-3p and let-7g-5p.

[0011] In one specific embodiment, the miRNA expression detection agent is a Q-PCR detection agent.

[0012] In one specific embodiment, the diagnosis kit further comprises an exosome capture device.

[0013] In one specific embodiment, the miRNA expression detection agent is used to detect the expression of miRNA in exosomes from serum.

[0014] In one specific embodiment, the diagnosis kit further comprises a negative control.

[0015] The scoring model used for diagnosing whether the subject has endometriosis is as follows:

[0016] logit(P) = β0 + β1 x X1 + β2 x X2 +... + βk x Xk

[0017] wherein P is the probability of being predicted to have endometriosis;

[0018] β0 is the intercept;

[0019] β1, β2,..., βk are the regression coefficients (weights) of each miRNA.

[0020] The present application uses the combined miRNA expression in serum exosomes as a diagnostic marker for endometriosis, with a sensitivity of 0.846, a specificity of 0.898, a false positive rate of 0.102, and an ROC curve AUC value of 0.921. It can be seen that the sensitivity and specificity of the combined miRNA are both high, and have good diagnostic value. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The expression of each miRNA in serum exosomes of endometriosis and healthy controls.

[0022] Figure 2 The ROC curve of the combined miRNA expression in serum exosomes as an index for diagnosing endometriosis. DETAILED DESCRIPTION

[0023] The principles and characteristics of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and are not intended to limit the scope of the present application.

[0024] 1. Detecting endometriosis patient exosome differential miRNA by using porous affinity microfluidic exosome capture chip

[0025] The chip holder is made of PDMS and foamed nickel, and the holder is modified by streptavidin and biotinylated exosome protein antibody to obtain a chip that can specifically capture exosomes.

[0026] The venous blood of the endometriosis patient is centrifuged at 3000 rpm / min for 10 min to obtain the patient's plasma, which is placed in a -80℃ refrigerator for use.

[0027] The patient's plasma is passed into the chip at a flow rate of 10 μl / min, and 100 μl of plasma is passed into each chip, and then washed with PBS three times. At this time, the exosomes have been captured on the holder in the chip.

[0028] The TRIZOL method was used to introduce into the chip, capture the RNA of the exosomes in the chip. After reverse transcription of the obtained RNA, Q-PCR was performed to obtain the cq value (u6 as an internal reference), and the relative abundance was expressed by 2^-ΔCt / 2^-ΔΔCt. The obtained cq value was statistically analyzed to obtain the differential expression result of miRNA.

[0029] The case group + control group n = 120; plasma exosomes were enriched by PAE-CM microfluidic chip, followed by RT-qPCR quantification.

[0030] The results are shown in Figure 1 7 miRNAs were found to be directionally stable and significantly different between cases and controls: miR-10b-5p, miR-126-3p, miR-122-5p, miR-15b-5p, miR-20b-5p, let-7g-5p, miR-328-5p. Except for miR-122-5p, the remaining miRNAs have not been reported in endometriosis studies.

[0031] 2. Diagnostic value of combined miR

[0032] The relative expression of the 7 miRNAs was used to diagnose endometriosis (sample size 124, including 65 patients and 59 healthy individuals), and the P values of different miRNAs in case and con groups were calculated by unmatched t test using Graph Prism. The specificity, sensitivity, true positive rate, false positive rate, AUC value and other key data of different miRNAs and 7 miRNAs were calculated using R packages such as pROC, and ROC curves were made.

[0033] The results are shown in Table 1, in which the combined miRNA as a detection agent detected 55 true positives, 53 true negatives, 6 false positives, and 10 false negatives; the sensitivity was 0.846, the specificity was 0.898, the false positive rate was 0.102, the false negative rate was 0.262, and the ROC curve was as shown in Figure 2 The AUC value was 0.921. It can be seen that the sensitivity and specificity of the combined miRNA are both high, and it has good diagnostic value.

[0034] The expression of the combined miRNA in diagnosing whether the subject has endometriosis is used in the scoring model as follows:

[0035] logit(P) = β0 + β1 × X1 + β2 × X2 +... + βk × Xk

[0036] Where P is the probability of being predicted to have endometriosis.

[0037] X1-Xk are miR-10b-5p, miR-126-3p, miR-122-5p, miR-15b-5p, miR-20b-5p, let-7g-5p, miR-328-5p in turn relative to the expression amount of healthy control;

[0038] β0 is the intercept, that is, when the value of all the miRNA included in the model is 0, the probability of the sample being predicted as case (group = 1)

[0039] β1, β2,..., βk are the regression coefficients of miR-10b-5p, miR-126-3p, miR-122-5p, miR-15b-5p, miR-20b-5p, let-7g-5p, miR-328-5p, respectively -0.66192, -0.94085, -0.91098, -0.85359, 0.385282, 1.514582, 0.768127, 0.295963.

[0040] Table 1 Key data of each miRNA and combined miRNA in diagnosing endometriosis

[0041]

[0042] The above description is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of an expression amount detection agent for miRNA in the manufacture of a diagnostic agent for endometriosis, the miRNA being a combination of miR-10b-5p, miR-126-3p, miR-122-5p, miR-15b-5p, miR-20b-5p, let-7g-5p, and miR-328-5p.

2. Use according to claim 1, characterized in that, The expression amount detection agent for the miRNA is a Q-PCR detection agent.

3. A diagnostic kit for endometriosis, characterized by, An expression amount detection agent for miRNA, the miRNA being a combination of miR-10b-5p, miR-126-3p, miR-122-5p, miR-15b-5p, miR-20b-5p, let-7g-5p, and miR-328-5p.

4. The endometriosis diagnostic kit according to claim 3, characterized in that, The expression amount detection agent for the miRNA is a Q-PCR detection agent.

5. The endometriosis diagnostic kit according to claim 4, characterized in that, Also included is an exosome capture device.

6. The endometriosis diagnostic kit according to claim 5, characterized in that, The expression amount detection agent for the miRNA is used to detect the expression amount of the miRNA in exosomes from serum.

7. The endometriosis diagnostic kit according to claim 6, characterized in that, Also included is a negative control.

8. The endometriosis diagnostic kit according to claim 7, characterized in that, The scoring model used in diagnosing whether a subject has endometriosis is as follows: logit(P) = β0 + β1 × X1 + β2 × X2 +... + βk × Xk; where P is the probability of being predicted to have endometriosis; X1-Xk are, in order, the expression amounts of miR-10b-5p, miR-126-3p, miR-122-5p, miR-15b-5p, miR-20b-5p, let-7g-5p, and miR-328-5p relative to healthy controls; β0 is the intercept, i.e., the probability of the sample being predicted to be a case (group = 1) when the values of all miRNAs included in the model are 0; β1, β2,..., βk are, in order, the regression coefficients of miR-10b-5p, miR-126-3p, miR-122-5p, miR-15b-5p, miR-20b-5p, let-7g-5p, and miR-328-5p, which are -0.66192, -0.94085, -0.91098, -0.85359, 0.385282, 1.514582, 0.768127, and 0.295963, respectively.

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