Surface hsa-miR-4516 marker for diagnosing fragile X syndrome, kit and application

Abstract

The invention relates to an application of hsa-miR-4516 as a marker for diagnosing fragile X syndrome, and a related detection reagent and a kit. According to the present invention, the significant up-regulation of the hsa-miR-4516 in the fragile X syndrome is identified, and the further data results show that the hsa-miR-4516 can effectively diagnose the FXS patient so as to make the diagnosis of the fragile X syndrome more convenient and easy.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological diagnosis, and specifically refers to a miRNA marker combination, a kit and an application thereof that can be used for diagnosing fragile X syndrome (FXS). Background Art

[0002] Fragile X syndrome (FXS) is a hereditary mental retardation syndrome with an incidence second only to congenital idiocy. The incidence rate accounts for 0.05% of all children. It is X-linked and accounts for 40% of X-linked mental retardation. Its penetrance varies according to gender, 80% in males and 30% in females. Clinically, the incidence of fragile X syndrome also varies according to gender, 1 / 4000 in males and 1 / 6000 in females. The neurodevelopmental disorders caused by FXS can bring about a variety of behavioral characteristics, such as delayed neuropsychomotor development (ADNPM), anxiety, aggression, self-mutilation, attention deficit disorder, social withdrawal and physical complications. The molecular genetic basis of the onset of more than 95% of fragile X syndrome patients is caused by dynamic mutations of the (CGG)n structure expansion of the FMR1 gene, and about 5% or less is caused by missense mutations and deletion mutations of the FMR1 gene that affect the normal structure of FMRP.

[0003] miRNAs (microRNAs) are a class of short non-coding RNAs that participate in post-transcriptional gene regulation by promoting messenger RNA (mRNA) silencing and affecting protein translation. Evidence from multiple studies suggests that miRNAs are involved in the pathogenesis of different diseases, and disease-specific miRNA profiles have been isolated from body fluids (circulating miRNAs), including miRNAs selectively expressed in blood and urine. miRNAs play a role in neurodevelopment and regulate important biological processes in central nervous system function. It has been found that dysregulated expression of miRNAs plays an important role in the pathogenesis of neurological diseases (see: Im HI, Kenny PJ. MicroRNAs in neuronal function and dysfunction. Trends Neurosci. 2012; 35(5): 325-334.). Due to the stability and accessibility of circulating miRNAs, their use as diagnostic and prognostic biomarkers for patient stratification has been valued and has improved the efficacy of targeted therapies for neurological diseases (such as fragile X syndrome, FXS).

[0004] Studies have also shown that miRNAs can be used as a new class of potential biomarkers for the diagnosis and prognosis of common central nervous system (CNS) diseases, including neurodevelopmental disorders, and provide new treatments (see: Jin XF, Wu N, Wang L, Li J. Circulating microRNAs: A novel class of potential biomarkers for diagnosing and prognosing central nervous system diseases Cell. Mol. Neurobiol. 2013; 33(5): 601–613.). However, so far, there are few reports on miRNAs biomarkers that can effectively diagnose FXS. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a serum miRNA marker for diagnosing fragile X syndrome (FXS).

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: a serum miRNA marker for the diagnosis of fragile X syndrome (FXS)

[0007] The present invention collects serum samples from case groups and healthy serum samples: the case group samples are patients clinically diagnosed with fragile X syndrome, and the healthy serum samples are from the physical examination population of the lecture. Through the detection of the two groups of serum samples, a miRNA significantly highly expressed in fragile X syndrome is screened and obtained: hsa-miR-4516. For hsa-miR-4516, it is further verified whether it can effectively diagnose FXS based on the verification samples collected again. After statistical calculation, ROC curve plotting is performed, showing that the AUC value of hsa-miR-4516 in distinguishing FXS patients from healthy people is 0.942. The results show that hsa-miR-4516 can effectively diagnose FXS patients as a biomarker.

[0008] The sequence of hsa-miR-4516 is: GGGAGAAGGGUCGGGGC (SEQ ID NO: 1).

[0009] In a first aspect, the present invention provides a serum miRNA marker for detecting fragile X syndrome: hsa-miR-4516.

[0010] In a second aspect of the present invention, a use of the miRNA markers described above in preparing a diagnostic kit for FXS is provided.

[0011] In a third aspect of the present invention, provided is the use of a reagent for detecting the expression level of the above miRNA in the preparation of a product for diagnosing fragile X syndrome, wherein the miRNA is hsa-miR-4516.

[0012] Preferably, the miRNA expression level is the expression level in a serum sample.

[0013] Preferably, the product is a test kit.

[0014] Preferably, the reagent also includes a specific primer pair for detecting the expression level of hsa-miR-4516. The sequence of the primer for detecting hsa-miR-4516 of the present invention can be obtained by conventional design methods in the art.

[0015] Specifically, the sequences of the specific primer pair for detecting hsa-miR-4516 are: 5'-GGACGGTAGCAAGCAAAGAGTGTGGCCCCGACCCT-3' and 5'-GGGATTCTGGAAGATGATGATGACGGGAGAAGGGT-3'. Further, the present invention also provides a universal primer pair, whose sequences are 5'-GGACGGTAGCAAGCAAAGAGTGTG-3' and 5'-GGGATTCTGGAAGATGATGATGAC-3'.

[0016] In the fourth aspect of the present invention, the present invention also provides a miRNA detection kit for diagnosing FXS, the kit comprising primers for detecting hsa-miR-4516; further, the kit also comprises other PCR basic reagents. The primers for detecting hsa-miR-4516 include a specific primer pair for detecting the expression level of hsa-miR-4516. Specifically, the sequence of the specific primer pair is: 5'-GGACGGTAGCAAGCAAAGAGTGTGGCCCCGACCCT-3' and 5'-GGGATTCTGGAAGATGATGATGACGGGAGAAGGGT-3'. Further, the present invention also provides a universal primer pair, the sequence of which is 5'-GGACGGTAGCAAGCAAAGAGTGTG-3' and 5'-GGGATTCTGGAAGATGATGATGAC-3'.

[0017] Preferably, the basic reagents include at least one of a positive quality control, a negative quality control, a poly-A tailing enzyme, a reverse transcriptase, dNTPs, a reverse transcription buffer, RNase-free water, a qPCR buffer, magnesium chloride, a DNA polymerase and a SYBR Green fluorescent dye.

[0018] In a fifth aspect of the present invention, the present invention further provides a use of a reagent for detecting hsa-miR-4516 in preparing a kit for diagnosing fragile X syndrome.

[0019] The technical effect of the present invention is as follows: through the development and application of serum miRNA markers and diagnostic kits, the miRNA markers provided by the present invention can quickly and accurately diagnose fragile X syndrome, thereby improving the diagnostic efficiency; making the diagnosis of fragile X syndrome more convenient and easy, and providing help for clinicians to quickly and accurately understand the patient's condition and improve the clinical treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Differences in the expression of related miRNAs in patients with fragile X syndrome (Case represents FXS patient samples, Control represents healthy samples; * indicates the degree of significant difference; ns indicates no significant difference).

[0021] Figure 2 Differential expression of hsa-miR-4516 in the validation set of patients with fragile X syndrome (Case represents FXS patient samples, Control represents healthy samples; * indicates the degree of significant difference; ns indicates no significant difference).

[0022] Figure 3 ROC curve of hsa-miR-4516 in diagnosing patients with fragile X syndrome. DETAILED DESCRIPTION

[0023] The following non-limiting embodiments may enable a person of ordinary skill in the art to more fully understand the present invention, but do not limit the present invention in any way. Any person familiar with the present technical field may make equivalent replacements or changes according to the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which shall fall within the protection scope of the present invention.

[0024] Example 1 Collection of samples

[0025] From 15 unrelated individuals collected: 8 male FXS patients and 7 male control individuals. The serum samples were taken out of the -80℃ refrigerator, placed in an ice bath at 0-4℃ until completely thawed, centrifuged at 4℃, 15000g for 10 minutes, and 200μL of the supernatant was taken and placed in a new RNase-free EP tube. The obtained blood samples of cases and healthy people were subjected to FXS molecular diagnosis, that is, the CGG repeat sequence in the FMR1 gene was detected using the fragile X PCR test. The control group showed normal FMR1 alleles.

[0026] Example 2 Extraction of small RNA

[0027] Using the kit method (PureLinkTM The miRNA isolation kit (Invitrogen) was used to extract small RNA according to the instructions, and the isolated small RNA samples were eluted in 40 μl of RNase-free water. The miRNA extracts were stored at -80 °C before processing. The RNA concentration was measured using a Nanodrop spectrophotometer, and an equal amount of miRNA (10 ng) was used in the RT-PCR quantitative reaction.

[0028] Example 3 Detection of differentially expressed miRNAs in FXS patients

[0029] The miRNAs (hsa-miR-575, hsa-miR-1248, hsa-miR-4516, hsa-miR-3609, hsa-miR-133b) that have been reported in the literature to be highly expressed in neurodevelopmental disorders were detected, and specific primers and universal primers were designed for RT-PCR detection to detect the expression levels of the above miRNAs in the two groups of samples in Example 1. Specifically, the specific primers designed were used to perform real-time quantitative PCR detection (RT-qPCR) on the test samples, using 0.8-1.02 μg of RNA. The expression level of miRNA was analyzed by real-time quantitative PCR in a 96-well plate using a real-time PCR system.

[0030] Table 1 shows the reaction system of RT-PCR detection of target miRNA; Table 2 lists the RT-PCR detection primers for detecting the corresponding miRNA. The reaction procedure of RT-PCR detection is: 45℃, 4min; 37℃, 5min, 1 cycle; 94℃, 30s; 37℃, 45s; 60℃30s, 1 cycle; 94℃, 30s; 72℃, 30s, 35cycles, 35 cycles. Three replicate wells were set for each sample to calculate the expression level of the miRNA to be tested. The results showed that the expression level of hsa-miR-4516 in FXS patients was significantly increased ( Figure 1 ), which can be considered as a potential diagnostic marker for the diagnosis of fragile X syndrome.

[0031] Table 1 Reaction system for RT-PCR detection of target miRNA

[0032] Total RNA 0.8-1.02 μg 10X PCR Buffer* 2.5μL 5X M-MLV Buffer** 5μL 20X SYBR Green I 1.25μL Reverse Transcriptase M-MLV 20U RNase inhibitor 4U Primer P1 (10 μM) 0.5μL Primer P2 (10 μM) 0.5μL Primer RP1 (2 μM) 0.5μL Primer RP2 (2 μM) 0.5μL <![CDATA[ddH2O]]> Up to 25 μL

[0033] *10X PCR Buffer (100mM Tris-HCl, pH 8.3, 500mM KCl, 15mM MgCl2)

[0034] **5X M-MLV Buffer (250mM Tris-HCl, pH 8.3, 375mM KCl, 15mM MgCl2, 50mM Dithiothreitol)

[0035] Table 2: RT-PCR detection primers

[0036]

[0037] Example 4: Further verification of the diagnostic effect of hsa-miR-4516 on FXS

[0038] Using clinical samples collected from other batches, a total of 85 unrelated individuals: 50 male FXS patients and 35 male control individuals. Referring to the detection method of Examples 1-3, the expression level of hsa-miR-4516 in FXS patient and control samples was detected ( Figure 2 ), draw the ROC curve ( Figure 3 ), and its AUC value was 0.942; the experimental results showed that hsa-miR-4516 can effectively diagnose fragile X syndrome; it makes the diagnosis of fragile X syndrome more convenient, and helps clinicians to quickly and accurately understand the patient's condition and improve the clinical treatment effect.

[0039] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting miRNA expression level in the preparation of a product for diagnosing fragile X syndrome, characterized in that: The miRNA is hsa-miR-4516.

2. The application according to claim 1, characterized in that: The reagent includes a specific primer pair for detecting the expression level of hsa-miR-4516.

3. The use according to claim 2, wherein the sequences of the specific primer pair are: 5'-GGACGGTAGCAAGCAAAGAGTGTGGCCCCGACCCT-3' and 5'-GGGATTCTGGAAGATGATGATGACGGGAGAAGGGT-3'.

4. The use according to claim 3, characterized in that: The miRNA expression level is the expression level in serum samples.

5. The use according to claim 1, characterized in that: The product is a detection kit.

6. A miRNA detection kit for fragile X syndrome, characterized in that: The miRNA is hsa-miR-4516, and the kit includes a reagent for detecting the expression level of hsa-miR-4516.

7. The kit according to claim 6, wherein the reagent comprises a specific primer pair for detecting the expression level of hsa-miR-4516, and the sequences of the specific primer pair are: 5'-GGACGGTAGCAAGCAAAGAGTGTGGCCCCGACCCT-3' and 5'-GGGATTCTGGAAGATGATGATGACGGGAGAAGGGT-3'.

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