Use of circular RNA in preparation of biomarker for diagnosing autism

Abstract

The use of a circular RNA in the preparation of a biomarker for diagnosing autism. Provided is a circular RNA that can be used for assisting the diagnosis of autism, wherein the circular RNA is a combination of at least two of hsa_circ_0054602, hsa_circ_0004566 and hsa_circ_0085145. By means of experiments, it is found that the combination of hsa_circ_0054602 and hsa_circ_0004566 has an optimal effect, with a sensitivity of 60.87% and a specificity of 94.79%.

Description

Application of circular RNA in the preparation of biomarkers for the diagnosis of autism Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of circular RNA in preparing biomarkers for diagnosing autism. Background Art

[0002] Autism spectrum disorder (ASD) is a group of pediatric psychiatric disorders caused by neurodevelopmental disorders, characterized by varying degrees of social impairment, language impairment, and stereotyped behavior. Currently, autism is the leading cause of disability in children under six years old. 75% of children with autism have intellectual disability, and even in adulthood, 50% to 70% still experience social maladjustment, are unable to care for themselves, and require lifelong care. Autism seriously compromises children's physical and mental health and places a heavy burden on society and families.

[0003] At present, there is no effective drug or method to completely cure ASD. It can only be treated with some drugs or methods to alleviate the core symptoms of patients, improve the development of the patient's nervous system and ability to take care of themselves, and thus improve the quality of life. Therefore, early detection of the disease and early diagnosis will help to take intervention measures early. The current clinical diagnosis method of ASD is mainly to use relevant scales (such as the Autism Behavior Scale, the Child Autism Rating Scale, the Autism Diagnostic Observation Scale, the Autism Diagnostic Interview Scale, etc.) to observe and evaluate the speech and behavior of children. It is subjective and prone to errors, resulting in missed or misdiagnosed diagnoses. Moreover, clinical evaluation and diagnosis must go through a systematic and complex process, which requires cooperation from multiple parties and is time-consuming. For ASD patients with atypical symptoms, it takes longer to diagnose, which will delay the best time for intervention. Therefore, early screening and diagnosis of ASD urgently need objective and rapid experimental indicators.

[0004] Circular RNA (circRNA) is an emerging class of specialized non-coding RNA molecules that lack a free 5' cap and 3' poly(A) tail, instead forming a covalently linked circular structure. CircRNAs are not easily degraded by RNA exonucleases and are more stable than linear RNAs. CircRNAs are abundant in the cytoplasm of eukaryotic cells, exhibiting specific organization, timing, and disease specificity, making them potential biomarkers with stable expression. Accurately determining a patient's epigenetic profile is a prerequisite for precision medicine. Detection of autism-associated circRNAs in children with autism is essential for the early and effective diagnosis of autism. However, there is currently no method for the early, objective diagnosis of autism. Developing a novel, non-invasive, and biologically objective autism detection method, with the goal of identifying objective, minimally invasive biomarkers for ASD diagnosis, would have significant socioeconomic and clinical value for the diagnosis and early intervention of ASD.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide the application of circular RNA in the preparation of biomarkers for diagnosing autism to solve the problems existing in the above-mentioned prior art. The present invention clarifies circular RNA for auxiliary diagnosis of autism. The circular RNA has the advantages of high sensitivity and strong specificity, and is of great value for the early diagnosis of autism.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides the use of circular RNA in preparing a biomarker for auxiliary diagnosis of autism, wherein the circular RNA is a combination of at least two of hsa_circ_0004566, hsa_circ_0054602 and hsa_circ_0085145.

[0009] The present invention also provides a primer set for detecting circular RNA in the preparation of an autism biomarker detection reagent, wherein the circular RNA is a combination of at least two of hsa_circ_0004566, hsa_circ_0054602, and hsa_circ_0085145, and the nucleotide sequence of the primer set is shown in SEQ ID NOs: 1-6.

[0010] The present invention also provides the use of a primer set for detecting circular RNA in preparing a kit for assisting in the diagnosis of autism, wherein the circular RNA is a combination of at least two of hsa_circ_0004566, hsa_circ_0054602, and hsa_circ_0085145, and the nucleotide sequence of the primer set is shown in SEQ ID NOs: 1-6.

[0011] Preferably, the nucleotide sequences of hsa_circ_0004566, hsa_circ_0054602 and hsa_circ_0085145 are shown in SEQ ID NOs: 9-11, respectively.

[0012] Preferably, the circular RNA is a combination of hsa_circ_0004566 and hsa_circ_0054602.

[0013] The present invention discloses the following technical effects:

[0014] The present invention provides the use of different combinations of three circular RNAs (hsa_circ_0004566, hsa_circ_0054602, and hsa_circ_0085145) for the auxiliary diagnosis of autism. Experimental results show that the combination of hsa_circ_0054602 and hsa_circ_0004566 is the most effective, with a sensitivity of 60.87% and a specificity of 94.79%. This invention addresses the current lack of an early, objective, highly sensitive, and specific diagnostic marker for autism. Through experimental discovery, a biomarker for autism has been discovered, providing new insights and diagnostic markers for the early identification and auxiliary diagnosis of autistic patients. The discovery of this biomarker facilitates the early detection and treatment of autistic patients and has significant clinical value and socioeconomic significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 shows the comparison of relative expression levels of serum circRNA between the case group and the control group;

[0017] Figure 2 shows the ROC curve of a single circRNA. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1

[0024] 1. Detection of circular RNA expression in serum

[0025] 1.1 Extraction of total RNA from serum

[0026] RNA was extracted from the serum of ASD patients using the Trizol method in accordance with strict and standardized RNA extraction procedures. The specific steps are as follows:

[0027] ①Put 200μL of serum into a 1.5mL enzyme-free EP tube, add 1000μL of Trizol, and let it stand for 5 minutes;

[0028] ② Add 200 μL of chloroform and shake vigorously for 15 seconds, then place on ice for 2 minutes;

[0029] ③ Centrifuge at 10,000 rpm for 15 min at 4°C and transfer the supernatant to a new enzyme-free EP tube;

[0030] ④ Add 500 μL of isopropanol, mix well, and place at -20°C for 10 min;

[0031] ⑤ Centrifuge at 10,000 rpm for 12 min at 4°C and remove the supernatant;

[0032] ⑥Add 1000 μL 75% alcohol and mix well;

[0033] ⑦ Centrifuge at 8000 rpm for 5 min at 4°C and remove the supernatant;

[0034] ⑧ Dry for 8-10 minutes, add 20μL DEPC water;

[0035] ⑨50-60℃ water bath, 5min.

[0036] 1.2 Determination of serum total RNA purity and concentration

[0037] The measurement steps are as follows:

[0038] ①Open the microplate reader, wash the test plate twice with DEPC water, and wipe it clean;

[0039] ②2 μL DEPC water for zero calibration;

[0040] ③ 2μL of the sample to be tested is dropped into the detection hole for detection; OD 260 / OD 280 If the value is within the range of 1.8 to 2.0, the extracted RNA is qualified.

[0041] 1.3 CircRNA Reverse Transcription

[0042] The total volume of the reverse transcription reaction was set to 10 μL. The reagents and amounts required for the reaction are shown in Table 1. The reaction conditions were: 25°C for 5 min; 42°C for 60 min; 95°C for 5 min; and 4°C forever.

[0043] Table 1 Reagents used

[0044] 1.4 Circular RNA Real-time Fluorescence Quantitative PCR

[0045] qPCR was used to detect hsa_circ_0004566, hsa_circ_0054602, hsa_circ_0085145, and GAPDH. Primers were designed using Primer Premier 5.0 software, and primer information is shown in Table 2. The total reaction volume was 20 μL, and the required reagents are shown in Table 3. Reaction conditions are shown in Table 4, and melting curve analysis was performed. The sequences of the three circular RNAs are documented in the circBase database (http: / / circbase.org / ).

[0046] Table 2 Primer sequence information

[0047] Table 3 Reagents required for the reaction

[0048] Table 4 Reaction conditions

[0049] The sequence information of hsa_circ_0004566, hsa_circ_0054602, and hsa_circ_0085145 is shown in Table 5 below:

[0050] Table 5

[0051] 1.5 Data Processing

[0052] GAPDH was used as the internal reference gene and the ΔCt method was used to calculate the expression level of the target circ RNA. -△△Ct Calculate the relative expression of target circRNA, ΔCt = Ct (target circRNA) - Ct (GAPDH), ΔΔCt = ΔCt (病例组) -ΔCt (对照 组) .

[0053] 2. Results and Analysis

[0054] 2.1 Expression levels of circular RNA in serum of ASD patients

[0055] As shown in Figure 1, compared with the control group, the expression of hsa_circ_0054602 in the serum of the ASD case group was increased, which was 1.61 times that of the control group; the expression of hsa_circ_0004566 and hsa_circ_0085145 was decreased, which were 0.55 times and 0.80 times that of the control group, respectively. The differences were statistically significant (P < 0.05).

[0056] 2.2 The value of single circular RNA in diagnosing ASD

[0057] To evaluate the potential of hsa_circ_0054602, hsa_circ_0004566, and hsa_circ_0085145 as diagnostic biomarkers for autism, receiver operating characteristic (ROC) curve analysis was performed. The ROC curve is a composite indicator of sensitivity and specificity for continuous variables. It uses a graphical method to reveal the relationship between sensitivity and specificity. The ROC curve calculates a range of sensitivities (SEN) and specificities (SPE) by setting multiple cutoff values ​​for the continuous variable. The curve is then plotted with sensitivity as the ordinate and (1-specificity) as the abscissa. The larger the area under the curve, the higher the diagnostic accuracy. On the ROC curve, the point closest to the upper left of the plot represents the cutoff value with the highest sensitivity and specificity. The area under the ROC curve (AUC) ranges from 1.0 to 0.5. When the AUC is > 0.5, the closer the AUC is to 1, the better the diagnostic performance. The AUC had low accuracy when it was 0.5-0.7, moderate accuracy when it was 0.7-0.9, and high accuracy when it was above 0.9. As shown in Figure 2, the AUCs for hsa_circ_0054602, hsa_circ_0004566, and hsa_circ_0085145 were 0.79 (95% CI: 0.74-0.83, P < 0.001), 0.74 (95% CI: 0.68-0.79, P < 0.001), and 0.68 (95% CI: 0.63-0.74, P < 0.001), respectively. The sensitivities were 65.8%, 89.1%, and 89.1%, respectively, and the specificities were 81.3%, 55.2%, and 45.3%, respectively. It can be seen that hsa_circ_0054602, hsa_circ_0004566 and hsa_circ_0085145 can be used as reliable and potential biomarkers for the diagnosis of autism, providing guidance for the study of autism-related circular RNA.

[0058] 2.3 Analysis of diagnostic efficacy of combined trials

[0059] The sensitivity and specificity of hsa_circ_0054602, hsa_circ_0004566, and hsa_circ_0085145 alone are not ideal. While sensitivity or specificity is high, the Youden index and kappa values ​​are relatively low. To improve diagnostic efficiency, specificity should be increased by combining two or three indicators in a tandem test. In a tandem test, a sample is considered positive only when all indicators are positive. This improves specificity but reduces sensitivity. Across all samples, the combination of hsa_circ_0054602 and hsa_circ_0004566 achieved the best diagnostic performance, with an AUC of 0.80 (95% CI: 0.73-0.84, P < 0.001), a sensitivity of 60.87%, a specificity of 94.79%, a Youden index of 0.56, a concordance rate of 78.19%, and a kappa value of 0.56 (see Table 6).

[0060] Table 6 Evaluation of the effectiveness of different serial methods of various indicators in diagnosing ASD Note: A: hsa_circ_0054602; B: hsa_circ_0004566; C: hsa_circ_0085145.

[0061] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The use of circular RNA in the preparation of biomarkers for assisting the diagnosis of autism, characterized in that: The circular RNA is a combination of at least two of hsa_circ_0004566, hsa_circ_0054602 and hsa_circ_0085145.

2. Use of a primer set for detecting circular RNA in preparing an autism biomarker detection reagent, characterized in that: The circular RNA is a combination of at least two of hsa_circ_0004566, hsa_circ_0054602 and hsa_circ_0085145, and the nucleotide sequence of the primer set is shown in SEQ ID NO: 1-6.

3. Use of a primer set for detecting circular RNA in preparing a kit for assisting in the diagnosis of autism, characterized in that: The circular RNA is a combination of at least two of hsa_circ_0004566, hsa_circ_0054602 and hsa_circ_0085145, and the nucleotide sequence of the primer set is shown in SEQ ID NO: 1-6.

4. The use according to any one of claims 1 to 3, characterized in that: The nucleotide sequences of hsa_circ_0004566, hsa_circ_0054602 and hsa_circ_0085145 are shown in SEQ ID NOs: 9-11, respectively.

5. The use according to any one of claims 1 to 3, characterized in that: The circular RNA is a combination of hsa_circ_0004566 and hsa_circ_0054602.

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