Application of a plasma circRNA biomarker in diagnosis and treatment of craniosynostosis

Abstract

The application discloses application of a plasma circRNA biomarker in diagnosis and treatment of craniosynostosis. The marker has_circ_0002572 provided by the application can effectively diagnose craniosynostosis, and meanwhile, can inhibit premature closure of craniosynostosis by subcutaneous injection of an AAV vector overexpressing has_circ_0002572 at a craniosynostosis site, so that the effect of early treatment of craniosynostosis is achieved. The plasma circRNA biomarker provided by the application provides a new thought for a diagnosis and treatment target of craniosynostosis, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of a plasma circRNA biomarker in the diagnosis and treatment of craniosynostosis. Background Technology

[0002] Craniosynostosis is the second most common craniofacial abnormality. Premature closure of the skull can lead to skull deformities and restrict brain growth. In severe cases, it can cause abnormal skull growth, increased intracranial pressure, delayed brain development, and impaired cognitive function. Approximately 15-30% of cases are accompanied by other clinical symptoms, which may include hand and foot deformities, skeletal and cardiac defects. Currently, the main treatment for craniosynostosis is surgery, but surgery is highly invasive and carries significant risks. Therefore, finding non-invasive, safe, and more effective treatment methods is crucial for the early diagnosis and treatment of craniosynostosis.

[0003] With the development of high-throughput RNA sequencing technology and advancements in biotechnology, many non-coding RNAs have been discovered to perform various biological functions in the human body and participate in the occurrence and development of various diseases. circRNAs are a mysterious subclass of endogenous long non-coding RNAs that regulate genes at the transcriptional or post-transcriptional levels. Due to their closed-loop structure, circular RNAs can evade degradation by exonucleases and are much more stable than linear RNAs. Advances in detection technology have made the detection of circRNAs more feasible, opening up possibilities for their application in the diagnosis and treatment of craniosynostosis (CSI). Studies have shown that circRNAs have multiple functions, including acting as miRNA sponges, regulating gene transcription, adsorbing RNA-binding proteins, and regulating protein translation. Therefore, studying the mechanisms of action of circRNAs in CSI and developing circRNA-based diagnostic and therapeutic tools is of great significance for improving the early diagnosis and treatment of CSI. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing an application of plasma circRNA biomarkers in the diagnosis and treatment of craniosynostosis.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A plasma circRNA biomarker for the auxiliary diagnosis and / or treatment of craniosynostosis, wherein the plasma circRNA biomarker is hsa_circ_0002572; the nucleic acid sequence of hsa_circ_0002572 is shown in SEQ ID NO:1.

[0007] Application of the reagent for detecting the expression level of the plasma circRNA biomarker in the preparation of auxiliary diagnostic reagents for craniosynostosis.

[0008] As a preferred embodiment of the present invention, the reagent for detecting the expression level of the plasma circRNA biomarker of claim 1 is the specific detection primer for hsa_circ_0002572.

[0009] As a further preferred embodiment of the present invention, the specific detection primer sequences of hsa_circ_0002572 are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0010] Application of hsa_circ_0002572 or substances that overexpress hsa_circ_0002572 in the preparation of drugs for the treatment of craniosynostosis.

[0011] As a preferred embodiment of the present invention, the substance overexpressing hsa_circ_0002572 is a recombinant expression vector overexpressing hsa_circ_0002572.

[0012] In a preferred embodiment of the present invention, the substance overexpressing hsa_circ_0002572 is a viral particle overexpressing hsa_circ_0002572, preferably an AAV particle overexpressing hsa_circ_0002572; the viral particle, particularly the AAV particle, is capable of transducing and expressing hsa_circ_0002572 in mammalian cells. The AAV particle contains a nucleic acid inserted between a pair of AAV inverted terminal repeat (ITR) sequences, the nucleic acid encoding hsa_circ_0002572.

[0013] Viral particles overexpressing hsa_circ_0002572 can be prepared using conventional techniques in the field, or they can be prepared by a biotechnology company, as long as they can overexpress hsa_circ_0002572 in mammals.

[0014] Multiple AAV particles as described are administered subcutaneously to the site of craniosynostosis in mammals in need of treatment for craniosynostosis.

[0015] A pharmaceutical composition for treating craniosynostosis comprises viral particles overexpressing hsa_circ_0002572, preferably comprising AAV particles overexpressing hsa_circ_0002572.

[0016] As a preferred embodiment of the present invention, it also includes conventional pharmaceutical excipients for preparing the viral particles overexpressing hsa_circ_0002572 into a pharmaceutical formulation.

[0017] A diagnostic kit for craniosynostosis, comprising reagents for detecting the expression level of the plasma circRNA biomarker, preferably comprising specific detection primers for hsa_circ_0002572 and primers for detecting internal reference genes, and more preferably comprising primers shown in SEQ ID NO:2-5.

[0018] Beneficial effects:

[0019] Advantages and beneficial effects of the present invention:

[0020] The plasma circRNA biomarker provided by this invention can effectively diagnose craniosynostosis (CSSI). Furthermore, by overexpressing hsa_circ_0002572, it can inhibit the differentiation of mesenchymal stem cells into osteoblasts, thereby suppressing premature closure of the craniosynostosis and achieving early diagnosis and treatment. The hsa_circ_0002572 blood biomarker provided by this invention offers a new approach to the diagnosis and treatment of CSISI and has broad application prospects. Attached Figure Description

[0021] Figure 1 This shows the differential expression of circRNAs in the plasma of patients with craniosynostosis. (A) Volcano plot of differentially expressed circRNAs; (B) Expression profile of abnormally low-expression circRNAs with human-mouse homology.

[0022] Figure 2 This refers to the dynamic expression of circRNAs during days 1 and 7 of mesenchymal stem cell-induced osteogenic differentiation.

[0023] Figure 3 The effect of siRNAs has_circ_0002572 and hsa_circ_0008616 on osteogenic differentiation of hMSCs.

[0024] Figure 4 This shows the expression of has_circ_0002572 in the cranial sutures of mice with premature cranial closure and wild-type mice.

[0025] Figure 5 This is the diagnostic efficacy of has_circ_0002572 in the population. (A) Concentration of has_circ_0002572 in plasma of different treatment groups, where CON: healthy controls; CS: patients with craniosynostosis. (B) Receiver operating characteristic (ROC) analysis results of has_circ_0002572.

[0026] Figure 6The study investigated the treatment of craniosynostosis in mice with AAV that overexpressed has_circ_0002572 in vivo. (A) qRT-PCR experiments confirmed that the AAV did indeed overexpress has_circ_0002572 in mice. (B) The therapeutic effect of the AAV on craniosynostosis mice. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings.

[0028] Unless otherwise specified, the conditions and methods used in the implementation process are in accordance with standard procedures. All test materials used are commercially available ordinary products, unless otherwise specified.

[0029] Example 1: Screening for circRNAs associated with premature craniosynostosis

[0030] 1.1 Sample Collection

[0031] This study collected blood samples from eligible children with craniosynostosis and healthy controls at Nanjing Children's Hospital. After data analysis, 5 healthy controls and 5 children with craniosynostosis were selected as experimental subjects. Informed consent was obtained from the guardians of all subjects.

[0032] 1.2 Extraction of plasma RNA

[0033] RNA was extracted from each serum sample using an RNA extraction kit, following the instructions in the manufacturer's manual.

[0034] 1.3 Screening of circRNA expression profiles

[0035] We commissioned Shanghai Kangcheng Biotechnology Co., Ltd. to construct circRNA expression profiles using the Arraystar Human Circular RNA Array V2.0 chip and screened for differentially expressed circRNAs. Significant differences were defined as |log2FC|>1.5 and FDR≤0.05.

[0036] 1.4 Experimental Results

[0037] like Figure 1 As shown, a total of 134 dysregulated circRNAs were identified, of which 74 were upregulated and 60 were downregulated. Figure 1 A). Of the 60 downregulated circRNAs, 12 circRNAs showed human-mouse homology ( Figure 1 B).

[0038] Example 2: Screening for circRNAs associated with osteogenic differentiation of mesenchymal stem cells

[0039] 2.1 Cell Culture

[0040] Human mesenchymal stem cells (hMSCs) were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in a cell culture incubator containing 5% CO2. hMSCs were seeded into 6-well plates, and the medium was replaced with osteogenic induction medium after 24 hours. Cells were collected after different induction times.

[0041] 2.2 qRT-PCR

[0042] Total RNA was extracted from cells using TRIzol reagent according to the instructions. Aliquots of 500 ng of total RNA were reverse transcribed into cDNA using a reverse transcriptase kit. Real-time quantitative PCR was performed using SYBR Green mixture and LightCycler 480. Primers for specific transcripts of 12 circRNAs containing human-mouse homologous circRNAs are shown in Table 1.

[0043] Table 1

[0044]

[0045]

[0046] 2.3 Experimental Results

[0047] like Figure 2 As shown, among the 12 downregulated circRNAs with human-mouse homology, only hsa_circ_0008616 and hsa_circ_0002572 decreased with increasing osteogenic differentiation time at different time points induced by hMSCs.

[0048] Example 3: Effect of has_circ_0002572 on osteogenic differentiation capacity of hMSCs in vitro

[0049] 3.1 Cell Culture and Transfection

[0050] hMSCs were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco) at 37°C in a cell culture incubator with 5% CO2. hMSCs were seeded in 6- or 12-well plates. After reaching 40% confluence, they were knocked down by siRNAs of has_circ_0002572 and hsa_circ_0008616, respectively. Twenty-four hours later, the osteogenic induction medium was replaced with a pre-prepared solution for induction. The cells were then cultured at 37°C in a 5% CO2 cell culture incubator, with the medium changed every two days. ALP staining was performed after 7 days of culture, and Alizarin Red staining was performed after 14 days to assess the effects of different treatments on osteogenic differentiation of hMSCs.

[0051] 3.2 Experimental Results

[0052] like Figure 3 As shown, knockdown of hsa_circ_0002572 cells significantly increased mineralization levels and enhanced cellular alkaline phosphatase activity.

[0053] Example 4: Expression of has_circ_0002572 in the cranial sutures of mice with premature cranial closure and wild-type mice.

[0054] 4.1 Sample Collection

[0055] Six- to eight-week-old female C57BL / 6J mice and Twist+ / - mice were purchased from Jicui Pharmaceutical (Jiangsu, China). WT female mice were mated with Twist+ / - male mice, and the offspring, after genotyping, were selected for further experiments at 9 days of age. All animal procedures were conducted in accordance with the protocols approved by the Animal Protection and Use Committee of Nanjing Medical University.

[0056] 4.2 Paraffin sections

[0057] Five skulls each from WT mice and Twist+ / - mice were selected at 9 days of age and fixed in 4% paraformaldehyde for 48 hours. After decalcification, the bone tissue was prepared into 8-micrometer thick paraffin sections.

[0058] 4.3 circRNA in situ hybridization

[0059] The Cy3-labeled has_circ_0002572 probe was designed and synthesized by Shanghai Gemma Genetics. The probe signal was detected using a fluorescence in situ hybridization kit according to the instructions. Images were acquired and analyzed using a Nikon confocal microscope.

[0060] 4.4 Experimental Results

[0061] like Figure 4As shown, has_circ_0002572 is expressed in wild-type mice and Twist+ / - mice with premature cranial suture closure. Compared with WT mice, has_circ_0002572 is significantly downregulated in the coronal suture of Twist+ / - mice.

[0062] Example 5: Diagnostic efficacy of has_circ_0002572 in the population

[0063] 5.1 Sample Collection

[0064] This study collected blood samples from eligible children with craniosynostosis and healthy controls at Nanjing Children's Hospital. After data analysis, 81 healthy controls and 67 children with craniosynostosis were selected as experimental subjects. Informed consent was obtained from the guardians of all subjects.

[0065] 5.2 qRT-PCR

[0066] RNA was extracted from each serum sample using an RNA extraction kit according to the instructions. 500 ng of total RNA was reverse transcribed into cDNA using a reverse transcriptase kit. Real-time quantitative PCR was performed using SYBR Green mixture and a LightCycler 480.

[0067] The specific detection primer sequences for hsa_circ_0002572 are as follows:

[0068] F:5'-TCTAACAAGAGGTCCCCAGC-3'(SEQ ID No.2)

[0069] R:5'-CAGGAAAGGTGGCGTGAAGTA-3'(SEQ ID No.3)

[0070] The GAPDH internal reference primer sequence is as follows:

[0071] F:5'-CAATGACCCCTTCATTGACC-3'(SEQ ID No.4)

[0072] R:5'-TTGATTTTGGAGGGATCTCG-3(SEQ ID No.5)

[0073] 5.3 Statistical Analysis

[0074] Plot the ROC curve using pROC analysis in R and calculate its AUC area.

[0075] 5.4 Experimental Results

[0076] like Figure 5As shown in Figure A, compared with healthy controls, the level of hsa_circ_0002572 was downregulated in plasma samples from patients with craniosynostosis (P<0.001). Using hsa_circ_0002572 as the detection variable, ROC curves were plotted and AUC values ​​were calculated. The results, shown in Figure B, indicate an AUC value of 0.885, a specificity of 0.801 at the optimal cutoff point, and a sensitivity of 0.851, suggesting that applying hsa_circ_0002572 to the diagnosis of craniosynostosis has good diagnostic efficacy.

[0077] Example 6: Evaluation of the therapeutic effect of AAV overexpressing has_circ_0002572 on craniosynostosis mice.

[0078] 6.1 Sample Collection

[0079] WT female mice were mated with Twist+ / - male mice, and the offspring were selected as Twist+ / - mice for subsequent experiments after genotyping. All animal procedures were conducted in accordance with the protocols approved by the Animal Protection and Use Committee of Nanjing Medical University.

[0080] 6.2 Subcutaneous injection of AAV overexpressing has_circ_0002572

[0081] The AAV targeting and overexpressing has_circ_0002572 was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. On days 1 and 5 after birth, progeny Twist+ / - mice were injected with AAV-oe-circ0002572 and AAV-oe-Vector into the coronal suture of Twist1+ / - mice using a microsyringe, and the development of the coronal suture was observed.

[0082] 6.3 micro-CT

[0083] After euthanasia of mice with CO2, the skulls were dissected from the skin and evaluated using a SkyScan 1176 high-resolution micro-CT imaging system. Each skull was scanned individually at a resolution of 18 μm. Images were reconstructed using NRecon.

[0084] 6.4 HE staining

[0085] Skulls were collected from WT and Twist+ / - mice 12 days post-surgery and fixed in 4% paraformaldehyde for 48 hours. After decalcification, the bone tissue was prepared into 8-micron thick paraffin sections. Staining was then performed using an HE staining kit (Beyotime, C0105S). Images were acquired and analyzed using a pathological slide scanner.

[0086] 6.5 qRT-PCR

[0087] Skull fragments from AAV-oe-circ0002572 and AAV-oe-Vector treated Twist+ / - mice were collected 12 days post-surgery. Total RNA was extracted from the cranial suture tissue using TRIzol reagent according to the manufacturer's instructions. Aliquots of 500 ng of total RNA were reverse transcribed into cDNA using a reverse transcriptase kit. Real-time quantitative PCR was performed using SYBR Green Mixture and LightCycler 480. Primers for the specific transcripts of circ_0002572 and gapdh are shown in Table 2.

[0088] Table 2

[0089]

[0090] 6.6 Experimental Results

[0091] like Figure 6 As shown, qRT-PCR experiments confirmed that this AAV did indeed overexpress has_circ_0002572 in mice. Figure 6 A). Micro-CT and histological analysis results showed that, compared with AAV-oe-Vector treatment, AAV-oe-circ0002572 treatment significantly reduced Twist1. + / - Premature closure of the coronal suture in mice Figure 6 B). The above results suggest that applying hsa_circ_0002572 to the early treatment of craniosynostosis has a good therapeutic effect.

Claims

1. Application of a reagent for detecting the expression level of plasma circRNA biomarkers in the preparation of auxiliary diagnostic reagents for craniosynostosis, wherein the plasma circRNA biomarker is hsa_circ_0002572; the nucleotide sequence of hsa_circ_0002572 is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The reagent used to detect the expression level of plasma circRNA biomarkers is the specific detection primer for hsa_circ_0002572.

3. The application according to claim 2, characterized in that, The specific detection primer sequences for hsa_circ_0002572 are shown in SEQ ID NO:2 and SEQ ID NO:

3.

4. Application of hsa_circ_0002572 or substances that overexpress hsa_circ_0002572 in the preparation of drugs for the treatment of craniosynostosis.

5. The application according to claim 4, characterized in that, The substance that overexpresses hsa_circ_0002572 is a recombinant expression vector that overexpresses hsa_circ_0002572.

6. The application according to claim 4, characterized in that, The substance overexpressing hsa_circ_0002572 is a viral particle overexpressing hsa_circ_0002572; the viral particle is capable of transducing mammalian cells and expressing hsa_circ_0002572 in mammalian cells; the viral particle is an AAV particle.

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