Application of plasma circRNA biomarker in diagnosis and treatment of cranial premature closure

Abstract

The invention discloses application of a plasma circRNA (Ribonucleic Acid) biomarker in diagnosis and treatment of cranial premature closure. The marker hascirc0002572 provided by the invention can be used for effectively diagnosing the premature closure of the cranial suture, and meanwhile, an AAV vector of overexpressing the hascirc0002572 can be subcutaneously injected into the premature closure part of the cranial suture to inhibit the premature closure of the cranial suture, so that the effect of early treatment of the premature closure of the cranial suture is achieved. The plasma circRNA biomarker provided by the invention provides a new thought for diagnosis and treatment targets of the early closure of the cranium, and has a wide application prospect.

Description

Technical Field

[0001] The present 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 Art

[0002] Craniosynostosis is the second most common craniofacial abnormality. Premature skull closure can cause skull deformity and restrict brain growth. In severe cases, it can lead to abnormal skull growth, increased intracranial pressure, delayed brain development, and impaired cognitive function. About 15-30% of cases are accompanied by other clinical symptoms, which may include hand and foot deformities, bone and heart defects, etc. Currently, the main treatment for craniosynostosis is surgical treatment, but the surgery is traumatic and has a high risk. Therefore, it is particularly important to find non-invasive, safe and more effective treatments for the early diagnosis and treatment of craniosynostosis.

[0003] With the development of RNA high-throughput sequencing technology and the advancement of biotechnology, it has been found that many non-coding RNAs perform multiple biological functions in the human body and are involved in the occurrence and development of multiple diseases. CircRNA is a mysterious subclass of endogenous long non-coding RNA that regulates genes at the transcriptional or post-transcriptional level. Due to their closed-loop structure, circular RNAs are able to escape degradation by nucleases and are much more stable than linear RNAs. With the advancement of detection technology, the detection of circRNAs has become more feasible, which provides the possibility for their application in the diagnosis and treatment of craniosynostosis. 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 mechanism of action of circRNAs in craniosynostosis and developing diagnostic and therapeutic tools based on circRNAs are of great significance for improving the early diagnosis and treatment capabilities of craniosynostosis. Summary of the invention

[0004] The purpose of the present invention is to provide a plasma circRNA biomarker for use in the diagnosis and treatment of craniosynostosis based on the above-mentioned deficiencies in the prior art.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A plasma circRNA biomarker for 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] Use of a reagent for detecting the expression amount of the plasma circRNA biomarker in the preparation of an auxiliary diagnostic reagent for craniosynostosis.

[0008] As a preferred embodiment of the present invention, the reagent for detecting the expression amount of the plasma circRNA biomarker according to claim 1 is the specific detection primer of 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] Use of hsa_circ_0002572 or a substance overexpressing hsa_circ_0002572 in the preparation of a drug for treating 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] As 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, especially the AAV particle, can transduce mammalian cells and express hsa_circ_0002572 in mammalian cells. The AAV particle comprises a nucleic acid inserted between a pair of AAV inverted terminal repeat sequences (ITRs), and the nucleic acid encodes hsa_circ_0002572.

[0013] Virus particles overexpressing hsa_circ_0002572 can be prepared using conventional technical means in the art, or can be commissioned to a biological company for preparation, as long as hsa_circ_0002572 can be overexpressed in mammals.

[0014] A plurality of the AAV particles are subcutaneously administered to a mammal in need thereof at the site of craniosynostosis to treat craniosynostosis.

[0015] A pharmaceutical composition for treating craniosynostosis, comprising 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 comprises preparing the virus particles overexpressing hsa_circ_0002572 as conventional pharmaceutical excipients for pharmaceutical preparations.

[0017] A craniosynostosis auxiliary diagnosis kit comprises a reagent for detecting the expression amount of the plasma circRNA biomarker, preferably comprising the specific detection primers of hsa_circ_0002572 and primers for detecting an internal reference gene, and further preferably comprising the 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 the present invention can effectively diagnose craniosynostosis, and can inhibit the differentiation of mesenchymal stem cells into osteoblasts by overexpressing hsa_circ_0002572, inhibit premature closure of craniosynostosis, and achieve the effect of early diagnosis and treatment of craniosynostosis. The hsa_circ_0002572 blood marker provided by the present invention provides a new idea for the diagnosis and treatment of craniosynostosis and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 3. Differential expression of circRNAs in the plasma of patients with craniosynostosis. (A) Volcano plot of differentially expressed circRNAs; (B) Expression profile of abnormally low-expressed circRNAs with human and mouse homology.

[0022] Figure 2 It is the dynamic expression of circRNA during the 1st and 7th days of osteogenic differentiation of mesenchymal stem cells.

[0023] Figure 3 The effects of siRNA of has_circ_0002572 and hsa_circ_0008616 on osteogenic differentiation of hMSCs.

[0024] Figure 4 This is the expression of has_circ_0002572 in the cranial sutures of craniosynostosis mice and wild-type mice.

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

[0026] Figure 6AAV that overexpresses has_circ_0002572 in vivo is used to treat craniosynostosis mice. (A) qRT-PCR experiments confirmed that the AAV indeed overexpressed has_circ_0002572 in mice. (B) The therapeutic effect of the AAV on craniosynostosis mice. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the embodiments and drawings.

[0028] Unless otherwise specified, the conditions and methods used in the implementation process are carried out in a conventional manner. The test materials used are all commercially available common products unless otherwise specified.

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

[0030] 1.1 Sample collection

[0031] This study collected blood samples from children with craniosynostosis and normal controls who met the requirements at Nanjing Children's Hospital. After sorting out the data, 5 healthy controls and 5 children with craniosynostosis who met the requirements were selected as experimental subjects. The guardians of all research subjects signed informed consent.

[0032] 1.2 Extraction of plasma RNA

[0033] RNA was extracted from each serum sample using an RNA extraction kit, and the operation steps were carried out according to the instructions.

[0034] 1.3 Screening of circRNA expression profiles

[0035] We commissioned Shanghai Kangcheng Bioengineering Co., Ltd. to establish the expression profile of circRNA using the Human circular RNAArray V2.0 chip from Arraystar and screen out circRNAs with differences. Significant differences were defined as |log2FC|>1.5 and FDR≤0.05.

[0036] 1.4 Experimental Results

[0037] like Figure 1 As shown in Figure 2, a total of 134 dysregulated circRNAs were identified, of which 74 circRNAs were upregulated and 60 circRNAs were downregulated ( Figure 1 A). Among the 60 circRNAs downregulated, 12 circRNAs had human and mouse homology ( Figure 1 B).

[0038] Example 2: Screening of 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) in a 37°C cell culture incubator containing 5% CO2. hMSCs were seeded in a 6-well plate, and the medium was replaced with osteogenic induction medium after 24 hours. After different induction times, the cells were collected.

[0041] 2.2qRT-PCR

[0042] Total RNA was extracted from cells using TRIzol reagent according to the instructions. An aliquot of 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 LightCycler 480. The primers for the specific transcripts of the 12 circRNAs with human and 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 and mouse homology, only hsa_circ_0008616 and hsa_circ_0002572 decreased with the increase of osteogenic differentiation time when hMSCs were induced to differentiate into osteoblasts at different times.

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

[0049] 3.1 Cell culture and transfection

[0050] hMSCs were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) in a 37°C cell culture incubator containing 5% CO2. hMSCs were seeded in 6- or 12-well plates, and after the cell confluence reached 40%, they were transfected with siRNA knockdown of has_circ_0002572 and hsa_circ_0008616, respectively. After 24 hours, the prepared osteogenic induction solution was replaced for induction, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2, with the solution changed every two days. ALP staining was performed after 7 days of culture, and Alizarin red staining was performed after 14 days of culture to detect the effects of different treatments on the osteogenic differentiation of hMSCs.

[0051] 3.2 Experimental Results

[0052] like Figure 3 As shown in the results, it was found that the mineralization level of cells knocked down by hsa_circ_0002572 was significantly increased, and the cellular alkaline phosphatase activity was enhanced.

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

[0054] 4.1 Collecting samples

[0055] Female C57BL / 6J mice and Twist+ / - mice aged 6–8 weeks were purchased from Jicui Yaokang (Jiangsu, China). WT female mice were mated with Twist+ / - male mice, and after genotyping of the offspring mice, WT mice and Twist+ / - mice at 9 days after birth were selected and collected for subsequent experiments. All animal procedures were performed in accordance with protocols approved by the Animal Care and Use Committee of Nanjing Medical University.

[0056] 4.2 Paraffin sections

[0057] Five skulls of WT mice and Twist+ / - mice were selected from 9-day-old mice and fixed in 4% paraformaldehyde for 48 hours. After decalcification of the bone tissue, 8-micron-thick paraffin sections were prepared.

[0058] 4.3 circRNA in situ hybridization

[0059] The Cy3-labeled has_circ_0002572 probe was designed and synthesized by Shanghai Genema Gene. 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 4Figure 2. Expression of has_circ_0002572 in wild-type mice and Twist+ / - craniosynostosis mice. has_circ_0002572 was significantly downregulated in the coronal suture of Twist+ / - mice compared with WT mice.

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

[0063] 5.1 Collecting samples

[0064] This study collected blood samples from children with craniosynostosis and normal controls who met the requirements at Nanjing Children's Hospital. After sorting out the data, 81 healthy controls and 67 children with craniosynostosis who met the requirements were selected as experimental subjects. The guardians of all research subjects signed informed consent.

[0065] 5.2qRT-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 mix and LightCycler 480.

[0067] The specific detection primer sequences of 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 sequence of the GAPDH internal reference primer 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] Use R's pROC analysis to draw the ROC curve and calculate its AUC area

[0075] 5.4 Experimental Results

[0076] like Figure 5As shown in A, hsa_circ_0002572 was downregulated in plasma samples of patients with craniosynostosis compared with healthy controls (P<0.001). Using hsa_circ_0002572 as the detection variable, the ROC curve was drawn and its AUC value was calculated. The results are shown in 5B, showing that the AUC value was 0.885, the specificity of the optimal critical point was 0.801, and the sensitivity was 0.851, indicating that the application of hsa_circ_0002572 in the diagnosis of craniosynostosis has a good diagnostic efficacy.

[0077] Example 6: AAV overexpressing has_circ_0002572 in vivo and evaluating its therapeutic effect on craniosynostosis mice.

[0078] 6.1 Collecting samples

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

[0080] 6.2 Subcutaneous injection of AAV overexpressing has_circ_0002572

[0081] AAV targeting overexpression of has_circ_0002572 was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. On the first and fifth days after birth of the offspring Twist+ / - mice, AAV-oe-circ0002572 and AAV-oe-Vector were injected into the coronal suture of Twist1+ / - mice by microinjection to observe the development of the coronal suture.

[0082] 6.3 micro-CT

[0083] After euthanizing the mice with CO2, the skulls were peeled 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] The skulls of WT and Twist+ / - mice were collected 12 days after surgery and fixed in 4% paraformaldehyde for 48 hours. After decalcification of the bone tissue, 8-micron thick paraffin sections were made. Then, HE staining kit (Biyuntian, C0105S) was used for staining. Images were acquired and analyzed using a pathological section scanner.

[0086] 6.5qRT-PCR

[0087] The skulls of Twist+ / - mice treated with AAV-oe-circ0002572 and AAV-oe-Vector were collected 12 days after surgery, and total RNA was extracted from the cranial suture tissue using TRIzol reagent according to the instructions. An aliquot of 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 LightCycler 480. The 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 the AAV indeed overexpressed has_circ_0002572 in mice ( Figure 6 A). Micro-CT and histological analysis results showed that AAV-oe-circ0002572 treatment significantly reduced Twist1 expression compared with AAV-oe-Vector treatment. + / - Premature closure of the coronal suture in mice Figure 6 B). The above results suggest that the application of hsa_circ_0002572 in the early treatment of craniosynostosis has a better therapeutic effect.

Claims

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

1.

2. Use of a reagent for detecting the expression of the plasma circRNA biomarker according to claim 1 in the preparation of an auxiliary diagnostic reagent for craniosynostosis.

3. The use according to claim 2, characterized in that: The reagent for detecting the expression amount of the plasma circRNA biomarker according to claim 1 is the specific detection primer of hsa_circ_0002572.

4. The use according to claim 2, characterized in that: The specific detection primer sequences of hsa_circ_0002572 are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

5. Use of hsa_circ_0002572 or substances overexpressing hsa_circ_0002572 in the preparation of drugs for the treatment of craniosynostosis.

6. The use according to claim 5, characterized in that: The substance overexpressing hsa_circ_0002572 is a recombinant expression vector overexpressing hsa_circ_0002572.

7. The use according to claim 5, characterized in that: The substance overexpressing hsa_circ_0002572 is a virus particle overexpressing hsa_circ_0002572; the virus particle is capable of transducing mammalian cells and expressing hsa_circ_0002572 in mammalian cells; the virus particle is preferably an AAV particle.

8. A pharmaceutical composition for treating craniosynostosis, characterized in that: A viral particle overexpressing hsa_circ_0002572, preferably an AAV particle overexpressing hsa_circ_0002572.

9. The pharmaceutical composition according to claim 9, characterized in that It also includes preparing the hsa_circ_0002572-overexpressing virus particles into conventional pharmaceutical excipients for pharmaceutical preparations.

10. A craniosynostosis auxiliary diagnosis kit, characterized in that: A reagent for detecting the expression amount of the plasma circRNA biomarker according to claim 1, preferably comprising a specific detection primer for hsa_circ_0002572 and a primer for detecting an internal reference gene, and further preferably comprising the primers shown in SEQ ID NO: 2-5.

Images