Use of circcasc15 in neuroblastoma treatment and diagnosis

Abstract

The application belongs to the field of biological medicine, and relates to the use of circCASC15 in the preparation of a drug for preventing and / or treating neuroblastoma and the preparation of a reagent for diagnosing neuroblastoma, wherein circCASC15 is circCASC15-1 and / or circCASC15-2, the sequence of circCASC15-1 is SEQ ID NO:1, and the sequence of circCASC15-2 is SEQ ID NO:2. By promoting the expression of circCASC15, the proliferation and migration of neuroblastoma cells are significantly inhibited. In addition, the expression of circCASC15-1 and circCASC15-2 is reduced in neuroblastoma cell lines and neuroblastoma patient tissues, and therefore the expression level of circCASC15-1 and circCASC15-2 can be used for the diagnosis of neuroblastoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the use of circCASC15 in the treatment and diagnosis of neuroblastoma, specifically the use of circCASC15 in the prevention and / or treatment of neuroblastoma and in the diagnosis of neuroblastoma. Background Technology

[0002] Neuroblastoma (NB) is the most common solid tumor in children under one year old, originating from the sympathetic nervous system. Neuroblastoma exhibits clinical and biological heterogeneity, including local recurrence and distant metastasis, spontaneous regression, and varying treatment responses. While low- and intermediate-risk NB can be cured with a combination of chemotherapy and surgical resection, the prognosis for high-risk (HR) NB remains poor, with a survival rate of less than 50% despite aggressive treatment. MYCN Amplification is also common in NB patients and contributes to a significant number of pediatric cancer-related deaths. Therefore, there is an urgent need to explore new treatment modalities to improve the prognosis of NB patients and address the challenges associated with their treatment.

[0003] New treatment options are urgently needed for neonatal non-coding (NB), and circular RNAs (circRNAs) show great potential in the diagnosis and therapeutic targeting of various cancers. Nucleic acid-based therapies are still in their early stages of potential NB treatment research. Circular RNAs are a unique class of endogenous non-coding RNAs characterized by the formation of a covalently circular structure through a non-canonical 3' to 5' end-joining process (i.e., backsplicing). Circular RNAs have shown great potential as molecular targets for tumor therapy. First, circular RNAs can effectively induce the expression of innate immune genes, providing protection against viral infections (BoothBJ, Nourreddine S, Katrekar D, Savva Y, Bose D, Long TJ). , et al. RNA editing:expanding the potential of RNA therapeutics. Mol Ther 2023;31:1533–49). Notably, circRNA has been explored for use as a SARS-CoV-2 vaccine in mice and macaques (Qu L, Yi Z, Shen Y, Lin L, Chen F, Xu Y). , et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell2022;185:1728–44.e16. Furthermore, nanoformulation delivery of circRNA can improve translation efficiency in cells and mouse adipose tissue (Liu X, Zhang Y, Zhou S, Dain L, Mei L, Zhu G. Circular RNA: an emerging frontier in RNA therapeutic targets, RNAtherapeutics, and mRNA vaccines). J Control Release (2022;348:84–94). However, very few tumor suppressor circRNAs have been discovered to date.

[0004] Summary of the Invention In order to provide a new method for treating and diagnosing neuroblastoma, in one aspect, the present invention provides the use of circCASC15 in the preparation of a medicament for the prevention and / or treatment of neuroblastoma, wherein circCASC15 is circCASC15-1 and / or circCASC15-2, wherein the sequence of circCASC15-1 is SEQ ID NO:1 and the sequence of circCASC15-2 is SEQ ID NO:2.

[0005] Preferably, circCASC15 is circCASC15-2, and the sequence of circCASC15-2 is SEQ ID NO:2.

[0006] Preferably, the drug includes a reagent that promotes circCASC15 expression.

[0007] More preferably, the reagent that promotes circCASC15 expression includes a circCASC15 overexpression construct.

[0008] Preferably, the vector for the circCASC15 overexpression construct is an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, or a retroviral vector.

[0009] Preferably, the drug is used in combination with a second drug.

[0010] More preferably, the second drug includes an mTOR pathway inhibitor.

[0011] More preferably, the mTOR pathway inhibitor includes rapamycin-type drugs.

[0012] More preferably, circCASC15-2 and everolimus are used in combination.

[0013] Preferably, the second drug includes CASC15-004.

[0014] On the other hand, the present invention provides the use of circCASC15 in the preparation of reagents for the diagnosis of neuroblastoma, characterized in that the circCASC15 is circCASC15-1 and / or circCASC15-2, the sequence of circCASC15-1 is SEQ ID NO:1, and the sequence of circCASC15-2 is SEQ ID NO:2.

[0015] Preferably, the reagent includes qPCR amplification primers for detecting the expression level of circCASC15, wherein the qPCR amplification primers for circCASC15-1 include the forward primer of SEQ ID NO:3 and the reverse primer of SEQ ID NO:4, and the qPCR amplification primers for circCASC15-2 include the forward primer of SEQ ID NO:5 and the reverse primer of SEQ ID NO:6.

[0016] This invention is the first to discover that circCASC15 expression is downregulated in neuroblastoma cells compared to normal cells. Overexpression of circCASC15 significantly inhibits the proliferation, migration, and invasion of neuroblastoma cells, while knockout of circCASC15 enhances these abilities. Furthermore, analysis of clinical and case characteristics of neuroblastoma patients revealed that, compared to L2 or M stage NB tumors, L1 / MS stage NB tumors showed higher levels of circCASC15-1 and circCASC15-2, and the expression of circCASC15-1 and circCASC15-2 was significantly higher in the non-HR group than in the HR group. MYCN The amplification status was associated with low expression of circCASC15-1 and circCASC15-2. In patients with overall survival (OS) and free sclerosis (EFS), high expression of circCASC15-1 and circCASC15-2 was associated with better prognosis. These results demonstrate the important role of circCASC15 in neuroblastoma and its promising application in gene therapy, providing new insights for the diagnosis and treatment of neuroblastoma patients. Furthermore, the circCASC15 of this invention can be administered locally to the lesion site, effectively reducing systemic toxicity and improving drug safety. Attached Figure Description

[0017] Figure 1 circCASC15-1 expression and INRGS staging ( Figure 1 A) INRG risk level ( Figure 1 B) and MYCN Amplification status ( Figure 1 The correlation between circCASC15-2 expression and INRGS staging (C) Figure 1 D), INRG risk level ( Figure 1 E) and MYCN Amplification status ( Figure 1 The correlation of F); circCASC15-1 ( Figure 1 G) and circCASC15-2 ( Figure 1 H) Kaplan-Meier (KM) plots of overall survival (OS) in NB patients with high and low expression; circCASC15-1 ( Figure 1 I) and circCASC15-2 ( Figure 1 J) EFS KM plots of NB patients in the high-expression and low-expression groups. *p<0.5, **p<0.1, ***p<0.01, ****p<0.001.

[0018] Figure 2 Schematic diagram of circRNA derived from lncRNA CASC15 ( Figure 2 AB). circCASC15-1 ( Figure 2 C) and circCASC15-2 ( Figure 2 Sanger sequencing results of the sequence following D). The circCASC15-1 nucleotide sequence in SH-SY5Y cells treated with RNase R (RNase R+) or untreated with RNase R (RNase R-) was quantified by RT-qPCR analysis. Figure 2 E) and circCASC15-2 ( Figure 2 The relative expression of F) was *p<0.5, **p<0.1, ***p<0.01, ****p<0.001. For SH-SY5Y cells ( Figure 2 G) and SK-N-BE(2) cells ( Figure 2 FISH analysis of circCASC15-1 and circCASC15-2 in H), where specific FISH probes for circCASC15-1, circCASC15-2, U6 and 18S were modified with Cy3, U6 was used as a nuclear marker, 18S was used for cytoplasmic localization, the nuclei were stained with DAPI, and the scale bar represents 10 μm.

[0019] Figure 3 : Knockdown groups in SK-N-BE(2) cells ( Figure 3A) and overexpression group ( Figure 3 The expression of circCASC15-1 in B) was observed; in each knockdown group of SK-N-BE(2) cells ( Figure 3 C) and overexpression group ( Figure 3 Expression of circCASC15-2 in D); knockdown groups in SH-SY5Y cells ( Figure 3 E) and overexpression group ( Figure 3 Expression of circCASC15-1 in F); knockdown groups in SH-SY5Y cells ( Figure 3 G) and overexpression group (G) Figure 3 Expression of circCASC15-2 in H). *p<0.5, **p<0.1, ***p<0.01, ****p<0.001.

[0020] Figure 4 RTCA assay was performed on SK-N-BE(2) cells to determine the knockdown of circCASC15-1 groups ( Figure 4 A) Knock down circCASC15-2 group ( Figure 4 B) and overexpression group ( Figure 4 C) proliferation status; RTCA assay of SK-N-BE(2) cells in each knockdown group of circCASC15-1 ( Figure 4 D) Each group of circCASC15-2 knockdowns ( Figure 4 E) and overexpression group ( Figure 4 The migration rate of F) was determined by colony formation assay in SK-N-BE(2) cells by knocking down circCASC15-1 groups ( Figure 4 G), each knockdown of circCASC15-2 groups ( Figure 4 H) Inhibition of SK-N-BE(2) cell colony formation ability (left panel, showing three replicates for each group), right panel shows the number of SK-N-BE(2) cell colonies; 4I: Colony formation assay to determine the effect of each overexpression group on SK-N-BE(2) cells (top panel, showing three replicates for each group), SK-N-BE(2) cell colony number is shown in the figure below. * P <0.5, ** P <0.1, *** P <0.01, **** P <0.001.

[0021] Figure 5 RTCA measurements were performed on each group of SH-SY5Y cells with knocked-down circCASC15-1. Figure 5 A) Each knockdown of circCASC15-2 groups ( Figure 5 B) and overexpression group ( Figure 5 C) proliferation status; RTCA assay of SH-SY5Y cells in each knockdown group of circCASC15-1 ( Figure 5 D) Each group of circCASC15-2 was knocked down ( Figure 5 E) and overexpression group ( Figure 5 The migration rate of F); the effect of circCASC15-1 overexpression group (OE-CASC15-1) and circCASC15-2 overexpression group (OE-CASC15-2) on the colony-forming ability of SH-SY5Y cells (F) Figure 5 G, where each group shows images of three replicates; colony numbers of SH-SY5Y cells ( Figure 5 H). *p<0.5, **p<0.1, ***p<0.01.

[0022] Figure 6 : A schematic diagram of establishing a NB model using NSG mice ( Figure 6 A); OE-circCASC15-1 group ( Figure 6 B) and OE-circCASC15-2 group ( Figure 6 C) Representative images of luciferase expression in primary xenograft tumors of mice 14 and 28 days after injection of the adenovirus construct, where NSG mouse #8 in the AD-NC-2 group died on day 27; OE-circCASC15-1 group (n=3 per group) Figure 6 D) and OE-circCASC15-2 groups (n=3 per group) Figure 6 Quantification of luciferase intensity (biofluorescence) in E) is given as mean ± SEM.

[0023] Figure 7 : RT-qPCR was used to measure the knockdown of si-circCASC15-2 groups in SK-N-BE(2) cells. Figure 7 A) and the overexpression group OE-circCASC15-2 group ( Figure 7 B) Relative expression levels of CASC15-003 and CASC15-004. Ns represents non-significant. *p<0.5, **p<0.1, ***p<0.01.

[0024] Figure 8 The overexpression group of CASC15-003 in SK-N-BE(2) cells was determined by RT-qPCR. Figure 8 A) and the CASC15-003 overexpression group in SH-SY5Y cells ( Figure 8 The expression level of CASC15-003 in C) was measured by RT-qPCR; the CASC15-004 overexpression group in SK-N-BE(2) cells was determined. Figure 8 B) and CASC15-004 overexpression group in SH-SY5Y cells ( Figure 8 The expression level of CASC15-004 in D) was detected; the proliferation of CASC15-003 overexpression group and CASC15-004 overexpression group in SH-SY5Y cells was detected by RTCA. Figure 8 E) and migration ( Figure 8 F). *p<0.5, **p<0.1, ***p<0.01.

[0025] Figure 9 : Proliferation of SK-N-BE(2) cells overexpressing CASC15-003 and CASC15-004 was detected by RTCA. Figure 9 A) and migration ( Figure 9 B); Figure 9 C shows the colony formation detection of the CASC15-003 overexpression group in SK-N-BE(2) cells (left, with three replicates shown for each group) and the number of cell colonies (right). Figure 9 D represents the colony formation detection of the CASC15-004 overexpression group in SK-N-BE(2) cells (left, with images of three replicates for each group shown) and the number of cell colonies (right). Ns indicates no significant difference. *p<0.5, **p<0.1, ***p<0.01.

[0026] Figure 10 Expression levels of Ki-67, Akt, pAkt S473 and mTOR were determined by IHC staining and analysis.

[0027] Figure 11 : Figure 11 A represents the high-throughput sequencing results of SH-SY5Y cells overexpressing circCASC15-2 and control samples; Figure 11 BD represents Western blot data of the CASC15-004 overexpression group (OE-004), the circCASC15-2 overexpression group (OE-circ2), and each circCASC15-2 knockdown group in SK-N-BE(2) cells. The Ladder in lane 1 of the figure is used to display the molecular weight of the proteins.

[0028] Figure 12KEGG pathway analysis results of SH-SY5Y cells overexpressing circCASC15-2 revealed the potential functional pathways of circCASC15-2. Figure 12 A) and signal pathways ( Figure 12 B); GO annotations for upregulated target mRNAs of circCASC15-2 in the GO terminology category of biological processes (BP). Figure 12 C).

[0029] Figure 13 : SK-N-BE(2) cells CASC15-004 overexpression group (OE-004) ( Figure 13 A) and circCASC15-2 overexpression group (OE-circ2) Figure 13 B) Results of co-immunoprecipitation of Sin1 and other proteins. Immunoprecipitation (IP) was performed using anti-Sin1, anti-Rictor, and anti-GβL antibodies. IB analysis was performed on the samples using the specified antibodies. IgG was used as the NC. The Ladder in lane 1 of the figure is used to display the protein molecular weight. Figure 13 C: Schematic diagram of the inhibitory effect of CASC15-004 and circCASC15-2 on mTORC2 activity.

[0030] Figure 14 : SK-N-BE(2) The results of co-immunoprecipitation of Sin1 and other proteins in each knockdown group of circCASC15-2 cells. The Ladder in lane 1 of the figure is used to show the molecular weight of the proteins.

[0031] Figure 15 The proliferation rate of SK-N-BE(2) cells treated with circCASC15-2 and CASC15-004 in combination was determined by RTCA. Figure 15 A) The proliferation rate of SK-N-BE(2) cells treated with a combination of circCASC15-2 and 15µM everolimus ( Figure 15 B). Schematic diagram of the progress of circCASC15-2 in collaboration with CASC15-004 and everolimus in regulating NB by damaging mTORC2 signal transduction ( Figure 15 C).

[0032] Figure 16 Dose-response curves of SK-N-BE(2) cells (OE-circ) overexpressing circCASC15-2 and control SK-N-BE(2) cells (OE-NC) after treatment with different concentrations of everolimus.

[0033] In the above figures, si-circ1 1# or si-circ CASC15-1 1# represents cells transfected with ASO containing the sequence si-circ CASC15-1 1#; si-circ1 2# or si-circ CASC15-1 2# represents cells transfected with ASO containing the sequence si-circ CASC15-1 2#; si-circ1 3# or si-circ CASC15-1 3# represents cells transfected with ASO containing the sequence si-circ CASC15-1 3#; si-circ2 1# or si-circ CASC15-2 1# represents cells transfected with ASO containing the sequence si-circ CASC15-2 1#; si-circ2 2# or si-circ CASC15-2 2# represents cells transfected with ASO containing the sequence si-circ CASC15-2 2#; and si-circ2 3# or si-circ CASC15-2 3# indicates cells transfected with ASO containing the sequence si-circ CASC15-2 3#; OE-circ-1 indicates cells transfected with a circCASC15-1 overexpressing lentiviral construct; OE-circ-2 indicates cells transfected with a circCASC15-2 overexpressing lentiviral construct; si-NC represents the knockdown control group; OE-NC represents the overexpression control group. OE-CASC15-1 indicates mice injected with the circCASC15-1 overexpressing adenovirus construct; OE-CASC15-2 indicates mice injected with the circCASC15-2 overexpressing adenovirus construct; OE-NC-1 and OE-NC-2 represent the control groups injected with empty AAV vector. OE-003 or OE-CASC15-003 represents the CASC15-003 overexpression group; OE-004 or OE-CASC15-004 represents the CASC15-004 overexpression group. Detailed Implementation

[0034] To make the technical solutions and effects of the present invention clearer, the present invention is further described below through embodiments. It should be understood that these are merely exemplary and not intended to limit the present invention. Materials similar to or the same as the types and models, properties, or functions of the reagents and instruments described below can be used in the implementation of the present invention. Unless otherwise specified, the reagents used in the present invention can be any suitable commercially available reagent. The instruments and reagents used in the present invention are used according to the instructions of the instrument / reagent manufacturer. The experimental methods involved in the present invention are conventional experimental methods in the art, and those skilled in the art can operate them based on known biological and chemical knowledge. Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which this invention relates.

[0035] The term "circCASC15-1" as used in this invention refers to the generation of lncRNA CASC15 (LINC00340, ENSG00000272168) via a backsplicing event. circCASC15-1 consists of two exons, namely exons 7 and 8 (…). Figure 2 A), whose nucleotide sequence is shown in SEQ ID NO:1, and whose circBank ID in the public database circBank (http: / / www.circbank.cn / index.html) is circBank ID: hsa_circLINC00340_029.

[0036] .

[0037] The term "circCASC15-2" as used in this invention refers to the generation of lncRNA CASC15 (LINC00340, ENSG00000272168) via a backsplicing event. circCASC15-2 consists of three exons, namely exons 7, 8, and 9 (…). Figure 2 B), whose nucleotide sequence is shown in SEQ ID NO:2, and whose circBank ID in the public database circBank is circBank ID: hsa_circLINC00340_033.

[0038] .

[0039] The term "tumor" as used in this invention refers to any malignant or benign growth and proliferation of neoplasms, as well as all precancerous and cancerous cells and tissues. The term "neuroblastoma" (NB) is a disease in which malignant (cancer) cells form in the nerve tissue of the adrenal glands, neck, chest, or spinal cord. It is an embryonic tumor of the sympathetic nervous system that originates from neuroblasts (pluripotent sympathetic nerve cells). Neuroblastoma is described in the NCBI-indexed book (StatPearls, 2024) as the most common extracranial solid tumor in children and is classified as an embryonic neuroendocrine tumor (Matthay KK, Maris JM, Schleiermacher G, et al. Neuroblastoma[J]. Nature Reviews Disease Primers, 2016, 2(1): 16078). In the fifth edition of the WHO Classification of Childhood Cancers in 2022, neuroblastoma (NB) is classified as a subtype of peripheral neuroblastic tumors (pNTs). Peripheral neuroblastomas include three basic histological types: neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN). The International Committee on Neuroblastoma Pathology classifies peripheral neuroblastomas into four histopathological types based on the grading of neuroblast differentiation and the degree of Schwannian stromal development: NB (Schwannian stroma-poor); GNB (Schwannian stroma-rich); GN (Schwannian stroma-dominant); and GNB (including both Schwannian stroma-rich and stroma-dominant composite types). The first three types represent the maturation process of neuroblastoma, while the last type is polyclonal."Neuroblastoma" is a general term used to refer to all types of peripheral neuroblastic tumors. It originates from primitive neural crest cells, with primary sites distributed along the neural crest developmental pathway, including the sympathetic ganglia of the neck, chest, abdomen, adrenal glands, and pelvis. It is most common in children under 5 years old, especially infants under 2 years old, and is relatively rare in children over 10 years old. It is the most common tumor in children under 1 year old (Sarnat HB, Chan ES, Ng D, et al. Maturation of metastases in peripheral neuroblastic tumors (neuroblastoma) of children[J]. JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY, 2023, 82(10): 853-864; Chinese Anti-Cancer Association Pediatric Oncology Committee, Chinese Medical Association Pediatric Surgery Branch Oncology Group. Expert Consensus on the Diagnosis and Treatment of Neuroblastoma in Children CCCG-NB-2021[J]. Chinese Journal of Pediatric Surgery, 2022, 43(07):588-598).

[0040] Pathological diagnosis of neuroblastoma includes: initial symptoms are often atypical, making early diagnosis difficult. Common symptoms include fatigue, loss of appetite, fever, and joint pain. Symptoms caused by the tumor depend on its location in the organ and whether metastasis has occurred. Local histological diagnosis of the tumor is possible, such as through biopsy or excision. Thin fibrovascular septa form nest-like structures. Based on the degree of differentiation of neuroblastoma cells within the nests, it can be classified as undifferentiated, poorly differentiated, and differentiated. Undifferentiated tumors often require IHC and / or molecular testing for confirmation due to the uniform "small round blue cell" morphology and lack of identifiable neurofelt structures. Poorly differentiated tumors show neurofelt structures in the background, and may exhibit Homer-Wright rosette structures. Less than 5% of the neuroblastoma cells show differentiated morphological characteristics, and the tumor cell nuclei have a "salt and pepper" appearance; rare cell morphologies may also be present, such as large and pleomorphic, spindle-shaped, and pseudo-striated muscle-like cells. Differentiated tumors have abundant cellular maturation, with more than 5% of cells exhibiting differentiated morphology, meaning that the nucleus (enlarged, eccentric, with vesicular chromatin and a single large nucleolus) and cytoplasm (eosinophilic or amphophilic, with a diameter greater than twice that of the nucleus) differentiate simultaneously. Furthermore, in cases where imaging confirms the presence of a tumor, combined with metastatic manifestations, such as bone metastasis or bone marrow metastasis, evidence of bone marrow metastasis can also confirm the diagnosis.

[0041] The staging and grading of neuroblastoma include:

[0042] According to the International Neuroblastoma Risk Group Staging System (INRGSS), neuroblastoma can be divided into L1, L2, M and Ms stages. According to the International Neuroblastoma Risk Group (INRG), neuroblastoma patients can be divided into high-risk and non-high-risk groups.

[0043] Table 1:

[0044]

[0045] Table 2:

[0046]

[0047] Table 3:

[0048]

[0049] MYCN Amplified NB and MYCN Non-amplified NBs exhibit many differences in gene expression. MYCN It participates in the regulation of multiple genes and also regulates the expression of some microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Dysregulation of many genes also leads to… MYCN The amplification. MYCN The expanded NB cells exhibit prolonged eukaryotic transcription, more active expression, and stronger invasive capabilities. MYCN Gene amplification is associated with high-risk grouping and poor prognosis in neuroblastoma, and is accompanied by... MYCN In children with expanded neonatal nephropathy (NB), the primary tumor lesion is mainly located in the retroperitoneal adrenal region, with a high rate of early distant metastasis. More than 50% of these children experience tumor progression during maintenance therapy, with a 3-year overall survival (OS) of only 19.7%. Approximately 25% of NB patients have [a condition that is not explicitly stated in the original text]. MYCN Gene amplification, accompanied by MYCN Children with amplified neonatal nephropathy (NB) urgently need new treatment methods such as targeted therapy to improve efficacy and prognosis (Zhao Qian et al., with...). MYCN A case series report of 133 cases of neuroblastoma with gene amplification, Chinese Journal of Evidence-Based Pediatrics, 2022, Vol. 17 Issue (3): 215-219. The term "treatment" as used in this invention includes relieving and / or eliminating symptoms associated with a particular disease or condition.

[0050] The term "prevention" as used in this invention refers to the treatment taken before the onset of a disease to avoid, minimize, or prevent the disease from developing or progressing.

[0051] As used in this invention, the term "diagnosis" refers to the detection and / or identification of a subject's case status, and the identification and / or determination of whether the subject suffers from a particular disease.

[0052] The term “combined use” or “combined drug use” as used in this invention includes the simultaneous, sequential, or alternating use of two or more drugs or drug components, including preparing two or more drugs or drug components into a pharmaceutical product present in one or more dosage units to obtain a suitable pharmaceutical product for combined use, and administering the pharmaceutical product to a mammal requiring combined use.

[0053] As used in this invention, the term "construction" refers to a recombinant gene molecule comprising one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule, and includes any construct containing: (1) nucleic acid sequences comprising regulatory and coding sequences not found together in nature (i.e., at least one nucleotide sequence is heterologous relative to at least one other nucleotide sequence); or (2) sequences encoding non-naturally adjacent functional RNA molecules or protein portions; or (3) non-naturally adjacent promoter portions. Representative constructs include any recombinant nucleic acid molecule, such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules derived from any source, capable of genome integration or autonomous replication, comprising nucleic acid molecules in which one or more nucleic acid molecules have been operatively linked. Constructs of this invention typically include essential elements for guiding the expression of a nucleic acid sequence of interest (e.g., a target nucleic acid sequence or a regulatory nucleic acid sequence) also contained in the construct. Such elements may include regulatory elements or regulatory sequences, such as a promoter operatively linked to a nucleic acid sequence of interest (to direct transcription of that nucleic acid sequence), and often also include a polyadenylation sequence. In some embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may also include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome. Two or more constructs may be contained in a single nucleic acid molecule, such as a single vector, or may be contained in two or more separate nucleic acid molecules, such as two or more separate vectors. An “expression construct” typically includes at least one regulatory sequence operatively linked to a nucleotide sequence of interest. In this way, for example, a promoter operatively linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in an organism or a portion thereof, including a host cell. For the purpose of carrying out this invention, conventional compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, Volumes 1, 2 and 3, J.F. S.A. S.B., D. W. S.S., and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0054] The term "mTOR pathway" as used in this invention refers to a signaling pathway that plays a crucial role in key cellular processes such as cell growth, protein translation, autophagy, angiogenesis, and cell metabolism. The mTOR pathway consists of two forms: mTORC1 and mTORC2, which include different protein components and exhibit different functions. mTORC1, in addition to mTOR and GβL, includes Raptor, which can be activated by phosphorylation of Akt at threonine 308 (T308). mTORC1 promotes metabolism-related growth. mTORC2, in addition to mTOR and GβL, includes Rictor and Sin1, and is considered a major kinase of Akt S473 substrates. mTORC2 regulates cell proliferation and survival by activating Akt.

[0055] The term "rapamycin class drugs" as used in this invention refers to rapamycin and its derivatives. Rapamycin and its derivatives share the same parent nucleus structure (as shown below), and are typically modified by altering the R group. Examples include temsirolimus, everolimus, ridaforolimus, umirolimus, and zotarolimus. Temsirolimus is a prodrug obtained by esterifying the 42-OH group of rapamycin with 2,2-dimethylolpropionate. Everolimus is obtained by etherifying rapamycin with ethylene glycol. Ridaforolimus is obtained by esterifying rapamycin with dimethylphosphonic acid. Umirolimus is a highly lipophilic rapamycin derivative; zotarolimus is obtained by replacing the 42-hydroxyl group of rapamycin with a tetrazolium ring. Rapamycin was the first marketed mTOR inhibitor, mediating its antiproliferative function by forming a complex with peptidyl-prolyl isomerase (PPIase) FKBP12. FKBP12 is a ubiquitous protein that acts as a receptor for immunosuppressive drugs. The mechanism of action of these inhibitors begins with the formation of a complex with FKBP12, which then binds to the mTOR FRB domain, inducing a conformational change that inhibits mTORC1 kinase activity. mTOR inhibition means blocking protein synthesis and cell growth, and amplifying autophagy by promoting tumor regression. Rapamycin and its derivatives inhibit mTORC1 activity through direct interactions with the components of mTORC1 (mTOR, GβL, and Raptor). However, long-term use of rapamycin-like drugs can modulate the expression of Akt, a downstream effector of mTORC2, leading to drug resistance.

[0056]

[0057] As used in this invention, the term "and / or" refers to and covers any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items can be used alone, or any combination of two or more listed items can be used. For example, A and / or B means A alone, B alone, and combinations of A and B.

[0058] One embodiment of the present invention provides the use of circCASC15 in the preparation of a medicament for the prevention and / or treatment of neuroblastoma, wherein the circCASC15 is circCASC15-1 and / or circCASC15-2, the sequence of circCASC15-1 is SEQ ID NO:1, and the sequence of circCASC15-2 is SEQ ID NO:2. In a preferred embodiment, the circCASC15 is circCASC15-2, and the sequence of circCASC15-2 is SEQ ID NO:2. In a preferred embodiment, the medicament comprises an agent that promotes circCASC15 expression. The agent that promotes circCASC15 expression can be any agent known in the art for promoting the expression of a target circular RNA, and in a preferred embodiment, the agent that promotes circCASC15 expression comprises a circCASC15 overexpression construct. In a preferred embodiment, the vector of the circCASC15 overexpression construct is an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, or a retroviral vector.

[0059] In a preferred embodiment, the medicament of the present invention can be used in combination with a second medicament. The second medicament refers to any medicament other than the medicament of the present invention that can be used for the prevention and / or treatment of neuroblastoma. The second medicament can be used simultaneously, sequentially, or alternately with the medicament of the present invention. When used simultaneously, the second medicament can be prepared in one unit or different units with the medicament of the present invention, without particular limitation. In a preferred embodiment, the second medicament comprises an mTOR pathway inhibitor. In a preferred embodiment, the mTOR pathway inhibitor is a rapamycin class drug. Rapamycin class drugs include rapamycin and its derivatives, wherein everolimus is a selective mTORC1 inhibitor that has been widely used in recent years to treat adult tumors. In a preferred embodiment, circCASC15-2 and everolimus are used in combination. In a preferred embodiment, the second medicament comprises CASC15-004. The nucleotide sequence of CASC15-004 is as follows:

[0060]

[0061] Another embodiment of the present invention provides the use of circCASC15 in the preparation of a reagent for diagnosing neuroblastoma, wherein circCASC15 is circCASC15-1 and / or circCASC15-2, the sequence of circCASC15-1 is SEQ ID NO:1, and the sequence of circCASC15-2 is SEQ ID NO:2. In a preferred embodiment, the reagent includes qPCR amplification primers for detecting circCASC15 expression levels, the qPCR amplification primers for circCASC15-1 including a forward primer of SEQ ID NO:3 and a reverse primer of SEQ ID NO:4, and the qPCR amplification primers for circCASC15-2 including a forward primer of SEQ ID NO:5 and a reverse primer of SEQ ID NO:6.

[0062] In the following embodiments of the invention, all data are expressed as mean ± standard error of mean (SEM) of at least three independent experiments, with each data point representing three measurements. Statistical analysis was performed using IBM SPSS software (version 2.0), and graphs were generated using Prism Graph Pad 8 for Windows. Student's t-test was used to compare the significance of differences between groups. Pearson's correlation coefficient was calculated to determine the correlation between gene expression levels. Kaplan-Meier method was used to assess overall survival (OS) and event-free survival (EFS). Groups were categorized according to the median expression levels of circCASC15-1 and circCASC15-2. A p-value less than 0.05 was considered statistically significant.

[0063] Example 1: Expression of circCASC15-1 and circCASC15-2 in NB clinical tissue samples

[0064] Patients and Samples

[0065] The 93 patients in this invention were all diagnosed with neuroblastoma (NB) using the International Neuroblastoma Pathological Classification (INPC) system at Beijing Children's Hospital, Capital Medical University, between January 2015 and June 2018. All samples underwent rigorous quality control. A total of 94 tumor samples were collected from the 93 NB patients. One patient had NB in ​​two different locations. All tumor samples were flash-frozen in liquid nitrogen within 30 minutes post-surgery. All NB samples were staged according to the International Neuroblastoma Risk Group Staging System (INRGSS) and microscopically evaluated by blinded experts. The tumor samples included 15 L1 stage samples, 27 L2 stage samples, 49 M stage samples, and 3 MS stage samples. Risk stratification was performed according to the International Neuroblastoma Risk Group (INRG) classification system. The tumor samples included 21 non-HR samples (including low-risk and intermediate-risk samples), 47 HR samples, and 26 unknown samples. Table 4 lists the patients' age, sex, and... MYCN Clinical information such as gene amplification status was collected. Follow-up of enrolled children continued until March 2023. Informed consent was obtained from all participants or their parents. This study was approved by the Ethics Committee of Beijing Children's Hospital (2020-K-59).

[0066]

[0067] RNA extraction, reverse transcription, and RT-qPCR analysis

[0068] Total RNA was isolated from the 94 tumor samples using TRIzol (15596018, Invitrogen). 30-50 mg of tissue was taken, 500 µl of TRIzol was added, and the mixture was homogenized on ice until no solid particles remained. The homogenized tissue solution was transferred to a new 1.5 mL EP tube. Another 500 µl of Trizol was added, and the mixture was thoroughly mixed by pipetting. The mixture was allowed to stand at room temperature for 5 min, then transferred to a 1.5 mL EP tube. 0.2 mL of chloroform (Beijing Tongguang Fine Chemical Co., Ltd.) was added to each 1.5 mL EP tube, and the mixture was vigorously shaken for 15 s, allowed to stand for 5 min, and then centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant was collected in a new EP tube. An equal volume of isopropanol was added, and the mixture was gently mixed by inverting the tube. The mixture was allowed to stand at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4℃. A white precipitate will appear at the bottom. Discard the supernatant, add 1 ml of 75% ethanol, wash the RNA once, centrifuge at 7500 rpm for 5 min at 4℃, add 30-50 µL of DEPC water, and after thorough dissolution, use a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) to assess the concentration and purity of the RNA sample. Generally, an A260 / A280 ratio between 1.8 and 2.2 is considered a preliminary indication of good total RNA quality. Perform reverse transcription immediately, and store the remainder at -80℃.

[0069] Then, using RT Master Mix (RR036A, TaKaRa) and random hexamer, 1 µg of extracted RNA was reverse transcribed into cDNA according to the manufacturer's instructions. CircCASC15-1 and circCASC15-2 were quantified using RT-qPCR (real-time quantitative PCR instrument, VIIA7 Dx) with a SYBR Green Supermix (#1725122, Bio-Rad). The following reaction conditions were used according to the manufacturer's instructions: 40 cycles of 95°C for 10 min, 95°C for 15 sec, and 60°C for 1 min. GAPDH was used as an internal control gene. Each experiment was performed independently in triplicate. Relative expression levels were calculated using 2-1. -ΔΔCTThe calculation method is as follows. The circCASC15-1 qPCR amplification primers include the forward primer circCASC15-1 Divergent F and the reverse primer circCASC15-1 Divergent R. The circCASC15-2 qPCR amplification primers include the forward primer circCASC15-2 Divergent F and the reverse primer circCASC15-2 Divergent R. The GAPDH qPCR amplification primers include the forward primer GAPDH F and the reverse primer GAPDH R. The specific sequences of the above primers are as follows:

[0070]

[0071] result

[0072] The above experimental results showed that, compared with L2 or M stage NB tumors, the levels of circCASC15-1 and circCASC15-2 were higher in L1 / MS stage NB tumors (P<0.01) (Figures 1A and 1D). The expression of circCASC15-1 and circCASC15-2 in the non-HR group was significantly higher than that in the HR group (P<0.001) (Figures 1B and 1E). MYCN Amplification status was associated with low expression of circCASC15-1 and circCASC15-2 (P<0.01). Figure 1 Low expression of circCASC15-1 and circCASC15-2 was associated with M-stage tumors, HR, and MYCN Amplification-related.

[0073] The samples were divided into high-expression and low-expression groups based on the median expression values ​​of circCASC15-1 and circCASC15-2. Kaplan-Meier (KM) plots showed that for patients in the OS group (Figures 1G and 1H) and EFS group (Figures 1I and 1J), high expression of both circCASC15-1 and circCASC15-2 was associated with better prognosis, while low expression was associated with poor prognosis. In summary, both circCASC15-1 and circCASC15-2 were expressed in clinical samples from NB patients. High expression of both circCASC15-1 and circCASC15-2 was associated with a good prognosis in NB patients.

[0074] Example 2: The structures of circCASC15-1 and circCASC15-2

[0075] Cell culture

[0076] The SH-SY5Y and SK-N-BE(2) human NB cell lines used in this embodiment and the following embodiments were purchased from the American Type Culture Collection (ATCC). All cell lines were negative for mycoplasma contamination by STR mapping. Cells were cultured in Duchenne modified Eagle medium (DMEM; 10-013-CV, Corning) supplemented with 10% fetal bovine serum (FBS; #35-081-CV, Corning) (hereinafter referred to as "medium 1"). CHLA255 cells obtained from UCLA were cultured in Iscove modified DMEM (10-016-CV, Corning) supplemented with 10% FBS (hereinafter referred to as "medium 2"). All cells were cultured in a humid environment of 5% CO2 and 37°C. SK-N-BE(2) was... MYCN Expanded cells (Peet AC, McConville C, Wilson M, et al. 1H MRS identify specific metabolite profiles associated with) MYCN -amplified andnon-amplified tumour subtypes of neuroblastoma cell lines[J]. NMR inbiomedicine, 2007, 20(7): 692-700; Farooqi AS, Dagg RA, Choi LMR, et al.Alternative lengthening of telomeres in neuroblastoma cell lines is associated with a lack of MYCN genomic amplification and with p53 pathway aberrations[J]. Journal of Neuro-Oncology, 2014, 119(1): 17-26). In November 1972, the SK-N-BE(2) neuroblastoma cell line was established from bone marrow aspiration of a child with diffuse neuroblastoma who had undergone multiple chemotherapy and radiotherapy. The cells showed moderate levels of dopamine-β-hydroxylase activity.

[0077] Sanger sequencing

[0078] Total RNA was extracted from SH-SY5Y cells according to the method in Example 1. One well of a six-well plate containing normal cultured cells was added to 1 ml of TRIzol and incubated at room temperature for 5 min. Subsequent RNA extraction steps were the same as those for tissue RNA extraction. circCASC15-1 and circCASC15-2 were each reverse transcribed into cDNA, which was then used as a template for RT-qPCR amplification. The RT-qPCR products were extracted and purified using a Wizard SV Gel and PCR Clean-Up System (REF A9281, Promega). The RT-qPCR amplicons were cloned and inserted into the pEASY-T1 vector (CT101, TransGenBiotech) for Sanger sequencing (sequencing was performed by Beijing Liuhe BGI Genomics Co., Ltd.).

[0079] The specific steps for cloning the RT-qPCR amplicon and inserting it into the pEASY-T1 vector are as follows:

[0080] 1. The cloning reaction system consists of 0.5-4 μl of PCR product and 2 μl of pEASY-T1 vector. The reaction temperature is 25℃ for 10 min, and the mixture is placed on ice after the reaction. The specific volume of the PCR product is calculated as follows: the PCR product concentration (ng / μl) is determined using a NanoDrop 8000 spectrophotometer (Thermo, catalog number ND-8000-GL). The mass of the PCR product is calculated according to the ratio of "1kb 20ng" in the PCR instrument manual. For example, if the qPCR product of circCASC15-2 is 96bp, and we calculate it as 100bp, then the mass of the PCR product is 2ng. Dividing the mass by the concentration of the PCR product gives the volume of the PCR product. Common methods in the art can be used to dilute or concentrate the PCR product to maintain the PCR product volume within the range of 0.5-4 μl.

[0081] 2. Transformation: Add the ligation product from step 1 to 50 µl of Transl-T1 competent cells (Transgold, CD501-02), gently swirl to mix, and incubate on ice for 20-30 min; heat shock at 42°C for 30 s, then immediately place on ice for 2 min; add 250 µl of LB liquid medium equilibrated to room temperature, and incubate at 37°C for 1 h at 200 rpm; take 8 µl of 500 Mm IPTG (GF101-01, Transgold) and 40 µl of 20 mg / ml X-gal (GF201-01, Transgold), and evenly spread the mixture on a prepared LB agar plate, incubate at 37°C for 30 min. After the IPTG and X-gal have been absorbed, take 200 µl of bacterial culture and evenly spread it on the plate, and incubate overnight at 37°C. To obtain a larger number of clones, centrifuge at 1500g for 1 min, discard part of the supernatant, retain 100-150µl, gently suspend the bacterial cells, and plate the entire bacterial suspension. The LB liquid medium was prepared as follows: 5g tryptone (OXOID, LP0137), 2.5g yeast extract (OXOID, LP0021B), and 5g sodium chloride, diluted to 500mL with autoclaved double-distilled water.

[0082] 3. On the second day, observe the LB plates and the growth of single colonies. Select the white single colonies as the target product. The LB plate is prepared by adding 7.5g of agarose (Sangon Biotech, A505255-0250) to LB liquid medium.

[0083] RNase R tolerance test

[0084] Total RNA was isolated from SH-SY5Y cells using TRIzol (15596018, Invitrogen). Total RNA (1 μg) was mixed with 3 U / μg ribonuclease R (RNase R, E0111-20D1, Lucigen) to degrade linear RNA; untreated RNA served as a control. RNA was incubated at 37°C for 25 min. Then, RNA was incubated at 70°C for 10 min. After RNase R inactivation, RNA was reverse transcribed into cDNA using the same method as in Example 1. The expression levels of circCASC15-1, circCASC15-2, and CASC15 mRNA were detected by RT-qPCR. GAPDH was used as an internal reference gene.

[0085] The primers for circCASC15-1 qPCR, circCASC15-2 qPCR, and GAPDH qPCR were the same as in Example 1. For the untreated RNA used as the circCASC15-1 control group, the qPCR primers included the forward primer circCASC15-1 Convergent F and the reverse primer circCASC15-1 Convergent R. For the untreated RNA used as the circCASC15-2 control group, the qPCR primers included the forward primer circCASC15-2 Convergent F and the reverse primer circCASC15-2 Convergent R. The specific sequences of the primers are as follows:

[0086]

[0087] Fluorescence in situ hybridization (FISH)

[0088] FISH assays were performed using a circRNA FISH assay kit (# C10910, RiboBio, Guangzhou, China). In short, cell slides (JingAn Biological, Shanghai, China) were placed in 24-well plates and cultured at 6 × 10⁶ wells. 4 NB cells were collected. Cells were then washed with phosphate-buffered saline (PBS; #KGB5001, KeyGen Biotech), fixed with 4% formaldehyde (#P1110, M&C Gene Technology) for 10 min, and permeabilized with 0.5% Triton X-100 (#T8787, Sigma) at 4°C for 5 min. Cells were then washed three times with PBS, pre-hybridized at 37°C for 30 min, and hybridized overnight at 37°C in the dark with U6 (lnc110101, RiboBio, Guangzhou, China), 18S (lnc110102, RiboBio, Guangzhou, China), circCASC15-1 (lnc1032281, RiboBio, Guangzhou, China) and circCASC15-2 (lnc1032135, RiboBio, Guangzhou, China) oligonucleotide probes. Cells were then washed according to standard procedures. Cells were then reverse stained with DAPI for 20 min. Finally, the cell slides were removed and imaged using a confocal laser scanning microscope (Leica SP8).

[0089] result

[0090] like Figure 2As shown in A and 2B, circCASC15-1 and circCASC15-2 are generated by lncRNA CASC15 (LINC00340, ENSG00000272168) through a backsplicing event. circCASC15-1 consists of exons 7 and 8, while circCASC15-2 includes exons 7-9. Upward arrows indicate splicing sites, solid triangles represent converging primers, and hollow triangles represent diverging primers. Figure 2 C and 2D were validated using Sanger sequencing of circCASC15-1 ( Figure 2 C) and circCASC15-2 ( Figure 2 The sequence following D). Figure 2 E and 2F show that the addition of RNase R exonuclease significantly reduced the content of linear CASC15 mRNA, while circCASC15-1 ( Figure 2 E) and circCASC15-2 ( Figure 2 The relative increase in F) showed no difference. This indicates that with the addition of RNase R exonuclease, the expression level of CASC15 mRNA decreased, while the expression levels of circCASC15-1 and circCASC15-2 did not decrease, confirming that circCASC15-1 and circCASC15-2 formed a closed loop structure. FISH results showed that circCASC15-1 and circCASC15-2 were mainly located in the cytoplasm and nucleus of the NB cell line (Figure 2G and H).

[0091] Example 3: circCASC15 inhibits the proliferation and migration of NB cells in vitro.

[0092] To observe the effect of circCASC15 on NB cell function, the inventors knocked down or upregulated the expression of circCASC15-1 and circCASC15-2 in SK-N-BE(2) cells and SH-SY5Y cells.

[0093] Construction and cell transfection of circCASC15 overexpression lentiviral construct and control lentiviral construct

[0094] The cDNA sequences of circCASC15-1 and circCASC15-2 were inserted into a lentiviral expression vector to construct circCASC15-1 overexpression lentiviral constructs and circCASC15-2 overexpression lentiviral constructs (empty lentiviral vector was used as a control lentiviral construct). The lentiviruses used were purchased from GeneChem (Shanghai, China). The circCASC15 overexpression lentiviral constructs and control lentiviral constructs were transfected into SK-N-BE(2) and SH-SY5Y NB cells, respectively, using HiTransGP infection enhancement medium (REVG005, GeneChem, Shanghai) at fold increases (MOI, MOI=20). Overexpression groups OE-circCASC15-1 and OE-circCASC15-2, and a control group OE-NC were obtained. The constructs in this example and the following examples were all constructed by GeneChem (Shanghai).

[0095] The specific steps for transfecting cells with lentiviral constructs are as follows: 1. One day before infection, transfect cells into 6-well cell culture plates at a rate of 5 × 10⁻⁶ cells / well. 5 1. Plant cells per well until the cell confluence reaches 40% and the cells are evenly distributed on the surface of each well, then infect the cells; 2. Replace the culture medium with 960 μl of fresh complete medium mixed with 40 μl of HitransGP infection enhancement solution (25×) 1 hour before virus infection; 3. Based on cell MOI=20 and virus titer (5×10⁻⁶), determine the appropriate viral load. 8 Add the appropriate amount of virus (TU / mL), calculated as follows: Virus volume = (MOI × cell number) / virus titer. After incubating at 37°C for 12 hours, replace with complete medium 1, adding 2 mL to each well for further incubation. The medium can be changed as needed to maintain cell viability. 4. Observe cell infection efficiency using a fluorescence microscope at 48 and 72 hours post-infection.

[0096] Knockdown of circCASC15-1 and circCASC15-2 expression in SK-N-BE(2) cells and SH-SY5Y cells

[0097] RNAiMAX (#13778150, Invitrogen) was used to transfect antisense oligonucleotides (ASO) purchased from RiboBio (Guangzhou, China) into human SK-N-BE(2) and SH-SY5Y cells according to the manufacturer's instructions. In short, 3 μl ASO (20 μM) and 5 μl RNAiMAX were mixed with 100 μl Opti-MEM (#11058021, Gibco) for 5 minutes. Then, 100 μl of the ASO suspension and 100 μl of the RNAiMAX suspension were added to new tubes, mixed thoroughly by pipetting, and incubated at room temperature for 15 minutes. Finally, 200 μl of transfection solution was added to each well of a 6-well plate containing cells. Forty-eight hours after transfection, cells were collected and used for experiments. Knockdown groups si-circCASC15-1 and si-circCASC15-2 were obtained, with untransfected ASO cells serving as the si-NC control group.

[0098] The specific sequence of the ASO is as follows:

[0099]

[0100] Cell proliferation, migration, and invasion were detected using a real-time cell analyzer (RTCA).

[0101] Cell proliferation and migration were monitored in real time using an xCELLigence RTCA-DP instrument (ACEA Bioscience, USA). Cell proliferation and migration rates were measured according to the instrument manufacturer's instructions. In the cell proliferation assay, SH-SY5Y and SK-N-BE(2) cells were seeded at a density of 4000 cells per well in electronic microplates (E-plates) (REF300600890) and cultured at 37°C and 5% CO2 for 72 hours. In the cell migration assay, 4 × 10⁴ cells of serum-free medium 1 were... 4 One NB cell was seeded into the top wells of a Cell Invasion and Migration (CIM) plate (REF5665817001), and complete culture medium 1 was added to the bottom wells. The plate was then incubated in RTCA for 48 hours, and cell counting and recording were performed automatically using the xCELLigence system. Cell index (CI) was used to represent cell proliferation and migration. CI was based on the change in electrical impedance divided by the background value. Each assay was performed three times.

[0102] Settlement formation experiment

[0103] Cells in logarithmic growth phase were digested with 0.25% trypsin (KGY0012, KeyGen Biotech) and isolated into single cells. These cells were then suspended in DMEM containing 10% FBS. A total of 800 cells were seeded into 6-well plates and cultured at 37°C and 5% CO2 for 14 days. Cells were then fixed with 4% paraformaldehyde for 10 minutes and stained with 1% crystal violet staining solution (#C0121, Beyotime, China) for 15 minutes. Finally, the cells were washed three times with PBS. A group containing more than 50 cells was considered a colony.

[0104] result

[0105] The relative expression of circCASC15-1 and circCASC15-2 in the overexpression and knockdown groups was assessed using RT-qPCR as described in Example 1. Figure 3 As can be seen from AH, for SK-N-BE(2) cells and SH-SY5Y cells, the expression levels of circCASC15-1 and circCASC15-2 in the overexpression group were higher than those in the control group, while the expression levels of circCASC15-1 and circCASC15-2 in the knockdown group were lower than those in the control group.

[0106] The proliferation of SK-N-BE(2) and SH-SY5Y cells with knockdown of circCASC15-1 or circCASC15-2 is shown in Figures 4A and 4B and Figures 5A and 5B. RTCA-based cell proliferation experiments showed that knockdown of circCASC15-1 or circCASC15-2 increased the proliferation of SK-N-BE(2) and SH-SY5Y cells. However, the proliferation capacity of SK-N-BE(2) and SH-SY5Y cells overexpressing OE-circCASC15-1 and OE-circCASC15-2 was lower than that of the OE-NC group (…). Figure 4 (C and Figure 5C). Furthermore, colony formation experiments also confirmed these results (4G-I, 5G-H).

[0107] Migration assays performed using RTCA showed that knockdown of circCASC15-1 or circCASC15-2 enhanced cell migration (Figures 4D and 4E). Figure 5 (D and 5E), but cell migration decreased after overexpression of circCASC15-1 or circCASC15-2 (Fig. 4F and Fig. 5F).

[0108] In summary, these results indicate that circCASC15-1 and circCASC15-2 can inhibit the proliferation and migration of NB cells, especially SK-N-BE(2) cells. SK-N-BE(2) cells are a type of... MYCN The results above further demonstrate that circCASC15-1 and circCASC15-2 express amplified cell lines. MYCN The relationship between amplification.

[0109] Example 4: Mouse model experiment

[0110] Construction of cells stably overexpressing luciferase

[0111] CHLA-255 cells were seeded in 24-well cell culture plates. Cell status was observed after 24 hours, preparing for lentiviral infection. First, the original culture medium was discarded, and 480 µl of fresh complete culture medium and 20 µl of HitransGP viral infection reagent (REVG005) were added. Then, cells containing... Luciferase Lentiviral cells (CON285) containing the luciferase gene (purchased from GeneChem, Shanghai, China) were added to 24-well cell culture plates and cultured. After 48 hours, puromycin (Lablead, China) was added to the cells at a final concentration of 4 µg / ml, and the cells were cultured further to screen for stable cell lines. During this period, the culture medium and puromycin were replaced with fresh medium every other day for 14 days. At this point, stable expression was achieved. Luciferase Gene cell lines were cultured for 7 days with a puromycin concentration reduced by half. Subsequently, we detected the results using a dual-luciferase reporter assay (Promega, E1910). Luciferase Gene expression status. Then, cells that stably overexpress luciferase are screened.

[0112] Construction of adenovirus-circCASC15 construct and control construct

[0113] The cDNA sequence of circCASC15-1 or circCASC15-2 was inserted into an adenovirus (AAV) vector to construct the adenovirus-circCASC15 construct. An empty AAV vector served as a control construct.

[0114] Mouse xenograft model and in vivo imaging

[0115] Twelve four-week-old female NSG mice were purchased from Sipeifu Co., Ltd. (Beijing). The mice were housed under specific pathogen-free (SPF) conditions. Two × 10⁶ mice were placed in each container. 6CHLA255 cells stably overexpressing the luciferase gene were suspended in 50 μL M Atrigel (#354230, Corning) and then injected into the left renal cortex of NSG mice. The tumor growth phase lasted 14 days, after which the mice were randomly divided into four groups. Subsequently, each group of xenograft mice was injected with 1 x 10n of the construct. 9 PFU / mouse (specific dosage calculated based on viral titer) for 14 days. The OE-circCASC15-1 group received the adenovirus-circCASC15-1 construct, while the NC-1 group served as a control and received an empty AAV vector. The OE-circCASC15-2 group received the adenovirus-circCASC15-2 construct, while the NC-2 group served as a control and received an empty AAV vector. Renal carcinoma volume was non-invasively measured weekly using the In VivoFX Pro visualization system (Bruker, Germany). Tumor size changes were observed for 4 weeks. VivoGlo™ luciferase (150 mg / kg) (P1043, Promega) was administered intraperitoneally, and luciferase activity was measured using FX Pro (Bruker, Germany). Data were acquired and analyzed using Carestream software. All animal welfare and experimental procedures were strictly performed in accordance with the "Guidelines of Beijing Municipal Committee for the Management of Laboratory Animals." Tumors were removed after euthanasia. Mice were euthanized via painless cervical dislocation. The mouse tumor model has received ethical approval (MDKN-2021-065).

[0116] result

[0117] Figure 6 A represents a schematic diagram of establishing a NB model using NSG mice. CHLA255 cells expressing luciferase (2 × 10⁻⁶) are used. 6 (1 cell / injection) was injected orthotopically into four-week-old female NSG mice. In vivo imaging and fluorescence intensity showed that on day 28, luciferase intensity increased at both primary and metastatic sites in the OE-NC-1 group (including mice 1, 2, and 3) (Figure 6B). In contrast, no significant fluorescence signal was observed in metastatic lesions of mice in the OE-circCASC15-1 group (mice 4, 5, and 6) on day 28. Figure 6 B). The above results indicate that circCASC15-1 can inhibit the formation of metastatic lesions and the proliferation of tumor cells in NB model mice. Figure 6D). Compared with OE-NC-2 (mice 7, 8, and 9), OE-circCASC15-2 (mice 10, 11, and 12) showed significantly enhanced tumor inhibition (Fig. 6E). Fluorescence intensity was significantly increased, and one mouse in the control group died (Fig. 6C). In contrast, the fluorescence intensity in the kidneys of mice in the OE-circCASC15-2 group was significantly reduced on day 28 (Fig. 6C). In conclusion, circCASC15-1 and circCASC15-2 can inhibit tumor growth. Among them, the effect of circCASC15-2 treatment was particularly significant. circCASC15-2 has an extra exon 9, while circCASC15-1 does not, which may be the reason for the functional difference between the two.

[0118] Results from in vitro cell experiments and mouse xenograft model experiments showed that CASC15 plays a tumor-suppressing role in NB tumors in this invention, which differs from previous reports. Yin Y, Zhao B, Li D, Yin G. Long non-coding RNA CASC15 promotes melanoma progression by epigenetically regulating PDCD4. Cell Biosci 2018;8:42. A report stated that CASC15 can promote the progression of melanoma. CASC15 has different and even opposite biological functions in different tumors. Even within the same tumor, different transcripts may play different roles in different cell types. This difference may be due to different cellular events, such as neuronal differentiation, proliferation, and migration, which are regulated by multiple molecular signaling pathways.

[0119] As a preferred embodiment of the present invention, the inventors further investigated the mechanism by which circCASC15-2 inhibits NB tumors.

[0120] Example 5: Mechanism of circCASC15-2 in inhibiting NB tumors

[0121] 1. circCASC15-2 positively regulates the expression of CASC15-003 and CASC15-004.

[0122] In Example 3, the expression of CASC15-003 and CASC15-004 in the circCASC15 overexpression group OE-circCASC15-2 and its control group OE-NC, as well as the knockdown group si-circCASC15-2 and its control group si-NC, was detected by RT-qPCR as described in Example 1. Each experiment was repeated at least 3 times. The forward primer CASC15-003 F and reverse primer CASC15-003 R for CASC15-003, and the forward primer CASC15-004 F and reverse primer CASC15-004 R for CASC15-004 are as follows:

[0123]

[0124] The primer sequences for CASC15-003 and CASC15-004 were synthesized by BGI Genomics.

[0125] result

[0126] The results showed that after circCASC15-2 was knocked down and overexpressed, the expression levels of CASC15-003 and CASC15-004 were lower or higher than those of the corresponding control groups, respectively. Figure 7 A and 7B). The above data indicate that circCASC15-2 can positively regulate the expression of CASC15-003 and CASC15-004 in SK-N-BE(2) cells.

[0127] 2. CASC15-004 inhibits tumor growth.

[0128] The lentiviral constructs overexpressing CASC15-003 (ENST00000606851.1, contract number GOSL0312880) and overexpressing CASC15-004 (ENST00000606197.1, contract number GOSL0312880) were both constructed by GeneChem (Shanghai, China).

[0129] experiment

[0130] SH-SY5Y and SK-N-BE(2) cells were transfected with lentiviral constructs overexpressing CASC15-003 and CASC15-004, respectively. NB cells transfected with empty lentiviral vectors constituted the control group OE-NC. The transfection steps and procedures were the same as in Example 3. The expression levels of CASC15-003 and CASC15-004 in the overexpression group and the control group SK-N-BE(2) and SH-SY5Y cells were determined by RT-qPCR as described in Example 1. Cell proliferation and migration were detected by RTCA as described in Example 3. The effects of CASC15-003 and CASC15-004 on cell colony formation ability were detected by colony formation assay as described in Example 3.

[0131] result

[0132] Depend on Figure 8 It can be seen that, compared with the control group, the expression levels of CASC15-003 and CASC15-004 in each expression group were significantly increased. Figure 8 AD). RTCA data showed that upregulation of CASC15-003 inhibited the proliferation of SH-SY5Y cells, but had no effect on SK-N-BE(2) cells (AD). Figure 9 A and Figure 8 E). In migration assays, CASC15-003 had no effect on either cell line. Figure 9 B and Figure 8F). However, overexpression of CASC15-004 significantly inhibited SK-N-BE(2) (B and Figure 8F). Figure 9 A and 9B) and SH-SY5Y cells ( Figure 8 The proliferation and migration of E and 8F cells were observed. Colony formation assays showed no difference in the number of SK-N-BE(2) cell colonies between the OE-CASC15-003 group and the control group (P=0.7). Figure 9 C). Conversely, the OE-CASC15-004 group formed fewer SK-N-BE(2) cell colonies than the OE-NC group (P<0.05). Figure 9 D).

[0133] The above results indicate that CASC15-004 can inhibit the growth of NB tumors.

[0134] 3. Xenograft mouse tumor tissue studies

[0135] IHC testing

[0136] After euthanizing the xenograft mice overexpressing circCASC15-2 in Example 4, tumor tissue was obtained, typically 3 mm thick. The tumor tissue was fixed in formalin and embedded in paraffin, then placed in an embedding mold. Gradual dehydration, clearing, and paraffin embedding were then performed. The tissue was embedded with the cut side facing down. After cooling, the paraffin block was removed from the mold and trimmed. The paraffin block was pre-cooled on a freezing stage for 20 minutes, then sectioned at 4 µm using a microtome. The sectioned tissue slides were then spread in a constant-temperature water bath in a slide spreader until flat and adhered to a glass slide. IHC analysis was then performed. The sections were stained with hematoxylin and eosin according to standard protocol. Sections were incubated overnight at 4°C with diluted primary antibodies against Ki67 (#9449, CST, 1:1000), Akt (#4691, CST, 1:200), phosphorylated Akt (Ser473) (#4060, CST, 1:100), and mTOR (#2983, CST, 1:100). They were then incubated with secondary antibody (K5007, DAKO) at room temperature for 50 minutes. Finally, the sections were imaged using a microscope (Nikon Eclipse E100). IHC results were determined by quantifying the proportion of brown staining on each section.

[0137] result

[0138] IHC results showed that the expression of Ki67, mTOR, Akt, and pAkt S473 were all decreased in the circCASC15-2 treatment group. Figure 10 ).

[0139] 4. CASC15-004 and circCASC15-2 inhibit the mTORC2 signaling pathway.

[0140] RNA sequencing (RNA-seq) analysis

[0141] RNA sequencing analysis was performed on three SH-SY5Y cell samples overexpressing circCASC15-2 and three negative controls (NC) from Example 3. Total RNA was isolated using TRIzol reagent. All libraries were prepared using Illumina HiSeq 4000 according to the manufacturer's recommendations. Sequence data were aligned to the hg19 reference using HISAT2 (GSE 112734). High-throughput sequencing was performed on the Illumina HiSeq 2000 platform, and RNA-seq transcript data were analyzed using DESeq2 and clusterProfiler software. Genes with a false discovery rate (FDR) < 0.05, p-value < 0.01, and fold change > 1.5 were considered differentially expressed genes (DEGs).

[0142] Pathway analysis

[0143] KEGG analysis was performed on the obtained DEG using KEGG Mapper (http: / / www.genome.jp / kegg / mapper.html). GO enrichment analysis (GO biological processes [GO:BP] analysis) was performed using the GO database (http: / / www.geneontology.org / ). A p-value less than 0.05 was considered statistically significant.

[0144] Protein extraction

[0145] According to the manufacturer's instructions, total protein was extracted from the cells using RIPA lysis buffer (#P0013B, Beyotime). SK-N-BE(2) cells overexpressing CASC15-004, SK-N-BE(2) cells overexpressing circCASC15-2, and SK-N-BE(2) cells with knockdown of circCASC15-2 expression were lysed in RIPA buffer supplemented with a phosphatase inhibitor tablet (#04906837001, Roche). The RIPA buffer consisted of 1.0% Nonidet P40 [N814616, Macklin]; 50 mM Tris base [B350, Genview], pH 7.4; 1 mM EDTA [E809185, Macklin]; 150 mM NaCl [S805275, Macklin]; a mixture of protease inhibitors containing PMSF [ST506, Beyotime]; Na3VO4 [S817660, Macklin] and a mixture of protease inhibitors [04693132001, Roche]). Protein concentration was determined using the bisquinolinic acid (BCA) protein assay kit (#23225, Thermo).

[0146] Western Imprint

[0147] Protein separation and antibody assay in the lysate were performed using the capillary automated system ProteinSimpleWes (Cat#SM-W004 and SM-W008). Antibodies were used against the following proteins: mTOR (#2983, CST, 1:100), phosphorylated mTOR (Ser2481) (#2974, CST, 1:100), phosphorylated mTOR (Ser2448) (#5536, CST, 1:100), Rictor (#2114, CST, 1:50), Sin1 (#12860, CST, 1:100), GβL (#3274, CST, 1:100), Akt (pan) (#4691, CST, 1:100), phosphorylated Akt (Ser473) (#4060, CST, 1:100), S6K (14485-1-AP, Proteintech, 1:100), and GAPDH (#5174, CST, 1:100). Primary antibody was detected using horseradish peroxidase (HRP)-bound anti-rabbit secondary antibody (DM-001, bio-techne), and the signal was visualized using Protein-Simple software (compass for SW-4.1.0).

[0148] Co-immunoprecipitation (co-IP) assay

[0149] The lysate was centrifuged at 14,000 rpm for 15 minutes, and the supernatant was collected. The supernatant was then incubated with anti-Sin1 antibody or control IgG (#2729S, CST) for 12 hours, followed by incubation with protein A-agarose beads (11719408001, Roche) at a constant rotation at 4°C for 4 hours. Immunoprecipitated proteins were then eluted from the beads. The eluted proteins were detected by Western blotting (IB) (i.e., the Western blotting described above).

[0150] result

[0151] The above experimental results demonstrate gene expression changes in SH-SY5Y cells overexpressing circCASC15-2. For example... Figure 11 As shown in Figure A, SH-SY5Y cells overexpressing circCASC15-2 contained 1861 downregulated genes and 1734 upregulated genes. DEG KEGG analysis revealed a significant enrichment of pathways related to cell proliferation and the cell cycle. Figure 12 A and 12C), consistent with the results of the cell culture and xenograft analyses above. DEG mapped to 10 KEGG pathways, of which 5 pathways were significantly enriched (A and 12C). Figure 12B). Increased Akt phosphorylation at S473 indicates enhanced mTORC2 activity. Similar results were obtained in IHC assays, showing decreased expression of Ki67, mTOR, Akt, and pAkt at S473 in the circCASC15-2 treatment group. Figure 10 ).

[0152] Western blot analysis was used to further investigate the levels of mTORC2, including its components mTOR, Rictor, Sin1, and GβL, as well as their substrates. Western blot data showed that overexpression of CASC15-004 reduced mTOR phosphorylation at Ser2448 and Ser2881. OE-CASC15-004 (hereinafter referred to as OE-004) significantly reduced the levels of mTOR, Rictor, and GβL, while OE-NC did not. Figure 11 B). Overexpression of circCASC15-2 reduces mTOR, Rictor, and GβL protein levels, and decreases mTOR phosphorylation at Ser2448 and Ser2481, as well as Akt phosphorylation at S473. Figure 11 C). Meanwhile, in circCASC15-2 knockout cells, the expression of mTOR, Rictor, Sin1, and GβL, as well as the phosphorylation of mTOR and Akt, were significantly upregulated. Figure 11 D).

[0153] Furthermore, co-IP experiments showed that overexpression of CASC15-004 reduced the interactions between Sin1 and Rictor, as well as between Sin1 and GβL. Figure 13 A). Furthermore, overexpression of circCASC15-2 can inhibit the formation of stable complexes between Sin1 and Rictor and GβL (A). Figure 13 B). Furthermore, after downregulation of circCASC15-2, Sin1 formed a complex with Rictor and GβL ( Figure 14 CASC15-004 and circCASC15-2 inhibited the assembly process of mTORC2. Figure 13 C).

[0154] These data indicate that CASC15-004 and circCASC15-2 inhibit mTORC2 by preventing its formation, thereby suppressing Akt signaling, especially signaling induced by serine 473 (Ser473) phosphorylation.

[0155] Example 6: Combined use

[0156] Combined use of CASC15-004 and circCASC15-2

[0157] Using the same method as in Example 3, the CASC15-004 overexpressing lentiviral construct was first transfected into SK-N-BE(2) cells at the infection fold (MOI, MOI=20). After 12 hours, the circCASC15-2 overexpressing lentiviral construct was transfected into SK-N-BE(2) cells to form the OE-circ2+OE-004 experimental group. The empty lentiviral vector was transfected into SK-N-BE(2) cells to form the OE-NC+OE-NC control group using the same method as in the OE-circ2+OE-004 experimental group. The circCASC15-2 overexpressing lentiviral construct and the empty lentiviral vector were transfected into SK-N-BE(2) cells to form the OE-circ2+OE-NC control group. The CASC15-004 overexpressing lentiviral construct and the empty lentiviral vector were transfected into SK-N-BE(2) cells to form the OE-004+OE-NC control group. SK-N-BE(2) cell proliferation was measured using an xCELLigence Real-Time Cell Analyzer (RTCA)-DP instrument in accordance with the manufacturer’s instructions.

[0158] All specific dosages involving lentiviruses are based on this formula: Virus volume = (MOI × number of cells) / Virus titer.

[0159] Combined use of circCASC15-2 and everolimus (S1120, Selleck, USA)

[0160] SK-N-BE(2) cells (OE-circ2, experimental group) transfected with the circCASC15-2 overexpressing lentiviral construct and SK-N-BE(2) cells (OE-NC, control group) transfected with the empty lentiviral vector were cultured at 37°C and 5% CO2. The following two sets of experiments were performed:

[0161] Experiment 1: 12 hours after transfection, cells were seeded into a proliferation assay plate compatible with the (RTCA)-DP instrument. After 4 hours of cell adhesion, an appropriate volume of everolimus was added to each well to achieve a drug concentration of 15 μM. An equal volume of dimethyl sulfoxide (DMSO) was added to the control group. SK-N-BE(2) cell proliferation was measured using the xCELLigence Real-Time Cell Analyzer (RTCA)-DP instrument according to the manufacturer's instructions. Since DMSO is the solvent for everolimus, it was used as the control group.

[0162] Experiment 2: Everolimus was added to each well of the experimental and control groups to achieve concentrations of 5 nM, 10 nM, 50 nM, 100 nM, and 1000 nM, respectively. After culturing for 48 hours, MTT (Solarbio, China) was added to each well. The treated cells were then cultured for another 4 hours, and the optical density at 490 nm was measured using a microplate reader (Clario Star, Germany).

[0163] result

[0164] RTCA showed that the combined effect of CASC15-004 and circCASC15-2 in inhibiting SK-N-BE(2) cell proliferation was stronger than that of inhibiting either RNA alone. Figure 15 A). The combination of everolimus and circCASC15-2 has a stronger ability to inhibit tumor cell proliferation than everolimus or circCASC15-2 alone. Figure 15 B), in addition, such as Figure 16 As shown, the slope of the dose-response curve for cells overexpressing circCASC15-2 treated with everolimus is significantly greater than that for cells not overexpressing circCASC15-2 treated with everolimus. This indicates that the difference in inhibitory effect between cells overexpressing circCASC15-2 and those not overexpressing circCASC15-2 increases with increasing everolimus concentration. These experimental results demonstrate that the combination of circCASC15-2 and everolimus exhibits a synergistic inhibitory effect.

[0165] These data collectively indicate that circCASC15-2 can not only cooperate with CASC15-004, but also with everolimus, to regulate NB progression by inhibiting mTORC2 signal transduction. Figure 15C). Negative feedback regulation mediated by mTORC1 includes inhibition of mTORC2 via S6K action. Although both mTOR and GβL are components of both mTORC1 and mTORC2, their interaction is crucial only for the integrity of the mTORC2 complex. Inhibition of mTORC1-S6K binding by rapamycin increases mTORC2 levels, thereby promoting Akt pathway activation. In other words, the Akt T308-mTORC1 and mTORC2-Akt S473 pathways form an autoactivation system, and mTORC2 plays a vital role in this cycle. circCASC15-2 inhibits the negative feedback loop between mTORC1 and mTORC2 by inhibiting mTORC2 assembly. This reduces the development of resistance compared to using rapamycin alone.

[0166] The above experiments demonstrate that circCASC15-1 and circCASC15-2 inhibit NB tumor growth both in vitro and in vivo. Furthermore, circCASC15-2, when used in combination with everolimus, a rapamycin derivative, exhibits a synergistic inhibitory effect on NB cell proliferation. This invention highlights the significant role of circCASC15-1 and circCASC15-2 in neuroblastoma and their potential applications in gene therapy. Simultaneously, circCASC15-1 and circCASC15-2 also serve as promising early diagnostic and prognostic biomarkers, providing new insights for the diagnosis and treatment of neuroblastoma patients.

[0167] The embodiments described above are exemplary and are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited thereto. For those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of circCASC15 in the preparation of a medicament for the treatment of neuroblastoma, characterized in that, The circCASC15 is circCASC15-2, and the sequence of circCASC15-2 is SEQ ID NO:

2.

2. The use according to claim 1, characterized in that, The drug is a reagent that promotes the expression of circCASC15.

3. The use according to claim 2, characterized in that, The reagent that promotes circCASC15 expression is a circCASC15 overexpression construct.

4. The use according to claim 3, characterized in that, The vector for the circCASC15 overexpression construct is an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, or a retroviral vector.

5. The use according to any one of claims 1-4, characterized in that, The drug also includes a second drug used in combination with circCASC15-2.

6. The use according to claim 5, characterized in that, The second drug is an mTOR pathway inhibitor.

7. The use according to claim 6, characterized in that, The mTOR pathway inhibitor is a rapamycin-type drug.

8. The use according to claim 7, characterized in that, The rapamycin-type drug in question is everolimus.

9. The use according to claim 5, characterized in that, The second drug is CASC15-004.

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