Intervention strategy targeting cacna2d2 protein and application thereof

Abstract

The application discloses an intervention strategy for targeting CACNA2D2 protein and application thereof, and belongs to the technical field of medicine and biotechnology.The application discloses for the first time that abnormal high expression of CACNA2D2 is related to poor prognosis of neuroblastoma, especially malignant progression, and provides that CACNA2D2 gene is used as a drug target to prepare a drug for treating neuroblastoma, and further provides application of CACNA2D2 siRNA in preparation of a drug for treating neuroblastoma.After down-regulation of CACNA2D2 gene expression, cell proliferation and clone formation ability of neuroblastoma cells are significantly inhibited.Therefore, the application provides a new direction for development of a drug for treating neuroblastoma, and provides a possibility for preparing a new drug for treating neuroblastoma, improving curative effect of patients, improving prognosis and survival.

Description

TECHNICAL FIELD

[0001] The application relates to the field of medicine and biotechnology, and particularly relates to application of an interfering siRNA targeting a CACNA2D2 gene in preparation of a neuroblastoma treatment drug. BACKGROUND

[0002] Neuroblastoma (NB) is a highly heterogeneous solid tumor originating from the sympathetic nervous system, and is also the most common extracranial malignant solid tumor in children, accounting for 8-10% of the total number of childhood malignant tumors and about 15% of the total number of childhood deaths due to malignant tumors. At present, neuroblastoma can be divided into low-risk, medium-risk and high-risk types according to risk rating. Although low-risk and medium-risk neuroblastoma patients can often achieve good treatment effects through comprehensive treatment, the cure rate is more than 90%, but the prognosis of high-risk neuroblastoma patients is poor, and even if various treatment schemes are combined, the five-year survival rate is less than 50%, and relapse and drug resistance are prone to occur in the later stage (Park JR, et al. JAMA. 2019 Aug 27; 322 (8): 746-755.).

[0003] Neuroblastoma is a major disease that endangers children's health, therefore, further research on new potential targets and intervention means for neuroblastoma treatment has a positive significance for promoting the clinical treatment of neuroblastoma.

[0004] The CACNA2D2 gene encodes a putative auxiliary subunit alpha2delta2 of voltage-dependent calcium channel, which is one of the auxiliary subunits of voltage-gated calcium channel (VGCC), participates in the trafficking of VGCC alpha1 subunit, changes the structure and function of VGCC, and further affects the calcium ion influx of cells. A large number of studies have shown that the change of calcium ion concentration caused by abnormal CACNA2D2 subunit is closely related to the occurrence of diseases such as epilepsy, for example, mutation of alpha2delta2 subunit easily leads to reduction of calcium channel current density and causes early infantile epileptic encephalopathy (Edvardson S, et al. J Med Genet. 2013 Feb; 50 (2): 118-123.); the characteristics of mouse absence epilepsy model is that CACNA2D2 exists in truncated mutation (Ivanov SV, et al. Am J Pathol. 2004 Sep; 165 (3): 1007-1018.).

[0005] Currently, there are few reports on the relationship between CACNA2D2 expression and tumors, and only one article shows that silencing CACNA2D2 can inhibit the proliferation of tumor cells in prostate cancer (Warnier M, et al. Oncogene. 2015 Oct 16; 34(42): 5383-5394.). There is no literature report on the role of CACNA2D2 gene in neuroblastoma, and the best applicable tumor type of the intervention strategy of inhibiting CACNA2D2 is unknown. Therefore, the role of CACNA2D2 in neuroblastoma, especially in high-risk patients, needs further study. SUMMARY

[0006] The purpose of the present application is to explore the genes related to the occurrence and development of neuroblastoma, and to apply it as a drug target for the preparation of a drug for treating neuroblastoma, so as to improve the curative effect, improve the prognosis and survival of patients.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application provides the application of voltage-gated calcium channel auxiliary subunit alpha 2 delta 2 (CACNA2D2) as a drug target in the preparation of a neuroblastoma treatment drug. Specifically, the drug is a drug that down-regulates the expression of CACNA2D2 gene.

[0009] Further, the CACNA2D2 gene nucleotide sequence is shown as SEQ ID NO: 1, and the protein sequence encoded by the CACNA2D2 gene is shown as SEQ ID NO: 2.

[0010] The present application found through pan-cancer database analysis that CACNA2D2 gene has the highest expression level in neuroblastoma, and the tumor cells are significantly higher than the normal adrenal tissue. More importantly, compared with neuroblastoma patients with low expression of CACNA2D2, the survival time of patients with high expression is significantly reduced. Therefore, we first propose that CACNA2D2 gene is a key gene for neuroblastoma, especially malignant progression, which provides a new treatment target for neuroblastoma, and neuroblastoma is a potential indication for CACNA2D2 targeted intervention strategy.

[0011] The present application achieves the effect of down-regulating CACNA2D2 by applying siRNAs targeting CACNA2D2 gene, so as to study the role of CACNA2D2 in neuroblastoma. The research results show that after down-regulating the expression of CACNA2D2, the cell proliferation and clonogenicity of neuroblastoma cells are significantly inhibited. The present application provides a new action target for the development of neuroblastoma treatment drugs.

[0012] Specifically, aiming at the CACNA2D2 gene as a target, a corresponding drug preparation for knocking down the expression of the gene is developed, and the purpose of resisting neuroblastoma is achieved by down-regulating the expression of the CACNA2D2 gene.

[0013] Further, the drug is an siRNA targeting CACNA2D2.

[0014] Further, the nucleotide sequence of the siRNA is shown in SEQ ID NO: 3 or SEQ ID NO: 4. The above two siRNAs for the CACNA2D2 gene can inhibit the expression of CACNA2D2, and further inhibit the proliferation and clonogenicity of neuroblastoma cells.

[0015] The research results of the present application show that down-regulation of CACNA2D2 expression can significantly inhibit the cell proliferation and clonogenicity of neuroblastoma SK-N-DZ and SK-N-BE(2) cells. Combined with the fact that SK-N-DZ and SK-N-BE(2) are both tumor cells derived from high-risk neuroblastoma patients amplified by MYCN, we first disclose that the siRNA molecule of CACNA2D2 can be used as an effective intervention means for the treatment of neuroblastoma, especially for high-risk patients.

[0016] Further, the drug further comprises a pharmaceutically acceptable excipient. The excipient is any preparation or carrier medium capable of delivering an effective dose of active substance, not interfering with the biological activity of the active substance and having no toxic side effects on the host or subject. The excipient includes a filler, a wetting agent, a binder, a disintegrating agent or a lubricant.

[0017] The drug can be prepared into a preparation according to the preparation method recorded in pharmacy. The preparation form of the drug described above is a liquid preparation or a solid preparation.

[0018] The present application has the following beneficial effects:

[0019] The present application first discloses that the abnormally high expression of CACNA2D2 is related to the poor prognosis of neuroblastoma, especially malignant progression, provides a drug for treating neuroblastoma by taking CACNA2D2 gene as a drug target, and further provides the use of CACNA2D2 siRNA in preparing a neuroblastoma treatment drug. Down-regulation of CACNA2D2 can effectively inhibit the cell proliferation and clonogenicity of neuroblastoma, providing a new direction for the development of current neuroblastoma treatment drugs. The present application provides the possibility for preparing a new neuroblastoma drug, improving patient efficacy, improving prognosis and survival. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1To analyze the expression of CACNA2D2 gene in pan-cancer (A) and in tissues (B), the expression level of CACNA2D2 gene in neuroblastoma is the highest, and is significantly higher than that in normal tissues.

[0021] Figure 2 To analyze the relationship between the expression of CACNA2D2 gene and the prognosis of neuroblastoma patients, high expression of CACNA2D2 gene is closely related to low survival rate of neuroblastoma patients.

[0022] Figure 3 The effect of two siRNAs (SEQ ID NO: 3; SEQ ID NO: 4) targeting CACNA2D2 gene on the expression level of CACNA2D2 protein, wherein A is for SK-N-BE (2) cells, and B is for SK-N-DZ cells, both of which have a significant effect of down-regulating the protein level of CACNA2D2.

[0023] Figure 4 The effect of two siRNAs (SEQ ID NO: 3; SEQ ID NO: 4) targeting CACNA2D2 gene on the proliferation of neuroblastoma cells, wherein A is for SK-N-BE (2) cells, and B is for SK-N-DZ cells, the proliferation of neuroblastoma cells is significantly inhibited.

[0024] Figure 5 The effect of two siRNAs (SEQ ID NO: 3; SEQ ID NO: 4) targeting CACNA2D2 gene on the clonogenic ability of neuroblastoma cells, wherein A is for SK-N-BE (2) cells, and B is for SK-N-DZ cells, the clonogenic ability of neuroblastoma cells is significantly inhibited. DETAILED DESCRIPTION

[0025] The application will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the application and are not used to limit the scope of the application.

[0026] The experimental methods not specified in the examples are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0027] The application of CACNA2D2 siRNA described in the application can refer to conventional drug configuration methods and actual development. The drug dosage form and biological preparation are any dosage form recognized by medicine, such as powder, injection, capsule, tablet or oral liquid.

[0028] Example 1

[0029] Based on the DepMap database (https: / / depmap.org / portal / ), the expression of CACNA2D2 gene in pan-cancer was analyzed, and the results are shown in FIG. 1A. The results showed that compared with other cancer types, the expression level of CACNA2D2 gene in neuroblastoma was the highest. Figure 1

[0030] Based on the GEO dataset (GSE3526, GSE7307, GSE8514, GSE16476), the expression of CACNA2D2 gene in neuroblastoma and normal tissue was analyzed, and the results are shown in FIG. 2B. The results showed that compared with normal tissue, CACNA2D2 gene was significantly highly expressed in neuroblastoma. Figure 1

[0031] Example 2

[0032] Based on the GEO dataset (GSE16476), the relationship between high and low expression of CACNA2D2 gene and prognosis of neuroblastoma patients was analyzed, and the results are shown in FIG. 3. The results showed that compared with neuroblastoma patients with low expression of CACNA2D2, the survival time of patients with high expression was significantly decreased, suggesting that high expression of CACNA2D2 gene was closely related to malignant progression of neuroblastoma patients. Figure 2

[0033] Example 3

[0034] Two CACNA2D2 siRNAs (SEQ ID NO: 3; SEQ ID NO: 4) targeting different sequences and negative control NC were introduced into SK-N-DZ and SK-N-BE(2) cells (purchased from the Chinese Academy of Sciences Cell Bank) by lipofection, and after 72 hours, the cells were collected, lysed with 4% SDS lysis buffer to extract proteins, and then Western Blot was used for protein level detection.

[0035] The nucleotide sequences of the two siRNAs are as follows:

[0036] 5'-TCAAGAACAAGGTCAACTATT-3'(SEQ ID NO: 3);

[0037] 5'-CGTGGCTCTGAATGACATCAA-3'(SEQ ID NO: 4).

[0038] The results are shown in FIG. 4. Figure 3 ​​​The results show that all the CACNA2D2 siRNAs (SEQ ID NO: 3; SEQ ID NO: 4) can effectively inhibit the protein expression of CACNA2D2.

[0039] Example 4:

[0040] Two CACNA2D2 siRNAs (SEQ ID NO: 3; SEQ ID NO: 4) targeting different sequences and a negative control NC were introduced into neuroblastoma SK-N-DZ and SK-N-BE(2) cells (purchased from the Chinese Academy of Sciences Cell Bank) by lipofection, and 4000 cells were inoculated into a 96-well plate after digestion and counting with a hemocytometer the next day after treatment. The cell proliferation ability was investigated by SRB assay after the first, third, fifth and seventh days after treatment.

[0041] The results are shown in Table 2. Figure 4 The results show that the CACNA2D2 siRNAs can significantly inhibit the proliferation of neuroblastoma SK-N-DZ and SK-N-BE(2) cells.

[0042] Example 5:

[0043] Two CACNA2D2 siRNAs (SEQ ID NO: 3; SEQ ID NO: 4) targeting different sequences and a negative control NC were introduced into neuroblastoma SK-N-DZ and SK-N-BE(2) cells (purchased from the Chinese Academy of Sciences Cell Bank) by lipofection, and 10000 cells were inoculated into a six-well plate for clonal culture after digestion and counting with a hemocytometer the next day after treatment.

[0044] The results are shown in Table 3. Figure 5 The results show that the CACNA2D2 siRNAs can significantly inhibit the clonogenicity of neuroblastoma SK-N-DZ and SK-N-BE(2) cells.

[0045] It is worth noting that the above examples are part of the embodiments of the present application and should not be understood as limiting the present application. The above examples use siRNA interference technology to verify that down-regulation of CACNA2D2 gene expression can inhibit the proliferation and clonogenicity of neuroblastoma cells. On this basis, other techniques for inhibiting gene expression also fall within the scope of the present application.

Claims

1. The use of siRNA targeting voltage-gated calcium channel auxiliary subunit α2δ2 in the preparation of a neuroblastoma treatment drug, characterized in that, The siRNA nucleotide sequence is shown as SEQ ID NO: 3 or shown as SEQ ID NO:

4.

2. Use according to claim 1, characterized in that, The medicine also includes a pharmaceutically acceptable excipient.

3. Use according to claim 2, characterized in that, The preparation form of the medicine is a liquid preparation or a solid preparation.

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