Use of ALDH18A1 in the preparation of a drug for treating Ewing's sarcoma

Abstract

The present invention discloses the application of ALDH18A1 in the preparation of a drug for treating Ewing's sarcoma. The amino acid sequence of the ALDH18A1 is shown as SEQ ID NO.1. After knocking down the expression of ALDH18A1, the proliferation ability, colony formation ability and self-renewal ability of Ewing's sarcoma cells can be significantly inhibited, and the tumorigenicity of Ewing's sarcoma cells and the growth rate of transplanted tumors can be inhibited. The present invention preliminarily reveals that ALDH18A1 plays an important role in the occurrence and progression of Ewing's sarcoma, provides a new candidate target for the treatment of Ewing's sarcoma, and ALDH18A1 may inhibit the proliferation and self-renewal of Ewing's sarcoma through the EWS-FLI1-PARP1 positive feedback loop.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of ALDH18A1 in preparing drugs against Ewing's sarcoma. Background Art

[0002] Ewing sarcoma is a highly aggressive malignant tumor, and its incidence ranks second among pediatric bone tumors. Currently, the treatment of Ewing sarcoma still relies on multidisciplinary treatment, combining high-intensity chemotherapy with surgery / radiotherapy to control the primary site and metastatic lesions of the disease. Although the 5-year survival rate of patients with localized Ewing sarcoma has reached 70-80%, the 5-year survival rate of patients with metastasis and recurrence is still less than 30%. As a highly aggressive malignant tumor with a high recurrence rate, there is an urgent need to find new therapeutic targets.

[0003] As a tumor with clear carcinogenic factors, the oncogenic factor EWS-FLI1 fusion gene of Ewing sarcoma activates downstream target genes in the form of phase separation, leading to extensive epigenetic remodeling and driving a large number of metabolic reprogramming processes. As a tumor with a low gene mutation rate, Ewing sarcoma lacks viable alternative therapeutic targets. Therefore, genetic transcriptional regulation driven by its oncogenic fusion protein is a new direction for targeted therapy of Ewing sarcoma. Epigenetics and tumor metabolism, as a pair of important features that are inseparable and cross-talking in tumors, have attracted much attention in recent years and may be a new direction for targeted therapy of Ewing sarcoma. Studies have shown that a large amount of prolyl hydroxylation mediated by the side metabolic effect in proline metabolism plays an important role in epigenetic modification, and aldehyde dehydrogenase 18 family member A1 (ALDH18A1), as a key enzyme in glutamate, arginine and proline metabolism, plays an unclear role in the progression of Ewing sarcoma and needs to be studied.

[0004] Based on this, studying the expression and prognostic significance of ALDH18A1 in Ewing sarcoma and the biological process of its regulation is of great significance for revealing the transcriptional regulation mechanism of Ewing sarcoma and finding new therapeutic targets for Ewing sarcoma. Summary of the invention

[0005] In view of this, an object of the present invention is to provide an application of an ALDH18A1 inhibitor in the preparation of an anti-Ewing's sarcoma drug.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. Application of ALDH18A1 inhibitors in the preparation of drugs against Ewing's sarcoma.

[0008] Preferably, the amino acid sequence of ALDH18A1 is as shown in NCBI reference sequence number NP_001310342.1.

[0009] Preferably, the ALDH18A1 inhibitor is dsRNA, antisense nucleic acid, shRNA, microRNA that takes the ALDH18A1 gene or its transcript as the inhibitory target or silencing target, or a construct that can express or form the dsRNA, antisense nucleic acid, shRNA, microRNA.

[0010] Preferably, the sense strand of the shRNA is as shown in SEQ ID NO.3, and the antisense strand of the shRNA is as shown in SEQ ID NO.4.

[0011] Preferably, the sense strand of the shRNA is as shown in SEQ ID NO.5, and the antisense strand of the shRNA is as shown in SEQ ID NO.6.

[0012] Preferably, the ALDH18A1 inhibitor is used in the preparation for prolonging the survival time of patients with Ewing's sarcoma.

[0013] Preferably, the ALDH18A1 inhibitor can inhibit the cell proliferation, colony formation ability, sphere formation ability and in vivo tumorigenicity of Ewing's sarcoma cells.

[0014] Preferably, the ALDH18A1 inhibitor can inhibit the expression level of the downstream target genes of the EWS-FLI1 fusion gene in Ewing's sarcoma cells.

[0015] Preferably, the downstream target genes are PARP1, EZH2, PTPL1, CCND1, VEGFA.

[0016] Preferably, the ALDH18A1 inhibitor can reduce the number of mitochondria in Ewing's sarcoma cells.

[0017] The beneficial effects of the present invention are as follows: Through research, it is found that ALDH18A1 is highly expressed in Ewing's sarcoma tissues. The high expression of ALDH18A1 can be used as a prognostic prediction marker for patients with Ewing's sarcoma, and the cell division-related pathway is highly enriched in patients with Ewing's sarcoma with high expression of ALDH18A1. By knocking down the expression of ALDH18A1, the proliferation ability of Ewing's sarcoma cells can be significantly inhibited. Knocking down ALDH18A1 can also significantly inhibit the colony formation ability of Ewing's sarcoma cells, and knocking down ALDH18A1 can significantly reduce the self-renewal ability of Ewing's sarcoma cells; knocking down ALDH18A1 can significantly inhibit the tumorigenicity of Ewing's sarcoma cells and the growth rate of transplanted tumors; after intervening in the expression of ALDH18A1, the downstream target genes of EWS-FLI1 are significantly down-regulated. ALDH18A1 may play its regulatory role through PARP1 in Ewing's sarcoma cells, and ALDH18A1 may affect PARP1 by regulating the number of mitochondria. The present invention preliminarily reveals that ALDH18A1 plays an important role in the occurrence and progression of Ewing's sarcoma, provides a new candidate target for the treatment of Ewing's sarcoma, and ALDH18A1 may inhibit the proliferation and self-renewal of Ewing's sarcoma through the EWS-FLI1-PARP1 positive feedback loop. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

[0019] Figure 1 For the mRNA expression difference of ALDH18A1 in Ewing's sarcoma tissues and normal tissues in the GEO dataset GSE17614;

[0020] Figure 2 For the relationship between the mRNA expression of ALDH18A1 in Ewing's sarcoma tissues and the prognosis of patients in the GEO datasets GSE17674 ( Figure 2 , A), GSE17679 ( Figure 2 , B), GSE63155 ( Figure 2 , C);

[0021] Figure 3 For the pathway enrichment of patients with high expression of ALDH18A1 in Ewing's sarcoma analyzed by WGCNA ( Figure 3 , A) and GO_BP ( Figure 3 , B) in the GEO dataset GSE17618;

[0022] Figure 4 For the effect on the proliferation ability of Ewing's sarcoma cells after knocking down ALDH18A1 ( Figure 4 , A - B), ( Figure 4 , C - D);

[0023] Figure 5 Effect of knocking down ALDH18A1 on the colony formation ability of Ewing's sarcoma cells ( Figure 5 , A-B);

[0024] Figure 6 Effect of knocking down ALDH18A1 on the spheroid formation ability of Ewing's sarcoma cells ( Figure 6 , A-B);

[0025] Figure 7 Effect of knocking down ALDH18A1 on the tumorigenic ability of Ewing's sarcoma cells in vivo ( Figure 7 , A-C);

[0026] Figure 8 Effect of knocking down ALDH18A1 on EWS-FLI1 in Ewing's sarcoma cells;

[0027] Figure 9 Effect of knocking down ALDH18A1 on the downstream target genes of the EWS-FLI1 oncoprotein in Ewing's sarcoma cells ( Figure 9 , A-B);

[0028] Figure 10 Predicted binding motif of PARP1 among differentially expressed genes in the GEO dataset ( Figure 10 , A), co-expression correlation analysis of PARP1 and ALDH18A1 ( Figure 10 , B), and box plot analysis of the differential expression of PARP1 mRNA in Ewing's sarcoma tissues and normal tissues in GSE17674 ( Figure 10 , C);

[0029] Figure 11 Effect of knocking down ALDH18A1 on the expression of PARP1 in Ewing's sarcoma cells ( Figure 11 , A-B);

[0030] Figure 12 Effect of knocking down ALDH18A1 on the change in the number of mitochondria in Ewing's sarcoma cells ( Figure 12 , A-B). Detailed implementation manners

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0032] Example 1. Relationship between the expression level of ALDH18A1 and Ewing's sarcoma patients

[0033] To study the relationship between the expression level of ALDH18A1 (amino acid sequence shown in NCBI reference sequence number NP_001310342.1) and patients with Ewing's sarcoma, the Ewing's sarcoma gene expression profile dataset (GSE17674) in the GEO dataset was used to compare the expression of ALDH18A1 in human Ewing's sarcoma tissues and human normal muscle tissues. The results are as Figure 1 shown. The results showed that ALDH18A1 was highly expressed in Ewing's sarcoma tissues.

[0034] Example 2. Relationship between the expression level of ALDH18A1 and the survival prognosis of patients with Ewing's sarcoma

[0035] To study the relationship between the expression level of ALDH18A1 and the survival prognosis of patients with Ewing's sarcoma, the GEO dataset was used to analyze the prognostic significance of ALDH18A1 in patients with Ewing's sarcoma by Kaplan-Meier and Cox regression. In the GSE17674, GSE17679, and GSE63155 datasets, combining the clinical survival information of patients and the ALDH18A1 mRNA expression level, ROC curves were plotted. According to the Youden index, the high and low expression status of ALDH18A1 was defined. The Kaplan-Meier survival curve was used to analyze the relationship between the expression level of ALDH18A1 and the survival prognosis of patients with Ewing's sarcoma. The results are as Figure 2 shown in A - C. The results showed that high expression of ALDH18A1 could be used as a predictive marker for the prognosis of patients with Ewing's sarcoma.

[0036] Example 3. Pathway enrichment in patients with high expression of ALDH18A1 in Ewing's sarcoma

[0037] To study the pathway enrichment in patients with high expression of ALDH18A1 in Ewing's sarcoma, the WGCNA algorithm was used to find gene sets closely related to high expression of ALDH18A1 in the GSE17618 dataset, and GO enrichment analysis was performed to screen (screening criteria: FDR < 0.25, P < 0.05) the pathway enrichment. The results are as Figure 3 shown in A - B. The results showed that cell division-related pathways were highly enriched in patients with Ewing's sarcoma with high expression of ALDH18A1.

[0038] Example 4. ALDH18A1 promotes cell proliferation, colony formation ability, sphere formation ability, and tumorigenicity in vivo of Ewing's sarcoma cells

[0039] Method for down-regulating ALDH18A1 expression: The shRNA sequences in Table 1 were ligated to the pLVshRNA-EGFP vector, and then co-transfected into 293T cells together with the PSPAX2 packaging plasmid and the PMD2.G envelope plasmid. The viral supernatant was collected 48 h and 72 h later, and then concentrated and purified. This lentivirus was used to infect the Ewing's sarcoma cell line, and after amplification, the stable transfected strain was cultured and screened in a medium containing 2 μg / mL puromycin.

[0040] Table 1. Sequence information of shRNA of ALDH18A1

[0041]

[0042] After screening the Ewing's sarcoma cells with stable knockdown of ALDH18A1 by puromycin, the knockdown efficiency was verified by fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western-Blot experiments at the mRNA level and protein level, and the results are shown in Figure 4 Figures A - B. The results showed that the knockdown of ALDH18A1 was successful at the mRNA level and protein level.

[0043] After down-regulating the expression of ALDH18A1, the Cell counting kit-8 (CCK-8), plate colony formation assay, sphere formation assay and nude mouse subcutaneous tumor transplantation assay were used to detect the changes in the proliferation, self-renewal and tumorigenicity in vivo of Ewing's sarcoma cells. It was found that knocking down the expression of ALDH18A1 could significantly inhibit the proliferation ability of Ewing's sarcoma cells ( Figure 4 Figures C - D); knocking down ALDH18A1 could significantly inhibit the colony formation ability of Ewing's sarcoma cells ( Figure 5 Figures A - B); knocking down ALDH18A1 could significantly reduce the self-renewal ability of Ewing's sarcoma cells ( Figure 6 Figures A - B); knocking down ALDH18A1 could significantly inhibit the tumorigenicity of Ewing's sarcoma cells and the growth rate of transplanted tumors ( Figure 7 Figures A - C).

[0044] Example 5. Effects of ALDH18A1 on the EWS-FLI1 oncoprotein and its downstream target genes in Ewing's sarcoma cells

[0045] The changes in the expression levels of the downstream target genes of the EWS-FLI1 oncoprotein in Ewing's sarcoma were detected by qRT-PCR after intervening with ALDH18A1 (Table 2). The results showed that the expression of EWS-FLI1 was down-regulated after knocking down the expression of ALDH18A1 in A673 ( Figure 8) Meanwhile, the downstream target genes of EWS-FLI1 were significantly downregulated, including PARP1, EZH2, PTPL1, CCND1, and VEGFA. Among them, CCND1, VEGFA, and PARP1 were the most significantly downregulated. Interestingly, PARP1 is involved in the regulation of the transcriptional activity of EWS-FLI1. After knocking down the expression of ALDH18A1 in SK-N-MC, the downstream target genes of EWS-FLI1 were significantly downregulated, including PARP1. The above results showed that after interfering with the expression of ALDH18A1, the downstream target genes of EWS-FLI1 were significantly downregulated( Figure 9 , A-B).

[0046] Table 2. Primer sequence information of EWS-FLI1 and its downstream target genes

[0047]

[0048] Example 6. Effect of knocking down ALDH18A1 on the expression of PARP1 in Ewing's sarcoma cells

[0049] To further investigate the molecular mechanism by which ALDH18A1 exerts its regulatory role, in the GSE63155 dataset, GSE63156 dataset, and GSE17679 dataset, patients were divided into high and low groups based on the median, and differential gene analysis was performed. Transcription factor motif enrichment analysis was performed on the differential genes. The enrichment results suggested that ALDH18A1 may exert its regulatory role through PARP1 (PARP1: NES value = 4.47; AUC value = 0.09)( Figure 10 , A). Meanwhile, the correlation between the expression of ALDH18A1 and PARP1 in Ewing's sarcoma patients was compared, and the results showed a positive correlation between their expressions( Figure 10 , B). Finally, in the GSE17614 dataset, it was also found that the mRNA expression level of PARP1 was significantly upregulated( Figure 10 , C). The above results suggest that ALDH18A1 may exert its regulatory role through PARP1 in Ewing's sarcoma cells.

[0050] The above bioinformatics analysis results showed that the differential expression of ALDH18A1 may exert its regulatory role by affecting PARP1. Next, after downregulating the expression of ALDH18A1 in the Ewing's sarcoma cell lines A673 and SK-N-MC, the expression of PARP1 was detected. The RT-PCR results showed that after downregulating ALDH18A1 in the Ewing's sarcoma cell lines A673 and SK-N-MC, the mRNA expression of PARP1 was also downregulated( Figure 11 , A). Similarly, the WB results showed that after downregulating ALDH18A1 in Ewing's sarcoma cells, the protein level of PARP1 was downregulated( Figure 11, B). The above results further suggest that ALDH18A1 may exert its regulatory role through PARP1 in Ewing's sarcoma cells.

[0051] Example 7. Effect on the change in the number of mitochondria in Ewing's sarcoma cells after knocking down ALDH18A1

[0052] PARP1 mainly exercises DNA damage repair and transcriptional regulation functions in the nucleus, and this process is regulated by the mitochondrial NAD + level. Next, it was detected whether knocking down ALDH18A1 in Ewing's sarcoma cells affected the number of mitochondria. The results showed that compared with the control group, the relative fluorescence intensity of Mito-Tracker Red was significantly decreased after knocking down ALDH18A1 ( Figure 12 , A - B). The above results suggest that the number of mitochondria in Ewing's sarcoma cells decreases after knocking down the expression of ALDH18A1, and ALDH18A1 may affect PARP1 by regulating the number of mitochondria.

[0053] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. Use of an ALDH18A1 inhibitor in the preparation of a drug for treating Ewing's sarcoma, characterized in that, The amino acid sequence of the said ALDH18A1 is as shown in NCBI reference sequence number NP_001310342.1, and the ALDH18A1 inhibitor is the shRNA shown in SEQ ID NO.3-4 or SEQ ID NO.5-6.

2. The application according to claim 1, characterized in that, Use of an ALDH18A1 inhibitor in the preparation of a medicament for prolonging the survival time of patients with Ewing's sarcoma.

3. The application according to claim 1, characterized in that, Use of an ALDH18A1 inhibitor in the preparation of a medicament for inhibiting the proliferation, colony formation ability, spheroid formation ability or in vivo tumorigenesis ability of Ewing's sarcoma cells.

4. The application according to claim 1, wherein Use of an ALDH18A1 inhibitor in the preparation of a medicament for inhibiting the expression level of downstream target genes of the EWS-FLI1 fusion gene in Ewing's sarcoma cells.

5. The application according to claim 4, wherein The said downstream target genes are PARP1, EZH2, PTPL1, CCND1, VEGFA.

6. The application according to claim 1, characterized in that Use of an ALDH18A1 inhibitor in the preparation of a medicament for reducing the number of mitochondria in Ewing's sarcoma cells.

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