Application of CYLD-NGFR regulation axis in preparation of drugs for targeted therapy of angiosarcoma

Abstract

The invention relates to an application of a CYLD-NGFR regulation axis in preparation of a drug for targeted therapy of angiosarcoma. The CYLD-NGFR regulation axis comprises a CYLD gene and an NGFR gene, and the nucleotide sequences of the CYLD gene and the NGFR gene are shown as SEQ ID NO.1-2. According to the invention, the activity of targeted inhibition of a CYLD-NGFR regulatory axis is found for the first time, and the proliferation, migration and invasion ability of vascular sarcoma cells can be effectively inhibited. The invention designs specific siRNA molecules, including si-CYLD, si-NGFR and a combination si-CN of si-CYLD and si-NGFR, and aims to accurately reduce the expression level of CYLD mRNA and NGFR mRNA. Experiments prove that si-CYLD and si-NGFR successfully intervene a CYLD-NGFR regulation axis by down-regulating the expression levels of CYLD and NGFR genes, so that the growth, migration and invasion behaviors of vascular sarcoma cells are remarkably inhibited. Therefore, the CYLD-NGFR regulatory axis is proved to be an effective therapeutic target, and a scientific basis is provided for developing a novel drug for targeted therapy of angiosarcoma.

Description

Technical Field

[0001] The invention relates to application of CYLD-NGFR regulatory axis in preparing a drug for targeted treatment of angiosarcoma, and belongs to the technical field of biomedicine. Background Art

[0002] Angiosarcoma is a rare soft tissue sarcoma with high heterogeneity and invasiveness, posing a serious threat to the life and health of patients. The incidence of this disease is relatively low worldwide and can occur at any age. It is more common in adults, and a few are congenital, accounting for 2%-4% of soft tissue tumors. Traditional treatments, such as surgery and chemotherapy, are often difficult to significantly improve patients' survival and quality of life due to the lack of individualized precision treatment strategies. In recent years, with the rapid development of molecular biology and genomics, researchers have discovered a variety of driver genes associated with the progression of angiosarcoma and gradually developed targeted drugs targeting these genes. However, the high heterogeneity of angiosarcoma, coupled with the rarity of cases, often makes single targeted therapy face the risk of failure. Therefore, in-depth exploration of new regulatory pathways that drive the malignant progression of angiosarcoma and combined intervention of multiple key genes in this pathway will help promote the development of clinical targeted treatment strategies for angiosarcoma, thereby improving the prognosis of patients.

[0003] Small interfering RNA (siRNA) is a double-stranded RNA molecule with a length of 20 to 24 nucleotides. It achieves gene silencing by specifically binding to the target mRNA and inducing its degradation. siRNA technology is widely used in gene function research and disease treatment. siRNA forms an RNA-induced silencing complex (RISC) by complementary pairing with the target mRNA, leading to the cleavage and degradation of the target mRNA, thereby inhibiting the expression of specific genes. This process regulates gene expression at the post-transcriptional level. siRNA technology has broad application prospects in the fields of cancer, genetic diseases, and viral infections. By targeting specific genes, siRNA can effectively inhibit the expression of disease-related genes and provide new treatment strategies. Therefore, designing siRNA with high knockdown efficiency and high specificity for the target gene will lay an important foundation for exploring the biological function of the target gene in the malignant progression of angiosarcoma and the development of targeted drugs. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides the application of the CYLD-NGFR regulatory axis in the preparation of drugs for the targeted treatment of angiosarcoma.

[0005] The technical solution of the present invention is as follows:

[0006] Application of the CYLD-NGFR regulatory axis as a drug target in the preparation of targeted drugs for the treatment of angiosarcoma.

[0007] Preferably according to the present invention, the CYLD-NGFR regulatory axis includes the CYLD gene and the NGFR gene.

[0008] Preferably according to the present invention, the mRNA nucleotide sequence of the CYLD gene is shown as SEQ ID NO.1, and the mRNA nucleotide sequence of the NGFR gene is shown as SEQ ID NO.2.

[0009] Preferably, according to the present invention, the targeted treatment drug for angiosarcoma uses the CYLD gene and the NGFR gene as intervention targets, based on interfering with CYLD mRNA and NGFR mRNA, and can efficiently and specifically inhibit the expression levels of the CYLD gene and the NGFR gene.

[0010] Preferably according to the present invention, the targeted therapeutic drug for angiosarcoma is siRNA, shRNA, microRNA or a chemical inhibitor.

[0011] Application of siRNA that specifically interferes with CYLD gene expression and siRNA that specifically interferes with NGFR gene expression in the preparation of targeted drugs for the treatment of angiosarcoma.

[0012] Preferably according to the present invention, the siRNA that specifically interferes with the expression of the CYLD gene is double-stranded, and its nucleotide sequence is as shown in SEQ ID NO.3-4; the siRNA that specifically interferes with the expression of the NGFR gene is double-stranded, and its nucleotide sequence is as shown in SEQ ID NO.5-6.

[0013] Preferably, according to the present invention, the molar ratio of the siRNA that specifically interferes with the expression of the CYLD gene to the siRNA that specifically interferes with the expression of the NGFR gene in the targeted drug for treating angiosarcoma is 1:1.

[0014] A targeted drug for treating angiosarcoma comprises siRNA that specifically interferes with CYLD gene expression and siRNA that specifically interferes with NGFR gene expression, and the molar ratio of the two is 1:1.

[0015] Preferably according to the present invention, the siRNA that specifically interferes with the expression of the CYLD gene is double-stranded, and its nucleotide sequence is as shown in SEQ ID NO.3-4; the siRNA that specifically interferes with the expression of the NGFR gene is double-stranded, and its nucleotide sequence is as shown in SEQ ID NO.5-6.

[0016] Beneficial effects:

[0017] 1. The present invention discovered for the first time that targeted inhibition of the activity of the CYLD-NGFR regulatory axis can effectively curb the proliferation, migration and invasion of angiosarcoma cells. The present invention designed specific siRNA molecules, including si-CYLD, si-NGFR and their combination si-CN, aiming to accurately reduce the expression levels of CYLD mRNA and NGFR mRNA. Experiments have shown that si-CYLD and si-NGFR successfully intervened in the CYLD-NGFR regulatory axis by downregulating the expression levels of the CYLD and NGFR genes, thereby significantly inhibiting the growth, migration and invasion of angiosarcoma cells. Therefore, the CYLD-NGFR regulatory axis has been confirmed to be an effective therapeutic target, providing a scientific basis for the development of new targeted drugs for the treatment of angiosarcoma.

[0018] 2. The present invention further reveals that, compared with interfering with the expression level of CYLD or NGFR alone, the use of si-CN to specifically reduce the expression levels of CYLD mRNA and NGFR mRNA simultaneously shows a more significant effect in inhibiting the proliferation, migration and invasion of angiosarcoma cells. This finding emphasizes the importance of synchronously targeting the CYLD and NGFR genes to maximize the interference with the CYLD-NGFR regulatory axis, thereby more effectively inhibiting tumor progression and metastasis. Therefore, the application strategy of si-CN provides strong support for the development of targeted therapeutic drugs for angiosarcoma, indicating its great potential in improving treatment effects and developing new therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 After si-CN or si-NC was transfected into ISO-HAS cells, reverse transcription-real-time fluorescence quantitative PCR was used to detect the changes in CYLD mRNA and NGFR mRNA levels, and the statistical significance of the differences among the groups was analyzed by Student's t test.

[0020] Figure 2 After transfecting equal doses of si-NC, si-CYLD, si-NGFR and si-CN into ISO-HAS cells, CCK-8 experiment was used to evaluate the effects of si-NC, si-CYLD, si-NGFR and si-CN treatment on the proliferation ability of ISO-HAS cells, and two-way analysis of variance was used to verify the statistical significance of the differences between different treatment groups.

[0021] Figure 3 To transfect equal amounts of si-NC, si-CYLD, si-NGFR, and si-CN into ISO-HAS cells, and determine the changes in cell migration and invasion abilities in each group by Transwell assay, one-way ANOVA was used to determine whether the differences among the groups were statistically significant. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0023] The ISO-HAS cell line is a cell line derived from human angiosarcoma and is available from Shanghai Baili Biotechnology Co., Ltd.

[0024] The si-CYLD sequence used in the present invention to specifically interfere with the expression of the CYLD gene is double-stranded, and its nucleotide sequence is as follows:

[0025] Forward: 5'-GGACAAGCUAGAACUCAUA-3'(SEQ ID NO.3),

[0026] Reverse: 5'-UAUGAGUUCUAGCUUGUCC-3' (SEQ ID NO. 4).

[0027] The si-NGFR sequence used in the present invention for specifically interfering with NGFR gene expression is double-stranded, and its nucleotide sequence is as follows:

[0028] Forward: 5'-GAGCACAUAGACUCCUUUA-3' (SEQ ID NO.5),

[0029] Reverse: 5'-UAAAGGAGUCUAUGUGCUC-3' (SEQ ID NO. 6).

[0030] The negative control si-NC is an existing published sequence.

[0031] Example 1

[0032] In previous studies, the inventors found that the expression levels of CYLD and NGFR genes in angiosarcoma were significantly upregulated, and this abnormal expression was closely related to the poor prognosis of patients. Further research on its mechanism showed that CYLD protein can directly bind to and stabilize NGFR protein, thereby promoting the development of angiosarcoma. Based on these findings, the inventors believe that it may be an effective method to develop new treatment strategies by targeted intervention of the CYLD-NGFR regulatory axis. At the same time, knocking down the expression levels of CYLD mRNA and NGFR mRNA will play a better anti-cancer role in angiosarcoma and improve the treatment effect of angiosarcoma patients. Therefore, this study proposes the possibility of using this regulatory axis as a potential target for the preparation of targeted drugs for angiosarcoma.

[0033] Example 2

[0034] The mRNA nucleotide sequences of the CYLD gene and the NGFR gene were obtained using the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. According to the above sequences, siRNAs that specifically knocked down the expression levels of CYLD mRNA and NGFR mRNA were designed, including si-CYLD and si-NGFR, and the negative control was si-NC. si-CYLD and si-NGFR were mixed in a molar ratio of 1:1 to obtain si-CN. The same dose (volume 3 μL, concentration 10 μM) of si-NC and si-CN were transfected into ISO-HAS cells, and the cells were cultured for 48 hours before RNA was extracted. Reverse transcription-real-time fluorescence quantitative PCR was used to detect the levels of CYLD mRNA and NGFR mRNA. The specific results are shown in the figure. Figure 1 shown.

[0035] Depend on Figure 1 It can be seen that transfection of si-CN can effectively knock down the levels of CYLD mRNA and NGFR mRNA in ISO-HAS cells, interfering with and inhibiting the expression of the CYLD-NGFR regulatory axis.

[0036] The specific implementation process is as follows:

[0037] (1) ISO-HAS cells were seeded into 6-well plates. When the cell growth density reached about 60%, si-NC and si-CN were transfected into ISO-HAS cells using the transfection reagent Lipofectamine RNAiMAX Reagent (ThermoFisher, 13778075). The siRNA dose used was the same (volume 3 μL, concentration 10 μM);

[0038] (2) After 48 hours of transfection, the cells were collected and RNA was extracted using an RNA extraction kit (TIANGEN, DP419). The integrity of the extracted RNA was evaluated by agarose gel electrophoresis, and the concentration of the extracted RNA was detected by NanoDrop2000.

[0039] (3) The above-mentioned RNA was used as a template and a reverse transcription fluorescence quantitative kit (TIANGEN, FP313) was used to synthesize cDNA. Power Green Master Mix (Thermo Fisher Scientific, 4367659) and QuantStudio TM 5System (Thermo Fisher Scientific) was used for fluorescence quantitative PCR analysis. GAPDH was selected as the internal reference gene in the experiment, and according to 2-ΔΔCT The relative expression levels of CYLD mRNA and NGFR mRNA were calculated using the formula.

[0040] Example 3

[0041] ISO-HAS cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of si-NC, si-CYLD, si-NGFR and si-CN, respectively. The CCK-8 experiment was used to detect the interference effect of the above siRNAs on ISO-HAS cell proliferation. The specific results are as follows Figure 2 shown.

[0042] Depend on Figure 2 It can be seen that compared with transfection of si-CYLD alone, transfection of si-CN significantly weakened the proliferation ability of ISO-HAS cells. In addition, the use of si-CN to specifically knock down the expression levels of CYLD mRNA and NGFR mRNA at the same time has a more significant inhibitory effect on the proliferation ability of ISO-HAS cells compared with the same dose of si-CYLD or si-NGFR to knock down the expression levels of CYLD mRNA or NGFR mRNA alone. This shows that the simultaneous knockdown of CYLD and NGFR mRNA expression levels can most effectively inhibit the CYLD-NGFR regulatory axis, thereby achieving the best effect of inhibiting the proliferation of angiosarcoma.

[0043] The specific implementation process is as follows:

[0044] (1) si-NC, si-CYLD, si-NGFR and si-CN were transfected into ISO-HAS cells respectively. The siRNA dose used in each group was the same (volume was 3 μL, concentration was 10 μM). The specific transfection method is detailed in Example 2; the transfected cells were collected and re-cultured in a 96-well plate at a density of 3,000 cells per well; the experiment was divided into 4 groups, each group including four transfected cells of si-NC, si-CYLD, si-NGFR and si-CN, and each cell was set up with 6 replicate wells;

[0045] (2) After the cells adhered (approximately 4-6 hours), the culture medium was aspirated from the 6 replicate wells of group 1, and then a mixed solution was prepared with CCK-8 reagent (Uelandy, C6005M) and culture medium at a ratio of 1:9. 100 μL of the mixed solution was added to each replicate well and incubated in a 37°C incubator for 2 hours. The culture plate was then gently shaken and the absorbance value was measured at a wavelength of 450 nm using an ELISA reader. This was used as the absorbance value of the initial seeded cells.

[0046] (3) At 24, 48, and 72 hours after the completion of the initial cell absorbance measurement, the absorbance values ​​of the remaining three groups of cell wells at a wavelength of 450 nm were measured using CCK-8 reagent, and the relative change level of cell viability in each group was calculated using the absorbance value of the initial seeded cells as a reference.

[0047] Example 4

[0048] The same dose (volume 3 μL, concentration 10 μM) of si-NC, si-CYLD, si-NGFR and si-CN were transfected into ISO-HAS cells, and the Transwell assay was used to detect the changes in cell migration and invasion abilities. The specific results are shown in the figure. Figure 3 shown.

[0049] Depend on Figure 3 It can be seen that at the same transfection dose, si-CN significantly inhibited the migration and invasion ability of ISO-HAS cells compared with the use of si-CYLD and si-NGFR alone. In addition, it was found that the simultaneous use of a mixture of si-CN that knocked down the expression levels of CYLD mRNA and NGFR mRNA significantly inhibited the migration and invasion ability of ISO-HAS cells compared with the use of the same dose of si-CYLD and si-NGFR alone. This shows that the simultaneous knockdown of the expression levels of CYLD mRNA and NGFR mRNA has the best inhibitory effect on the CYLD-NGFR regulatory axis, and is most effective in inhibiting the migration and invasion of angiosarcoma cells.

[0050] The specific implementation process is as follows:

[0051] (1) ISO-HAS cells were selected, and si-NC, si-CYLD, si-NGFR and si-CN were transfected into ISO-HAS cells respectively. The siRNA dose used was the same (volume was 3 μL, concentration was 10 μM), and the operation method was consistent with Example 2;

[0052] (2) Place a Transwell chamber (Corning, 3422) in a 24-well plate, which is pre-coated with Matrigel to evaluate cell invasion ability and uncoated with Matrigel to evaluate cell migration ability; add 600 μL of complete culture medium to each well;

[0053] (3) After collecting the transfected cells, resuspend them in serum-free medium and adjust the cell concentration to 20,000 cells per 200 μL. Then, add 200 μL of the cell suspension into the Transwell chamber and culture the cells in an incubator for 48 hours;

[0054] (4) The Transwell chamber was removed and the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes. The fixative was discarded and the chamber was washed with PBS. Then, the chamber was stained with 0.1% crystal violet dye and washed 2 to 3 times with PBS buffer. The non-migrated or invaded cells in the chamber were scraped off with a cotton swab, and the migrated or invaded cells were observed under an inverted microscope and photographed for record.

Claims

1. Application of the CYLD-NGFR regulatory axis as a drug target in the preparation of targeted drugs for the treatment of angiosarcoma.

2. The use according to claim 1, characterized in that The CYLD-NGFR regulatory axis includes the CYLD gene and the NGFR gene.

3. The use according to claim 2, characterized in that The mRNA nucleotide sequence of the CYLD gene is shown as SEQ ID NO.1, and the mRNA nucleotide sequence of the NGFR gene is shown as SEQ ID NO.

2.

4. The use according to claim 1, characterized in that The targeted therapy for angiosarcoma uses the CYLD gene and the NGFR gene as intervention targets, based on interfering with CYLD mRNA and NGFR mRNA, and can effectively and specifically inhibit the expression levels of the CYLD gene and the NGFR gene.

5. The use according to claim 1, characterized in that The targeted therapy drug for angiosarcoma is siRNA, shRNA, microRNA or chemical inhibitor.

6. Application of siRNA that specifically interferes with CYLD gene expression and siRNA that specifically interferes with NGFR gene expression in the preparation of targeted drugs for the treatment of angiosarcoma.

7. The use according to claim 6, characterized in that The siRNA that specifically interferes with the expression of the CYLD gene is double-stranded, and its nucleotide sequence is shown in SEQ ID NOs.3-4; the siRNA that specifically interferes with the expression of the NGFR gene is double-stranded, and its nucleotide sequence is shown in SEQ ID NOs.5-6.

8. The use according to claim 6, characterized in that The molar ratio of the siRNA that specifically interferes with the expression of the CYLD gene and the siRNA that specifically interferes with the expression of the NGFR gene in the targeted treatment drug for angiosarcoma is 1:

1.

9. A drug for targeted treatment of angiosarcoma, characterized in that: The targeted treatment drug for angiosarcoma comprises siRNA that specifically interferes with CYLD gene expression and siRNA that specifically interferes with NGFR gene expression, and the molar ratio of the two is 1:

1.

10. The targeted treatment drug for angiosarcoma according to claim 9, characterized in that: The siRNA that specifically interferes with the expression of the CYLD gene is double-stranded, and its nucleotide sequence is shown in SEQ ID NOs.3-4; the siRNA that specifically interferes with the expression of the NGFR gene is double-stranded, and its nucleotide sequence is shown in SEQ ID NOs.5-6.

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