WO2023029489A1

WO2023029489A1 - Oligonucleotide rna double-stranded molecule and use thereof in preparation of drug for treating malignant tumor

Info

Publication number: WO2023029489A1
Application number: PCT/CN2022/086705
Authority: WO
Inventors: 张小雷; 刘培庆; 熊小峰; 王延东; 巫瑞波; 欧阳淑敏; 石硕; 朱剑征; 葛阳
Original assignee: 中山大学
Priority date: 2021-09-03
Filing date: 2022-04-13
Publication date: 2023-03-09
Also published as: CN113980963A

Abstract

Provided are an oligonucleotide RNA double-stranded molecule and the use thereof in the preparation of a drug for treating a malignant tumor. The oligonucleotide RNA double-stranded molecule is siRNAs, namely siRNA-1 and siRNA-2, which is directed to VGF. The siRNAs have high selectivity and strong specificity for a VGF protein, have an obvious inhibitory effect on the VGF protein, and can significantly inhibit tumor proliferation, induce tumor cell apoptosis, inhibit tumor cell dryness, inhibit tumor cell migration and invasion and significantly reduce the tumor volume and the tumor weight in vivo.

Description

An oligonucleotide RNA double-stranded molecule and its application in the preparation of medicines for treating malignant tumors

technical field

The invention relates to the technical field of medicine, in particular to an oligonucleotide RNA double-stranded molecule and its application in the preparation of medicines for treating malignant tumors.

Background technique

Malignant tumors are a major killer of contemporary human health. At present, the commonly used clinical tumor treatment methods include: chemotherapy, radiotherapy, surgery, etc., but there are still many problems in tumor treatment, such as: tumor drug resistance, metastasis, postoperative metastasis, and toxic and side effects of drugs and radiation. In recent years, with the advancement of life sciences, the treatment of tumors has begun to develop into targeted therapy, and the use of small interfering nucleotide therapy is one of the targeted therapy methods.

Small interfering nucleotides (siRNA) are a class of double-stranded RNA molecules with a length of 20-25 base pairs, which can form an RNA-induced silencing complex (RISC) and silence specific genes. The biggest advantage of siRNA as a tumor treatment drug is that it can efficiently and specifically inhibit the expression level of homologous mRNA with good specificity. Although the siRNA effect in mammalian cells is relatively transient, the application of siRNA vectors has enabled the inheritance of RNA inhibitory effects in mammals. Therefore, small interfering nucleotide drugs have good application prospects.

Theoretically, siRNA drugs can be applied to any link in the process of tumorigenesis, development and metastasis with abnormally high expression of genes. The current strategy of small interfering nucleotide drugs in the field of tumor treatment mainly focuses on the following aspects. 1. Targeting tumor-related fusion gene transcription products; 2. Targeting overexpressed oncogenes or apoptosis inhibitors; 3. Targeting tumor drug resistance genes; 4. Targeting tumor angiogenesis factors and receptors; 5. Allele specificity inhibition.

VGF is a kind of secretable neuropeptide precursor protein that is rapidly induced by the treatment of mouse adrenal pheochromocytoma cells with nerve growth factor (neural growth factor inducible gene, NGF). Studies (The Journal of Neuroscience, 2003,23(34):10800-8) have shown that many neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophic factor-3 (neurotrophin- 3, NT-3) can also induce the expression of VGF in neurons. In addition, VGF is a kind of polypeptide with relatively high conservation, and there is about 85% sequence identity in the amino acid sequences of human, mouse, horse and bovine. Human VGF contains 615 amino acids and a molecular weight of about 68kD, which can be cleaved into different peptides with specific neuronal biological activities. The spliced peptide of VGF is associated with many neuroendocrine actions and plays an important role in many diseases, especially energy metabolism, pain, reproduction and cognition. Although the research on the molecular mechanism and signaling events of VGF-derived active neuropeptides to exert their physiological effects is still in its infancy. However, there are a few studies (Cancer Res, 2017, 11:3013-3026, Endocr Relat Cancer, 2019, 7: 643-658, Cell Stem Cell, 2018, 4: 514-528.e5) indicating that VGF plays a role in tumor formation. If small interfering nucleotide drugs targeting VGF can be developed, it is expected to treat malignant tumors by inhibiting VGF targets.

To sum up, drugs targeting VGF have good prospects, but there is no drug targeting VGF target protein at present, which greatly limits the development and application of drugs targeting VGF. As VGF is a promising target for tumor therapy, there is an urgent need to develop small nucleotide drugs that can block VGF and drug regimens that use VGF as a target to treat malignant tumors.

Contents of the invention

The purpose of the present invention is to aim at malignant tumors, especially melanoma, and the present invention provides an oligonucleotide RNA double-stranded molecule. The present invention finds that siVGF-1 and siVGF-2 nucleotide drug molecules can specifically reduce the expression of VGF in tumor cells, and can significantly inhibit tumor proliferation, induce tumor cell apoptosis, inhibit tumor cell stemness, inhibit tumor cell migration and Invasion, and can significantly reduce the volume and weight of tumors in the body, and significantly inhibit the growth and proliferation of tumors. Therefore, the nucleotide drug molecule has wide application in the preparation of anti-malignant tumor drugs.

Another object of the present invention is to provide an expression vector.

Another object of the present invention is to provide a host cell.

Another object of the present invention is to provide the application of the above-mentioned oligonucleotide RNA double-stranded molecule or its pharmaceutically acceptable salt or solvate in the preparation of a drug for inhibiting the activity of VGF protein.

Another object of the present invention is to provide the application of the above-mentioned oligonucleotide RNA double-stranded molecule or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for treating malignant tumors.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

An oligonucleotide RNA double-stranded molecule, its sequence is one or more of the following nucleotide sequences:

siVGF-1:

Sense strand: 5'-GAAUUACAUCGAGCACGUGCU-3' (as shown in SEQ ID NO: 1)

Antisense strand: (as shown in SEQ ID NO: 2)

siVGF-2:

Sense strand: 5'-GACGAUCGACAGCCUCAUUGA-3' (as shown in SEQ ID NO: 3)

Antisense strand: (as shown in SEQ ID NO: 4).

The present invention found that siRNA nucleotide sequences siVGF-1 and siVGF-2 with specific structures can specifically reduce the expression of VGF in tumor cells, and can significantly inhibit tumor proliferation, induce tumor cell apoptosis, inhibit tumor cell stemness, It can inhibit the migration and invasion of tumor cells, and can significantly reduce the volume and weight of tumors in the body, and obviously inhibit the growth and proliferation of tumors, and has a wide range of applications in the preparation of anti-malignant tumor drugs.

An expression vector contains the above-mentioned oligonucleotide RNA double-stranded molecule.

A host cell containing the above-mentioned oligonucleotide RNA double-stranded molecule, or containing the above-mentioned expression vector.

The application of the above-mentioned oligonucleotide RNA double-strand molecule or its pharmaceutically acceptable salt or solvate in the preparation of a drug for inhibiting the activity of VGF protein.

It is found through research that the oligonucleotide RNA double-stranded molecules siVGF-1 and siVGF-2 of the present invention can inhibit the invasion, infiltration, growth, proliferation and cloning of malignant tumor cells, especially melanoma cells.

Application of the above-mentioned oligonucleotide RNA double-strand molecule or its pharmaceutically acceptable salt or solvate in the preparation of a drug for treating malignant tumors.

Preferably, the drug is a drug that inhibits the growth and/or proliferation of malignant tumor cells.

Preferably, the drug is a drug that inhibits metastasis and/or invasion of malignant tumor cells.

Preferably, malignant tumors include but are not limited to: melanoma, gastric cancer, breast cancer, rectal cancer, non-small cell lung cancer, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, Anal cancer, extrahepatobiliary duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumor, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor, unknown primary cancer, central nervous system lymphoma, sub Cervical cancer, kidney cancer, laryngeal cancer, blood cancer, liver cancer, prostate tumor, salivary gland cancer, sarcoma, small bowel cancer, soft tissue sarcoma, uterine sarcoma, testicular cancer, ovarian cancer, rhabdoid tumor, synovial sarcoma, mesothelioma, skin cancer , oral cancer, fallopian tube tumor, peritoneal tumor, glioma, glioblastoma, myeloma.

Preferably, the drug is used in combination with a G protein inhibitor, and the G protein inhibitor includes but is not limited to GQ127.

Specifically, through experiments on cell models and subcutaneous cell xenografts in nude mice, it was found that siVGF-1 and siVGF-2 nucleotide RNA double-stranded molecules can specifically reduce the expression of VGF in tumor cells, and at the same time significantly inhibit tumor proliferation, induce Tumor cell apoptosis, inhibition of tumor cell stemness, inhibition of tumor cell migration and invasion, and can significantly reduce the volume and weight of tumors in vivo, and significantly inhibit tumor growth and proliferation. The combination of nucleotide drug molecule and G protein inhibitor GQ127 can significantly enhance its effect.

The term "pharmaceutically acceptable" means that a certain carrier, diluent or excipient, and/or the salt formed is usually chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form, and physiologically compatible with compatible with the receptor.

The term "acceptable salt" refers to the above-mentioned compounds or their stereoisomers, acidic and/or basic salts formed with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), including Quaternary ammonium salts, such as alkyl ammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base as appropriate (for example, equivalent). These salts may form precipitates in solution and be collected by filtration, or may be recovered after evaporation of the solvent, or may be obtained by freeze-drying after reaction in an aqueous medium.

More preferably, the pharmaceutically acceptable salt is a pharmaceutically acceptable inorganic or organic salt.

Specifically, pharmaceutically acceptable salts include, but are not limited to: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid Phosphate, Isonicotinate, Lactate, Salicylate, Acid Citrate, Tartrate, Oleate, Tannate, Pantothenate, Bitartrate, Ascorbate, Succinate, Horse Tonate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate (methanesulfonate salt), ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate; or ammonium salts (such as primary amine salts, secondary amine salts, tertiary amine salts, quaternary ammonium salts), Metal salts (eg sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts, copper salts, lithium salts, aluminum salts).

Preferably, the dosage form of the drug is injection, capsule, tablet, pill or granule.

Compared with the prior art, the present invention has the following advantages and effects:

The siVGF-1 and siVGF-2 oligonucleotide RNA double-stranded molecules provided by the present invention have high selectivity and strong specificity for VGF protein, have significant inhibitory effect on VGF protein, and can be used as VGF-specific nucleotide drug molecules ; In addition, the nucleotide sequence can significantly inhibit tumor proliferation, induce tumor cell apoptosis, inhibit tumor cell stemness, inhibit tumor cell migration and invasion, and can significantly reduce the volume and weight of tumors in vivo, and significantly inhibit tumor growth. Growth and proliferation. Therefore, the nucleotide drug molecule or its pharmaceutically acceptable salt or solvate has wide application in the preparation of medicines for treating malignant tumors.

Description of drawings

Figure 1 shows the test results of cell viability after silencing VGF gene with siVGF-1 and siVGF-2 in human cancer cell lines (lung cancer PC-9, gastric cancer AGS, triple negative breast cancer MDA-MB-231, colon cancer HCT-116) picture;

Figure 2 is the result of cell counting experiment of melanoma cell lines MP41 and 92-1 after silencing VGF gene with siVGF-1 and siVGF-2; Figure 2A is the result of MP41 cell experiment, and Figure 2B is the result of 92-1 cell experiment.

Figure 3 is the results of the EDU cell proliferation detection experiment of melanoma cell lines MP41 and 92-1 after silencing the VGF gene with siVGF-1 and siVGF-2; Figure 3A is the result of EDU staining of MP41 cells, and Figure 3B is the staining of 92-1 cells As a result, Figure 3C shows the technical statistics of MP41 proliferative active cells, and Figure 3D shows the technical statistics of 92-1 proliferative active cells.

Figure 4 shows the results of melanoma cell lines MP41 and 92-1 using siVGF-1 and siVGF-2 to silence the VGF gene and then detecting cell apoptosis by flow cytometry; the left side of Figure 4 is the result of flow cytometry analysis, and the right side is the result of flow cytometry Analyze statistics.

Fig. 5 is a graph showing the results of cell scratch experiments of melanoma cell lines MP41 and 92-1 after the VGF gene was silenced by siVGF-1 and siVGF-2.

Fig. 6 is a diagram showing the results of transwell cell invasion experiments of melanoma cell lines MP41 and 92-1 after VGF gene was silenced by siVGF-1 and siVGF-2.

Fig. 7 is a sphere formation experiment of melanoma cell lines MP41 and 92-1 after VGF gene was silenced by siVGF-1 and siVGF-2.

Figure 8 shows the results of siVGF used alone or in combination with the G protein inhibitor GQ127 in animal models to inhibit the growth of melanoma; the experiments were divided into Vehicle group, siControl group, siVGF-1 group, GQ127 10mg/kg group and GQ127 10mg/kg +siVGF-1 group, Figure 8A is the growth curve of tumor volume over time for each group, Figure 8B is the weight of tumor tissue at the end of the experiment for each group, and Figure 8C is a picture of representative tumor tissue for each group.

Detailed ways

The present invention is further explained below in conjunction with the examples and accompanying drawings, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

The sequence information mentioned in each embodiment of the present invention is as follows:

1. CDS region sequence of VGF gene (such as SEQ ID NO: 5):

2. VGF protein sequence (such as SEQ ID NO: 6):

3. siVGF-1: 5'-GAAUUACAUCGAGCACGUGCU-3'

4. siVGF-2: 5'-GACGAUCGACAGCCUCAUUGA-3'

Below in conjunction with embodiment, further set forth the present invention.

Embodiment 1: Construction and expression of siVGF cell line

1) 92-1 cells, MP41 cells, MDA-MB-231 cells, PC-9 cells, HCT-116 cells and AGS cells were respectively treated with RIPM1640 medium containing 10% serum in a 100mm cell culture dish (92-1 cells , MP41 cells, PC-9 cells, HCT-116 cells and AGS cells) or 10% serum DMEM high glucose medium (MDA-MB-231 cells) cultured until the cells grew to a confluence of 70-80%.

2) Digest the cells, inoculate each cell line into a 6-well plate with 100,000 cells per well, and culture overnight.

3) After each cell adheres to the wall, the cell culture medium is sucked off, and 800 μl of serum-free RIPM1640 medium or DMEM high-glucose medium is added to each well of the six-well plate.

4) According to the number of holes in the six-well plate to be processed, prepare the following solutions:

Solution 1: 3μL dhramofect+97μl opti-MEM for each aliquot, make several aliquots according to the number of wells to be treated.

Solution 2: 2.5μl VGF small interfering RNA + 97.5μl opti-MEM per aliquot, make several aliquots according to the number of wells to be treated.

Solution 1 and solution 2 were respectively incubated at room temperature for 5 min, and then solution 1 and solution 2 were mixed evenly, and incubated at room temperature for 20 min. After the incubation, 200 μl of the mixed solution was added to the six-well plate per well.

5) Place the six-well plate in a 37°C CO incubator for 6 hours, then suck out the medium in the cells, and replace it with 10% serum RIPM1640 medium or DMEM high-glucose medium for culture.

Example 2: siVGF inhibits the proliferation of cells such as gastric cancer, lung cancer, triple-negative breast cancer, and colorectal cancer

1) Cell transfection

According to the method in Example 1, MDA-MB-231, PC-9, HCT-116 and AGS cells were transfected to obtain siControl, siVGF-1 and siVGF-2, respectively.

2) Cell plating

The 15 kinds of cells obtained by transfection were inoculated into 96-well plates according to 2000 cells/well, and RIPM1640 medium (PC-9, HCT-116 and AGS cells) or DMEM high-glucose medium containing 10% serum was added to each well ( MDA-MB-231 cells) to 100 μl, and each cell was repeatedly inoculated into three wells. At the same time, 100 μl of RIPM1640 medium containing 10% serum was added to three wells, and DMEM with 10% serum was added to the other three wells as a blank control. The 96-well plate was placed in an incubator and cultured for 72 hours.

3) CCK8 cell proliferation detection kit to measure cell viability

Take out the 96-well plate and add 10 μl of CCK8 solution to each well. During the liquid addition process, it needs to be protected from light, and then placed in a 37°C incubator for dark incubation. At certain intervals of incubation, use a microplate reader to measure the OD value at 450nm. The measured OD value is between 0.8-1.5. If the OD value does not reach 0.8, continue to incubate in the dark. The total incubation time should be between 30min-4h. Measure the OD value of each well minus the average OD value of the corresponding blank medium to get the result. The result is shown in Figure 1. It can be seen from Figure 1 that the cell viability of each cancer cell line decreased after being treated with siVGF-1 and siVGF-2, suggesting that siVGF-1 and siVGF-2 can inhibit the growth and proliferation of various malignant tumor cell lines.

Example 3: si-VGF inhibits the growth and proliferation of melanoma cells

1) Cell transfection

According to the method in Example 1, the MP41 cells of siCONTROL, siVGF-1, siVGF-2 and the 92-1 cells of siCONTROL, siVGF-1, siVGF-2 were transfected

2) Cell plating

The 6 kinds of cells obtained by transfection were inoculated on a 6-well plate at 1×105 cells/well, and 1 ml of RIPM1640 medium containing 10% serum was added to each well, and 6 wells of each cell were repeatedly inoculated, and 3 wells were used Cells were counted after three days, and the remaining 3 wells were used for cell counts after five days.

3) Cell count

Three days later, the 6-well plate was taken out, the culture medium was sucked off, 1ml of PBS was added to each well for washing, and after the PBS was discarded, it was digested with 300μl of trypsin at room temperature for 1min. Then, 1 ml of RIPM1640 medium containing 10% serum was added to each well to terminate the digestion, and the cell suspension was collected separately, centrifuged at 2000 rpm for 2 min, and the supernatant was discarded. The cells were resuspended with 1ml of RIPM1640 medium containing 10% serum, and the cells were counted.

Five days later, the remaining 6-well plates were taken out, and the above steps were repeated for cell counting. The result is shown in Figure 2. It can be seen from Figure 2 that after treatment with siVGF-1 and siVGF-2 for 3 days and 5 days, the number of viable cells in MP41 and 92-1 was significantly reduced compared with the siControl group; siVGF-1 and siVGF-2 can inhibit the proliferation and growth of melanoma cells.

Example 4: Determination of siVGF inhibiting melanoma cell proliferation by EDU staining experiment

1) Sample processing:

92-1 and MP41 cells were cultured in six-well plates at 100,000/well. Cells were transfected with siControl, siVGF-1 and siVGF-2 according to the cell transfection method in Example 1(1). After 72 hours of transfection, EdU reagent was added and cultured for 2 hours.

2) Fixation and staining

The culture medium of the sample was discarded, and 4% paraformaldehyde was added to fix it for 15 minutes. After that, residual paraformaldehyde was washed away with PBS, and then treated with PBS solution containing 0.3% TritonX-100 for 15 min. After washing away the rupture agent, add 100 μl Hoechst 33342 to each well and incubate for 30 min. Hoechst 33342 was then washed away with PBS.

3) Imaging

The cells were photographed with a fluorescence microscope, and the results are shown in Figure 3. It can be known from Figure 3 that siVGF-1 and siVGF-2 can significantly inhibit the proliferation of MP41 and 92-1 cells.

Example 5: Detection of siVGF-induced apoptosis in tumor cells by flow cytometry

1) Sample processing:

92-1 and MP41 cells were cultured in six-well plates at 100,000/well. Cells were transfected with siControl, siVGF-1 and siVGF-2 according to the cell transfection method in Example 1(1). After 72 hours of transfection, the medium was aspirated and the cells were digested. The digested cells were collected in a centrifuge tube and centrifuged at 1000rpm for 1min. After centrifugation, add 400 μl of binding solution to each group of cells and place them on ice.

2) Cell staining

Add 2.5 μl of Annexin V-FITC dye to the cells, pipette evenly, and stand at 4°C in the dark for 15 minutes, then add 5 μl of PI dye, pipette gently, and let stand at 4°C for 5 minutes.

3) Quadrant division

Clean the flow cytometer, inject samples from the siControl group, and divide the quadrants with Green B on the abscissa and Red B on the ordinate.

4) Sorting apoptotic cells by flow cytometry

Set the number of cells to 10000 for flow cytometry sorting, the results are shown in Figure 4. It can be seen from Figure 4 that the results showed that the apoptosis of cells after treatment with siVGF-1 and siVGF-2 was significantly increased, suggesting that siVGF-1 and siVGF-2 can promote tumor apoptosis.

Example 6: siVGF reduces the migration ability of melanoma cells

1) Cell transfection

92-1 and MP41 cells were cultured in six-well plates at 100,000/well. Cells were transfected with siControl, siVGF-1 and siVGF-2 according to the cell transfection method in Example 1(1).

2) Scratches

After transfection, when the cells grow to a density between 90% and 100%, scratch the six-well plate along the ruler with a pipette tip, paying attention to make the scratch uniform. The medium was then aspirated and the cells were washed with PBS. Then add the culture medium again.

3) Microscope imaging

Put the six-well plate in the microscope, and take pictures of the scratches with a 4x lens. Three fields of view were taken for each well, and the results are shown in Figure 5. It can be seen from Figure 5 that after being treated with siVGF-1 and siVGF-2 for 24 hours, the scratch recovery ability of MP41 and 92-1 cells decreased, indicating that siVGF-1 and siVGF-2 inhibited the migration ability of MP41 and 92-1 cells.

Example 7: siVGF reduces the invasive ability of melanoma cells

1) Cell transfection

2) Cell plating

Inoculate the above six kinds of cells into the upper wells of the transwell plate at 1× ¹⁰⁶ cells/well, add RIPM1640 medium containing 10% serum to each well of 300 μl system, and at the same time, add 500 μl containing 10 to the lower well of the transwell plate % serum RIPM1640 medium, cultured for 24 hours, until the cells adhered to the wall.

3) Cell invasion

Take out the transwell plate, replace the medium in the upper well with serum-free RIPM1640 medium, and replace the medium in the lower well with RIPM1640 medium containing 20% serum, and culture for 48 hours.

4) Staining and imaging

Take away the wells on the transwell plate, suck away the medium in the lower wells of the transwell plate, add 300-400 μl of 4% paraformaldehyde to each well, and fix for 15 minutes. After fixation, absorb paraformaldehyde, add PBS to each well along the wall of the transwell plate, and rinse for 1-2 days. Then add 300-400 μl of crystal violet to each well, stain in the dark for 30 minutes, and absorb the crystal violet. It is then rinsed with ultrapure water to remove residual crystal violet, and allowed to air dry.

After the cells in the transwell plate were air-dried, the cell invasion was photographed with a 4x microscope, and the results are shown in Figure 6. It can be seen from Figure 6 that after treatment with siVGF-1 and siVGF-2, the number of MP41 cells and 92-1 cells invading into the lower hole of the transwell decreased, indicating that siVGF-1 and siVGF-2 can inhibit the infiltration of melanoma cells.

Example 8: siVGF inhibits melanoma cell stemness into spheres

1) Cell transfection

2) Tumor stem cell sphere formation experiment

The 6 kinds of cells obtained above were resuspended with sphere forming medium (DMEM-F12 medium containing 0.4% BSA+5 μg/ml insulin+20ng/ml EGF+20ng/ml FGF). Afterwards, 2000 cells per well were seeded into low-adsorption 6-well plates. Add sphere media to 1 ml per well.

Collect the cell suspension in each well every three days, discard the supernatant after centrifugation, gently resuspend the cells with new spheroidizing medium, and reseet them in 6-well plates. After 15 days, the state of cell sphere formation was recorded with a 20X microscope, and the results are shown in Figure 7. It can be seen from Figure 7 that, compared with the siControl group, the sphere-forming ability of the tumor cells in the siVGF-1 group and the siVGF-2 group was significantly reduced. It shows that siVGF-1 and siVGF-2 inhibit the stemness of MP41 and 92-1 cells.

Example 9: siVGF alone or in combination with the G protein inhibitor GQ127 inhibits the growth of melanoma in animal models

1) Construction of xenograft tumor model

MP41 was inoculated subcutaneously on both sides of the back of Balb/c nude mice at 3×10 ⁷ cells/only, when the tumor volume reached 100 mm3. The nude mice were randomly divided into 5 groups, 7 in each group, 5 groups in total, namely blank control group, siControl group, siVGF group, GQ127 group and GQ127+siVGF-1 group, and recorded as day 0.

3) Administration

From day 0, the blank group was injected intraperitoneally with cosolvent daily, and the GQ127 10mg/kg group was injected intraperitoneally with 10mg/kg of GQ127 daily. The siControl group, siVGF-1 group and GQ127 10mg/kg+siVGF-1 group were injected intratumorally with the transfection complex of siControl or siVGF:RNA transfection reagent=2:1 every three days. The tumor volume of nude mice was recorded every three days.

3) Determination of antitumor activity

After 18 days of administration, the nude mice were killed and the subcutaneous tumors on the back of the nude mice were taken out, and the tumor size and tumor weight were compared. The result is shown in Figure 8. It can be seen from Figure 8 that siVGF-1 alone significantly inhibited tumor growth, and the combination of G protein inhibitor GQ127 and siVGF-1 treated tumor-bearing mice had a more obvious tumor inhibitory effect. It shows that siVGF-1 significantly inhibits tumor growth, and the combination of G protein inhibitor GQ127 has obvious synergistic anti-tumor effect.

Based on the above results, the present invention proves the therapeutic effect of siVGF nucleotide fragments on various malignant tumors, especially melanoma, and lays the foundation for further clinical application development.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims

An oligonucleotide RNA double-stranded molecule is characterized in that its sequence is one or more of the following nucleotide sequences:

siVGF-1:

Sense strand: 5'-GAAUUACAUCGAGCACGUGCU-3'

Antisense strand: 5'-CUUAAUGUAGCUCGUGCACGA-3'

siVGF-2:

Sense strand: 5'-GACGAUCGACAGCCUCAUUGA-3'

Antisense strand: 5'-CUGCUAGCUGUCGGAGUAACU-3'.
An expression vector, characterized in that it contains the oligonucleotide RNA double-stranded molecule as claimed in claim 1.
A host cell, characterized in that it contains the oligonucleotide RNA double-stranded molecule as claimed in claim 1, or contains the expression vector as claimed in claim 2.
The use of the oligonucleotide RNA double-stranded molecule or its pharmaceutically acceptable salt or solvate in claim 1 in the preparation of a drug for inhibiting the activity of VGF protein.
The use of the oligonucleotide RNA double-stranded molecule of claim 1 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for treating malignant tumors.
The application according to claim 5, wherein the drug is a drug that inhibits the growth and/or proliferation of malignant tumor cells.
The use according to claim 5, characterized in that the drug is a drug that inhibits metastasis and/or invasion of malignant tumor cells.
The use according to claim 5, characterized in that the drug is used in combination with a G protein inhibitor.
The application according to claim 5, characterized in that the medicament further comprises one or more of pharmaceutically acceptable carriers, diluents or excipients.
The application according to claim 5, wherein the dosage form of the medicine is injection, capsule, tablet, pill or granule.