A double nucleic acid sequence for inhibiting melanoma growth and its application

Abstract

The present invention provides a dual nucleic acid sequence for inhibiting melanoma growth and its application, as well as the use of a substance that reduces the expression levels of the ITK and MAT2B genes in the preparation of a drug for treating or preventing melanoma. The substance that reduces the expression levels of the ITK and MAT2B genes includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, and substances for implementing lentiviral infection or gene knockout targeting the ITK and MAT2B genes. The present invention provides the use of the ITK and MAT2B genes in the treatment of melanoma. By specifically interfering with the ITK and MAT2B genes, the malignant progression of melanoma can be effectively inhibited. The ITK and MAT2B genes can be used as targets for the treatment of melanoma and have good clinical application value.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and medical technology, and in particular to a double nucleic acid sequence for inhibiting melanoma growth and its application. Background Art

[0002] Melanoma is a malignant tumor originating from melanocytes, with a continuously increasing incidence worldwide. Although melanoma is a rare malignancy in my country, it carries a high mortality rate and a significant economic burden. Among Asians and other populations of color, acral melanomas account for approximately 50% of cases, with primary sites most commonly located on the extremities, such as the soles of the feet, fingertips, and subungual areas. In contrast, skin melanomas account for approximately 90% of cases in Caucasians. The 5-year survival rates for stages I to IV of skin melanoma are 97%, 84%, 68%, 55%, and 17%, respectively. Melanoma often presents a poor prognosis and is prone to metastasis in early stages. In addition to chemotherapy, immunotherapy and targeted therapies have made significant progress. However, the Chinese population is predominantly characterized by acral melanomas, which have a low prevalence of genetic mutations, hindering the use of targeted agents. Traditional treatments have limited efficacy due to their poor sensitivity, significant side effects, and high drug resistance. There is an urgent need to explore novel treatment options to improve efficacy, prolong patient survival, and enhance quality of life.

[0003] The IL-2-induced T-cell kinase ITK (interleukin-2 inducible T-cell kinase) is expressed in melanoma and promotes its growth. Inhibiting ITK activity can inhibit melanoma cell growth, making ITK a potential drug target for melanoma. However, only one drug currently inhibits ITK in the treatment of hematological cancers, making the development of drugs targeting ITK of great significance.

[0004] The protein encoded by the MAT2B gene belongs to the MAT (methionine aminotransferase) family and is a regulatory subunit of MAT. MAT2B is highly expressed in malignant melanoma cell lines. Downregulation of the MAT2B gene inhibits cell proliferation, promotes apoptosis, and suppresses tumor growth in mice. MAT2B is a potential molecular target for malignant melanoma. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a double nucleic acid sequence for inhibiting melanoma growth and its application, so as to solve the problem of limited efficacy of existing melanoma treatment.

[0006] The present invention provides an application of a double nucleic acid sequence for inhibiting melanoma growth, and an application of a substance for reducing the expression levels of ITK gene and MAT2B gene in the preparation of a drug for treating or preventing melanoma.

[0007] The functions of the substance that reduces the expression levels of ITK gene and MAT2B gene include any one or more of the following: (1) inhibiting the proliferation of melanoma cells; (2) inhibiting the ability of melanoma cells to form colonies; and (3) reducing the ability of melanoma cells to migrate.

[0008] The substances that reduce the expression levels of ITK gene and MAT2B gene include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA targeting ITK gene and MAT2B gene, and substances for implementing lentiviral infection or gene knockout.

[0009] The substance for implementing gene interference is siRNA interference sequence:

[0010] wherein siITK: sense: ACU CAG AGG UGG UGG AAG A, the sequence is as shown in SEQ ID NO: 1;

[0011] antisense: UGA GUC UCC ACC ACC UUC U, sequence as shown in SEQ ID NO: 2;

[0012] siMAT2B: sense: GAC AAG AGA UGG AGA CAA A, sequence as shown in SEQ ID NO: 3;

[0013] antisense: CUG UUC UCU ACC UCU GUU U, the sequence is shown in SEQ ID NO:4.

[0014] The present invention has the following beneficial effects: providing the application of ITK gene and MAT2B gene in the treatment of melanoma, effectively inhibiting the malignant progression of melanoma by specifically interfering with ITK gene and MAT2B gene, and ITK gene and MAT2B gene can be used as targets for the treatment of melanoma, and have good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a qPCR effect diagram of the ITK gene and MAT2B gene in melanoma cells before and after interference in this Example 1.

[0016] Figure 2 This is a comparison chart of the proliferation ability of melanoma cells after interference with the ITK gene and the MAT2B gene in Example 2.

[0017] Figure 3 This is a comparison chart of melanoma cell apoptosis after interference with ITK gene and MAT2B gene in Example 3.

[0018] Figure 4This is a diagram showing the cloning analysis results of the proliferation ability of melanoma cells after interference with the ITK gene and the MAT2B gene in Example 4.

[0019] Figure 5 This is a Transwell result diagram of the migration ability of melanoma cells after interference with ITK gene and MAT2B gene in Example 5. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some representative embodiments of the present invention, rather than all embodiments. All other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0021] Example 1

[0022] In this example, the qPCR effects of ITK gene and MAT2B gene interference (shRNA lentivirus infection of A375 cells) in A375 melanoma cells were verified before and after.

[0023] This example includes a qPCR fluorescence quantitative PCR experiment, which includes the following methods:

[0024] RNA extraction

[0025] (1) Collect cells (6-well plate), centrifuge at 2000 rpm for 5 minutes, remove the supernatant, add 1 mL of Trizol reagent to the cell pellet, mix well, let it stand at room temperature for 5 minutes, and then transfer it to a new 1.5 mL tube.

[0026] (2) Add 200 μL of chloroform to each tube, invert the EP tube for 15 seconds, and let it stand at room temperature for 10 minutes. Centrifuge at 4°C and 12,600 rpm for 15 minutes.

[0027] (3) Transfer the upper layer of liquid to a new 1.5 mL EP tube, add an equal volume of pre-cooled isopropanol, mix well, and let stand at 4°C for 10 min. Centrifuge at 4°C, 12,600 rpm for 12 min, and discard the supernatant.

[0028] (4) Add 1 mL of 75% ethanol to wash the precipitate. Centrifuge at 12,000 rpm at 4°C for 5 min and discard most of the supernatant.

[0029] (5) Centrifuge at 12,000 rpm for 5 min at 4°C, discard the supernatant, and dry at room temperature.

[0030] (6) When the RNA precipitate becomes transparent, add RNase-free water until it is completely dissolved. Analyze the concentration and quality of the extracted RNA using a Nanodrop 2000 / 2000C spectrophotometer.

[0031] Reverse transcription to obtain cDNA:

[0032] RNA Reverse Transcription

[0033] (1) Add 1 μL of Oligo dT and 2 μg of Total RNA to a PCR tube and add RNase-Free H2O to 10 μL; mix well and centrifuge, then incubate at 70°C for 10 min; then place in an ice bath in an ice-water mixture to allow the Oligo dT and template to anneal.

[0034] (2) Prepare the reaction system in the above mixture according to the following proportions: 4 μL 5×RT buffer, 2 μL 10 mM dNTPs, 0.4 μL RNase (40 U / μL), 1 μL M-MLV-RTase (200 U / μL), and 2.6 μL RNase-Free H2O.

[0035] (3) The above system was reacted in a 42℃ water bath for 1 h, then in a 70℃ water bath for 10 min, and the obtained RT product cDNA was stored at -20℃ for later use.

[0036] Real-time PCR detection

[0037] (1) Prepare the reaction system according to the following proportions: SYBR premix ex taq 6.0 μL, primer mix (5 μM) 0.3 μL, template (reverse transcription product) 0.6 μL, RNase-Free H2O 5.1 μL.

[0038] (2) Real-time PCR was performed in two steps and a melting curve was prepared. The procedure was as follows: stage 1: 95°C for 30 s; stage 2: 95°C for 5 s, 60°C for 30 s, 40 cycles; stage 3: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s.

[0039] Data Analysis

[0040] Relative quantitative analysis ΔCt = target gene Ct value - reference gene Ct value; -ΔΔCt = average ΔCt value of NC group - ΔCt value of each sample; 2 -ΔΔCt Reflects the relative expression level of the target gene in each sample relative to the NC group.

[0041] Reference Figure 1As shown, the bar graph in Figure A shows the comparison before and after ITK gene inhibition. After the addition of ITK-siRNA, the expression of ITK gene was significantly inhibited (**p<0.01).

[0042] Figure B is a bar graph showing the comparison before and after MAT2B gene inhibition. After adding MAT2B-siRNA, the expression of MAT2B gene was significantly inhibited.

[0043] Example 2

[0044] In this example, after interfering with ITK gene and MAT2B gene using siITK and siMAT2B, the Celigo method was used to detect the number of cells and analyze the growth and proliferation of cells.

[0045] The specific method is:

[0046] (1) After trypsin digestion, cells in each group were resuspended in complete culture medium to form a cell suspension and counted.

[0047] (2) The cell density for plating is determined based on the cell growth rate. The culture system is 100 μL / well. Ensure that the number of cells added to each well is consistent during plating and culture in a 37°C, 5% CO2 incubator.

[0048] (3) Starting from the second day after plating, read the plates once a day using Celigo assay for 5 consecutive days.

[0049] (4) Adjust the input parameters of the analysis settings to accurately count the number of cells with green fluorescence in each scan. Analyze the data and plot a cell proliferation curve for 5 consecutive days.

[0050] Reference Figure 2 As shown, the horizontal axis represents the number of days. Figure 2 The results showed that the proliferation ability of tumor cells was significantly reduced, which proved that the interference of ITK gene and MAT2B gene in the embodiment can reduce the proliferation ability of melanoma cells.

[0051] Example 3

[0052] This example studies the apoptosis of melanoma cells after interfering with the ITK gene and the MAT2B gene.

[0053] In this example, cell apoptosis was detected by Annexin V-APC single staining method, and the specific method was as follows:

[0054] (1) Induce apoptosis when cells grow to a confluence of approximately 70% in a 6-well plate.

[0055] (2) For adherent cells, the supernatant cells need to be collected. After trypsin digestion, resuspend the cells in complete medium into a cell suspension and collect them in 5 mL centrifuge tubes. Set up three replicates for each group. For suspended cells, collect them directly.

[0056] (3) Centrifuge at 1300 rpm for 5 min, remove the supernatant, and wash the cell pellet with D-Hanks. Wash the cell pellet with binding buffer, centrifuge at 1300 rpm for 3 min, and then collect the cells.

[0057] (4) Resuspend the cell pellet in 200 μL of binding buffer. Add 10 μL of Annexin V-APC staining and incubate at room temperature (protected from light) for 10 min. Then, adjust the amount of binding buffer based on the cell number and analyze on a flow cytometer.

[0058] Reference Figure 3 The data showed that the number of apoptotic tumor cells increased after dual nucleic acid interference (*p<0.05), demonstrating that interference with the ITK and MAT2B genes in the present invention increased melanoma cell apoptosis.

[0059] Example 4

[0060] This example studies the proliferation ability of melanoma cells after interfering with the ITK gene and the MAT2B gene;

[0061] This example uses a cell clone detection method: the specific method is as follows:

[0062] (1) A375 cells were transfected with siITK and siMAT2B using Lipofectamine 2000 (Thermo Fisher) and cultured in a 37°C incubator.

[0063] (2) Count the cells and plate approximately 1000 cells / well in a 6-well plate, with each well containing 2 ml of culture medium.

[0064] (3) After 2 weeks, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and washed with PBS.

[0065] (4) Count the cell clones under the microscope and perform statistical analysis.

[0066] Reference Figure 4 As shown, dual-nucleotide knockdown of the ITK and MAT2B genes in A375 cells significantly reduced tumor cell proliferation, as shown in the statistical graph of relative clone numbers. **p<0.01. This confirms that knockdown of the ITK and MAT2B genes reduces melanoma cell proliferation.

[0067] Example 5

[0068] This example studies the migration ability of melanoma cells after interfering with ITK gene and MAT2B gene;

[0069] This example uses the Transwell cell migration ability detection method, which is as follows:

[0070] (1) A375 cells were treated and cultured in cell culture medium containing 10% FBS (fetal bovine serum).

[0071] (2) Wash with 1xPBS and digest with trypsin. Adjust the cell number to 3 x 10 5 cell / ml.

[0072] (3) Add 0.6 ml of DMEM medium containing 10% FBS to the wells of the plate and place the chamber in the wells with the bottom in contact with the medium.

[0073] (4) Add 3 x 10 4 The cells were cultured in an incubator at 37°C for 48 hours. The culture medium in the wells and chambers was removed by aspiration.

[0074] (5) Wash twice with PBS, fix with 4% paraformaldehyde for 30 min, stain with crystal violet for 30 min, and wash away excess dye with PBS.

[0075] (6) Wipe the cells in the chamber clean, place the cells at the bottom of the filter membrane under a microscope for observation, and use ImageJ software to count and statistically analyze the cells outside the chamber.

[0076] Reference Figure 5 As shown, after using double nucleic acid sequences to interfere with ITK gene and MAT2B gene in A375 cells, the migration ability of tumor cells was significantly reduced, which verifies that interfering with ITK gene and MAT2B gene can reduce the migration ability of melanoma cells.

[0077] Those skilled in the art may make various modifications to the above without departing from the spirit and scope of the present invention as defined by the claims. Therefore, the scope of the present invention is not limited to the above description, but is determined by the scope of the claims.

Claims

1. Use of a combination of ITK-siRNA and MAT2B-siRNA in the preparation of a drug for treating or preventing melanoma; The sequence of the ITK-siRNA is: sense: ACU CAG AGG UGG UGG AAG A, the sequence is shown in SEQ ID NO: 1; antisense: UGA GUC UCC ACC ACC UUC U, sequence as shown in SEQ ID NO: 2; The sequence of the MAT2B-siRNA is: sense: GAC AAG AGA UGG AGA CAA A, the sequence is shown in SEQ ID NO: 3; antisense: CUG UUC UCU ACC UCU GUU U, the sequence is shown in SEQ ID NO:

4.

2. The use according to claim 1, characterized in that The melanoma is skin melanoma.

3. The use according to claim 1, characterized in that The melanoma is acral melanoma.

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