Application of non-targeting interference nucleotide in preparation of anti-lung cancer drugs

Abstract

The invention discloses an application of a non-targeting interference nucleotide in preparation of an anti-lung cancer drug, and the non-targeting interference nucleotide is characterized in that the nucleotide achieves an effect of inhibiting lung cancer through non-targeting interference, and the sequence of the nucleotide is GCACGUGAUCGAGGGCUAdTdT and a complementary sequence UAGCCCUCGAUCACCGUGCdTdT of the nucleotide, or the sequence of the nucleotide is GCGUGUCAUAUACUACADTdT and a complementary sequence UGUAGUUAUUGGCACCGCdTdT of the nucleotide. The oligonucleotide can inhibit lung cancer representative cells A549 and even cause apoptosis of the lung cancer representative cells A549.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals for the treatment of tumors with nucleotides, and specifically to oligonucleotide fragments. Although the designed small interfering RNA (siRNA) generally interferes with its complementary target sequence gene in the traditional sense, the siRNA of this patent does not directly target and interfere with the expression of the specific gene IGF1R, but through an unknown mechanism, it effectively weakens the malignant progression of lung cancer without directly interfering with the expression of the IGF1R gene, thereby exerting an anti-tumor effect. Background Art

[0002] Tumors refer to masses formed by abnormal proliferation of cells in the human body. Malignant tumors, namely cancer, have become a major problem in the field of global public health due to their invasiveness and metastasis. Lung cancer, as one of the most deadly members of the cancer family, has not only attracted much attention due to its high morbidity and mortality, but also has become a hot spot and difficulty in medical research due to its complex pathogenic mechanism, diverse clinical manifestations and different responses to treatment.

[0003] From a historical perspective, human knowledge of tumors can be traced back to the ancient civilization period, but the scientific understanding and systematic research on lung cancer has only been carried out in recent centuries. With the invention of the microscope and the establishment of cell theory, scientists began to realize that tumors are not simple masses, but are composed of abnormally proliferating cells. These cells have lost their normal growth regulation mechanism and can divide indefinitely, eventually invading surrounding tissues and possibly spreading throughout the body through the blood or lymphatic system.

[0004] The pathogenesis of lung cancer is also complex, involving the interaction of multiple factors such as genetics, environment, and lifestyle. Smoking has been widely recognized as the most controllable risk factor for lung cancer. However, as research deepens, scientists have found that even among non-smokers, the incidence of lung cancer remains high, which suggests that in addition to smoking, air pollution, occupational exposure, genetic factors, and personal lifestyle habits also play an important role in the development of lung cancer.

[0005] In terms of treatment, the treatment of lung cancer has undergone a transformation from traditional surgical resection, radiotherapy and chemotherapy to targeted therapy and immunotherapy that have emerged in recent years. The advancement of these treatment methods has not only improved the survival rate of lung cancer patients, but also improved their quality of life. However, the prognosis of lung cancer is still not optimistic, especially for patients with advanced lung cancer, who have limited treatment options and a short survival time.

[0006] IGFs are a family of secreted peptide hormones that are essential for a variety of cellular and organismal functions. Their biological activities are mediated by IGF1R, a cell surface receptor associated with a variety of cellular signaling responses. IGF1R has anti-apoptotic, pro-survival effects and plays a key role in cancer development. IGF activity is highly associated with a variety of chronic lung diseases, especially lung cancer. IGF1R signaling is a key factor in cancer cell proliferation, survival, migration and drug resistance, making it an attractive therapeutic target. Currently, IGF1R is being evaluated as a pharmacological target in clinical trials in tumor patients. In addition, many anticancer drugs regulate cell fate by controlling the expression of the IGF1R gene. Although IGF1R is a promising therapeutic target, it is crucial to identify biomarkers that can predict responsiveness to IGF1R-directed therapies.

[0007] Small interfering RNA (siRNA) has a unique ability, that is, it can accurately identify and degrade the messenger RNA (mRNA) paired with it, thereby blocking its conversion into protein and achieving the silencing expression of specific genes. This feature gives siRNA a core position in gene expression regulation. In the development of cancer, the abnormal expression of certain key genes may cause uncontrolled cell proliferation, blocked apoptosis mechanisms, and accelerate the evolution of cancer. siRNA can accurately target these abnormally expressed genes and curb the deterioration of cancer by silencing their expression. In addition, cancer cells often develop resistance to traditional chemotherapy drugs, affecting the treatment effect. siRNA can silence the expression of genes related to drug resistance and enhance the sensitivity of cancer cells to chemotherapy drugs. As a powerful tool for gene therapy, siRNA can correct the incorrect expression of genes in cells and provide a new way for cancer treatment. At present, gene therapy strategies based on siRNA have shown initial results in clinical trials, such as the application of HT-101 and Elebsiran in the treatment of chronic hepatitis B. Summary of the invention

[0008] The purpose of the present invention is to provide a non-targeted nucleotide and its application in the preparation of anti-tumor drugs.

[0009] The object of the present invention is achieved by synthesizing a nucleotide sequence consisting of GCACGGUGAUCGAGGGCUAdTdT (SEQ ID NO.1) and its complementary sequence UAGCCCUCGAUCACCGUGCdTdT (SEQ ID NO.2), or GCGGUGUCCAAUAACUACAdTdT (SEQ ID NO.3) and its complementary sequence UGUAGUUAUUGGACACCGCdTdT (SEQ ID NO.4). The nucleotide can be used in the preparation of anti-lung cancer drugs.

[0010] The invention (advantages): The specific nucleotide designed by this innovative invention has a unique mechanism, that is, although it does not specifically interfere with the expression level of IGF1R, this nucleotide has shown a good tumor killing effect in cell experimental research. It can significantly inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 SEQ ID NO.1-4 did not interfere with IGF1R protein expression Figure 2 SEQ ID NO.1-4 promotes apoptosis of A549 cells DETAILED DESCRIPTION

[0012] The present invention is further described below, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0013] Example: The primers and interference fragments used were all written from (5´-3´), the control fragments UUCUCCGAACGUGUCACGUdTdT (SEQ ID NO.5) and ACGUGACACGUUCGGAGAAdTdT (SEQIDNO.6), GCACGGUGAUCGAGGGCUAdTdT (SEQ ID NO.1) and its complementary sequence UAGCCCUCGAUCACCGUGCdTdT (SEQID NO.2), or GCGGUGUCCAAUAACUACAdTdT (SEQ IDNO.3) and its complementary sequence UGUAGUUAUUGGACACCGCdTdT (SEQID NO.4).

[0014] 1. Cell Culture When the cell abundance reaches 80%-90%, the cells are passaged. First, the original solution in the T25 culture flask is discarded, and then 1 mL of PBS solution is added to wash the cells twice. Add 0.8 mL of 0.25% trypsin to rinse the culture flask and dry the liquid. Then add 0.8 mL of trypsin again, and place the culture flask in a cell culture incubator at 37°C for 2 minutes. After the incubation, the cells in the T25 culture flask can be seen to be in a quicksand state. Then add 5 mL of RPMI 1640 culture medium supplemented with 10% fetal bovine serum and 1% double antibody to terminate the digestion, and mix the cells with a Pasteur pipette. Finally, discard part of the cell solution and add an appropriate amount of culture medium, and continue to culture in a constant temperature incubator.

[0015] 2. siRNA interference verification (1) Cell inoculation: Digest the cells for 2 minutes according to the above steps, then add 5 mL of culture medium without double antibody to terminate digestion, and mix the cells by blowing. Pipette 12 μL of cell solution into the abalone counting plate, and count the cells in the counting plate under a microscope. According to the counting results, the cells were counted at 0.8×10 5 The cells were seeded at a density of 1.54 μg / mL in a 6-well plate and the 6-well plate was placed in a constant temperature incubator for culture.

[0016] (2) Transfection: The cell density should be maintained at 35%-55% on the day of transfection. Cell transfection was performed 18 hours after inoculation. Take four 1.5 mL EP tubes and label them as NC, siRNA-1, siRNA-1 and RFect. Add 100 μL, 100 μL, 100 μL and 200 μL of serum-free and dual antibody-free culture medium to the four EP tubes in order. Then, add 2 μL of the corresponding siRNA interference fragment to each of the EP tubes labeled NC, siRNA-1 and siRNA-1, and add 12 μL of RFect transfection reagent to the EP tube labeled RFect, and incubate at room temperature for 5 minutes. Then, add 100 μL of RFect diluent to each tube of siRNA solution (the total volume of the solution in each EP tube is 200 μL at this time) and incubate at room temperature for 20 minutes. Discard the original liquid in the 6-well plate and add 800 μL of dual antibody-free culture medium and the corresponding 200 μL of siRNA mixture to each well.

[0017] (3) Medium change: 24 hours after transfection, change the cell medium. Discard the transfection mixture in the 6-well plate and add 4 mL of culture medium containing serum and double antibody to each well.

[0018] (4) Total cell protein extraction: Cell protein was extracted 72 hours after transfection. Prepare lysis buffer containing protease inhibitors. Discard the original liquid in the 6-well plate. Add 1 mL of PBS to each well for 2-3 times, and discard the liquid in the well. Then add 200 µL of lysis buffer to each well of the 6-well plate, let it stand on ice for 15 minutes to fully lyse, and use a cell scraper to peel the cells, and then transfer them to a 1.5 mL EP tube. Vortex 3 times, 1 minute each time, and let it stand for 2 minutes. Centrifuge at 12000×g at 4℃ for 8 minutes. Obvious precipitation can be seen in the centrifuge tube. Pipette 160 µL of supernatant into a new EP tube. Prepare BCA mixed solution at a ratio of 50 (A): 1 (B) according to the instructions of the BCA kit to determine the protein concentration. Add 40 µL of 5× Loading buffer to each 160 µL of protein supernatant, heat in a metal bath at 95℃ for 5 minutes to denature the protein, and then store at -20℃.

[0019] (5) Western Blot: Prepare the gel using the Omni Easy™ One-Step PAGE Gel Rapid Preparation Kit (10%). Mix the concentrated gel and separation gel prepared according to the instructions, then add the separation gel to the lower layer of the glass plate, and then add the concentrated gel immediately. Slowly insert the comb vertically into the glass plate and let it stand at room temperature for 1 hour. After the gel solidifies, place the gel plate in the electrophoresis tank, add the newly prepared electrophoresis solution, and let it stand for a few minutes to check for leakage. Then slowly pull out the comb, vortex the protein sample to mix, centrifuge, and add the sample according to the protein concentration. After the sample is added, add the electrophoresis solution to the electrophoresis tank, set 120V, and perform electrophoresis for 100 minutes. Cut the PVDF membrane to the same size as the corresponding colloid size and soak it in methanol for 1 minute to activate it. After the electrophoresis is completed, perform wet transfer. From the negative electrode to the positive electrode, stack the sponge net, sponge pad, glue, PVDF membrane, sponge pad, and sponge net in order. Avoid bubbles during the whole process. Then place it in the transfer tank and add wet transfer solution to completely soak the sponge net. Set the transfer conditions to a constant current of 200mA and transfer for 150 minutes. Block with a quick blocking solution for 2 hours after the transfer. Wash with TBST 3 times for 10 minutes each time after blocking. Dilute the primary antibody of interest with the primary antibody diluent, incubate each membrane with the corresponding primary antibody solution, and incubate overnight in a 4°C refrigerator. Wash the membrane 3 times with TBST on the second day for 10 minutes each time. Dilute the required secondary antibody with the secondary antibody diluent and incubate with the corresponding secondary antibody for 2 hours. After incubation, use a chemiluminescent solution for development and detection.

[0020] 3. Cell Viability Detection Experiment (1) Cell inoculation: After digesting the cells for 2 minutes, add 5 mL of culture medium without double antibody to terminate digestion, and mix the cells by blowing. Pipette 12 μL of cell solution into the abalone counting plate, and count the cells in the counting plate under a microscope. According to the counting results, 2000 cells per well are inoculated into a 96-well plate.

[0021] (2) Transfection: Cell transfection was performed 18 hours after cell inoculation. Take four 1.5 mL EP tubes and label them as NC, siRNA-1, siRNA-1, and RFect. Add 100 μL, 100 μL, 100 μL, and 200 μL of serum-free and anti-dual antibody-free culture medium to the four EP tubes in order. Subsequently, add 2 μL of the corresponding siRNA interference fragment to each of the EP tubes labeled NC, siRNA-1, and siRNA-1, and add 12 μL of RFect transfection reagent to the EP tube labeled RFect, and incubate at room temperature for 5 minutes. Subsequently, add 100 μL of RFect diluent to each tube of siRNA solution (the total volume of the solution in each EP tube is 200 μL at this time), and incubate at room temperature for 20 minutes. Subsequently, add 800 μL of anti-dual antibody-free culture medium to the EP tubes labeled NC, siRNA-1, and siRNA-1, and gently pipette to mix. Discard the original liquid in the 96-well plate and add 50 µL of siRNA mixture to each well.

[0022] (3) Medium change: 24 hours after transfection, change the cell medium. Discard the transfection medium in the 96-well plate and add 200 µL of culture medium containing serum and double antibody to each well.

[0023] (4) CCK-8 experiment: Cell viability was detected 72 hours after transfection. After aspirating 80 µL of liquid from each well of the 96-well plate, 20 µL of mixed CCK-8 reagent was quickly added and placed in a constant temperature incubator for incubation. The absorbance value (450 nm) was detected every 15 minutes until the absorbance value of the control group was 1. The cell survival rate was calculated, and 6 replicate wells were set for each group. Cell survival rate = (OD value of the silencing group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. The viability of A549 cells was expressed as (x±s): 100%±0.87 for the NC group, 75.89%±1.57 for the siRNA-1 group, and 65.56%±1.39 for the siRNA-2 group. The one-way ANOVA test showed statistical differences.

[0024] 4. Cell apoptosis experiment (Hoechst33342 / PI) (1) Cell seeding: Cells were seeded at 0.8×10 5 The cells were seeded at a density of 1.54 μg / mL in a 6-well plate and the 6-well plate was placed in a constant temperature incubator for culture.

[0025] (2) Transfection: Cell transfection was performed 18 hours after cell inoculation. Take four 1.5 mL EP tubes and label them as NC, siRNA-1, siRNA-1, and RFect. Add 100 μL, 100 μL, 100 μL, and 200 μL of serum-free and dual-antibody-free culture medium to the four EP tubes in order. Subsequently, add 2 μL of the corresponding siRNA interference fragment to each of the EP tubes labeled NC, siRNA-1, and siRNA-1, and add 12 μL of RFect transfection reagent to the EP tube labeled RFect, and incubate at room temperature for 5 minutes. Subsequently, add 100 μL of RFect diluent to each tube of siRNA solution (the total volume of the solution in each EP tube is 200 μL at this time), and incubate at room temperature for 20 minutes. Discard the original liquid in the 6-well plate, and add 800 μL of dual-antibody-free culture medium and the corresponding 200 μL of siRNA mixture to each well.

[0026] (3) Medium change: 24 hours after transfection, change the cell medium. Discard the transfection mixture in the 6-well plate and add 4 mL of culture medium containing serum and double antibody to each well.

[0027] (4) Cell apoptosis experiment: HO-PI fluorescent double staining experiment was performed 72 hours after transfection to detect cell apoptosis. Preheat the cell culture medium in advance. Prepare HO-PI fluorescent double staining working solution (1mL culture medium + 5µL Hoechst33342 solution + 5µL PI solution) according to the instructions and vortex to mix. Discard the original liquid in the 6-well plate, wash each well with 1mL PBS 2-3 times, then add 1mL of the prepared HO-PI working solution to each well and incubate at room temperature and away from light for 15 minutes. After incubation, wash 1-2 times with PBS. To prevent the cells in the wells from drying, add 1mL PBS to each well, and then take pictures under an inverted fluorescence microscope. Apoptotic cells can be stained bright blue by Hoechst33342 solution, and late apoptotic cells can be stained red by PI solution. When calculating the apoptosis rate, select 3 different fields of view in the same well. The apoptosis rate of A549 cells in the NC group was 0.67%±0.06, 4.14%±0.13 in the siRNA-1 group, and 5.47%±0.14 in the siRNA-2 group, which were statistically different after one-way analysis of variance.

Claims

1. A non-targeted interfering nucleotide, characterized in that Its sequence is GCACGGUGAUCGAGGGCUAdTdT (SEQ ID NO.1) and its complementary sequence UAGCCCUCGAUCACCGUGCdTdT (SEQ ID NO.2), or GCGGUGUCCAAUAACUACAdTdT (SEQ ID NO.3) and its complementary sequence UGUAGUUAUUGGACACCGCdTdT (SEQ ID NO.4).

2. The use of the non-targeted interfering nucleotide according to claim 1, characterized in that Application in the preparation of drugs for treating lung cancer.

Images