Application of lncRNA DDB2-AS1 as target spot in preparation of anti-cancer drugs

Abstract

The invention relates to application of lncRNADDB2-AS1 as a target spot in preparation of anti-cancer drugs, and belongs to the technical field of biology. According to the present invention, the expression of the lncRNA DDB2-AS1 in lung cancer cells is down-regulated, and the lncRNA DDB2-AS1 is adopted to design the overexpression plasmid so as to achieve the overexpression of the lncRNA DDB2-AS1 in the lung cancer cells, such that the proliferation of the lung cancer cells can be effectively inhibited, the lung cancer cell apoptosis can be effectively promoted, and the cell cycle can be retarded; the result shows that the overexpression of the lncRNADDB2-AS1 can effectively treat the lung cancer, and a new therapeutic target is provided for the treatment of the lung cancer.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the application of lncRNA DDB2-AS1 as a target in the preparation of anticancer drugs. Background Art

[0002] Lung cancer, also known as primary bronchial lung cancer, is the most common lung malignancy originating from the trachea, bronchial mucosa or glands. The incidence of lung cancer is much higher than other types of cancer. Lung cancer is mainly divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for about 80-85% of the incidence of all lung cancers. At present, the main treatment methods include surgery, chemotherapy, radiotherapy, molecular targeted therapy and local interventional therapy. However, due to insufficient drug efficacy and limited induction of cell apoptosis, treatment failure often occurs. Therefore, surgery is still the first choice and the main method for the treatment of lung cancer. However, surgical treatment is not only traumatic, but also has many risks and postoperative complications. At the same time, for advanced patients with cancer that has spread, surgical treatment not only fails to achieve the purpose of treatment, but also brings greater trauma and risks to patients. Therefore, there is an urgent need for a non-surgical treatment method and drug that can effectively treat lung cancer. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide the use of lncRNA DDB2-AS1 as a target in the preparation of anticancer drugs, which can kill lung cancer cells and effectively treat lung cancer.

[0004] The present invention provides the use of lncRNA DDB2-AS1 as a target in the preparation of a drug for treating lung cancer, wherein the nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1.

[0005] Preferably, the lncRNA DDB2-AS1 is used to treat lung cancer by overexpression.

[0006] The present invention provides use of a lncRNA DDB2-AS1 overexpression reagent in preparing a drug for treating lung cancer, wherein the nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1.

[0007] The present invention provides use of a lncRNA DDB2-AS1 overexpression reagent in preparing a drug for inhibiting lung cancer cell proliferation. The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1.

[0008] The present invention provides use of a lncRNA DDB2-AS1 overexpression reagent in preparing a drug for promoting apoptosis of lung cancer cells. The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1.

[0009] The present invention provides use of a lncRNA DDB2-AS1 overexpression reagent in preparing a drug for blocking lung cancer cell cycle, wherein the nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1.

[0010] Preferably, the lncRNA DDB2-AS1 overexpression reagent includes a lncRNA DDB2-AS1 overexpression plasmid.

[0011] Preferably, the plasmid vector of the lncRNA DDB2-AS1 overexpression plasmid includes PCDNA3.1.

[0012] The invention provides a lncRNA DDB2-AS1 overexpression plasmid, which is obtained by inserting the lncRNA DDB2-AS1 into a transient overexpression plasmid PCDNA3.1, wherein the insertion position is a BamHI and XhoI restriction site between a T7 sequence and a bGH sequence; the nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1.

[0013] The present invention also provides the use of the lncRNA DDB2-AS1 overexpression plasmid in preparing a drug for treating lung cancer.

[0014] Beneficial effects of the present invention:

[0015] The present invention proposes that lncRNA DDB2-AS1 is down-regulated in lung cancer cells, and an overexpression plasmid is designed using lncRNA DDB2-AS1 to overexpress lncRNA DDB2-AS1 in lung cancer cells, which can effectively inhibit the proliferation of lung cancer cells, promote lung cancer cell apoptosis, and block the lung cancer cell cycle. This indicates that overexpression of lncRNA DDB2-AS1 can effectively treat lung cancer, providing a new therapeutic target for the treatment of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 2 is a diagram of the overexpression plasmid of lncRNA DDB2-AS1 and its overexpression effect in lung cancer cells; A is the map of the overexpression plasmid of lncRNA DDB2-AS1; B is the qRT-PCR result of the lncDDB2-AS1 RNA level in human lung cancer cells after lncRNA DDB2-AS1 was transfected into the cells; ***P<0.001.

[0017] Figure 2 The figure is a plate cloning experiment to verify the effect of lncRNA DDB2-AS1 overexpression on the proliferation of human lung cancer cells; A is the experimental result of plate cloning before and after lncRNA DDB2-AS1 transfection of human lung cancer cells; B is a statistical bar graph of the proliferation rate of human lung cancer cells before and after overexpression of lncRNA DDB2-AS1; **P<0.01, *P<0.05.

[0018] Figure 3 The diagrams are for apoptosis experiments and cell cycle experiments to verify the effect of lncRNA DDB2-AS1 overexpression on apoptosis and cell cycle of human lung cancer cells; A is a diagram showing that lncRNA DDB2-AS1 overexpression promotes apoptosis in human lung cancer cells; B is a diagram showing that lncRNA DDB2-AS1 overexpression effectively blocks the cell cycle of human lung cancer cells. DETAILED DESCRIPTION

[0019] The present invention provides the use of lncRNA DDB2-AS1 as a target in the preparation of a drug for treating lung cancer; the lncRNA DDB2-AS1 is preferably used to treat lung cancer by overexpression.

[0020] The present invention also provides the use of a lncRNA DDB2-AS1 overexpression reagent in preparing a drug for treating lung cancer, and the use of a lncRNA DDB2-AS1 overexpression reagent in preparing a drug for inhibiting lung cancer cell proliferation, promoting lung cancer cell apoptosis or blocking lung cancer cell cycle.

[0021] In the present invention, the transcript length of the lncRNA DDB2-AS1 is 3701 nt, and the nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1; the lncRNA DDB2-AS1 is overexpressed in lung cancer cells by an overexpression reagent, and the overexpression of lncRNA DDB2-AS1 can preferably inhibit lung cancer cell proliferation, promote lung cancer cell apoptosis and block lung cancer cell cycle to effectively treat lung cancer, the lung cancer is preferably non-small cell lung cancer, and the lung cancer cells are preferably non-small cell lung cancer cells, more preferably A549 cells or H460 cells.

[0022] In the present invention, the lncRNA DDB2-AS1 overexpression reagent preferably includes a lncRNA DDB2-AS1 overexpression plasmid, and the plasmid vector of the lncRNA DDB2-AS1 overexpression plasmid preferably includes PCDNA3.1. The overexpression reagent may also include a pharmaceutically acceptable carrier.

[0023] The present invention provides a lncRNA DDB2-AS1 overexpression plasmid, which is obtained by inserting lncRNA DDB2-AS1 into a transient overexpression plasmid PCDNA3.1, wherein the insertion position is at the BamHI and XhoI restriction sites between the T7 sequence and the bGH sequence. The lncRNA DDB2-AS1 overexpression plasmid map is as follows: Figure 1 As shown in A. The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.1.

[0024] The present invention also provides the use of the lncRNA DDB2-AS1 overexpression plasmid in preparing a drug for treating lung cancer.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] Example 1

[0029] In order to study the potential biological function of lncRNA DDB2-AS1, human lncRNA DDB2-AS1 was overexpressed in human lung cancer cells, and the regulatory effect of lncRNA DDB2-AS1 on lung cancer cell clone formation, lung cancer cell apoptosis and lung cancer cell cycle was detected to investigate its therapeutic effect on lung cancer.

[0030] The cells used in this example are A549 cells and H460 cells, both of which are kept in the laboratory; the overexpression plasmid used in this example is an overexpression plasmid containing the nucleotide sequence of lncRNA DDB2-AS1 shown in SEQ ID NO.1, which was designed and customized by Miaoling Biotechnology Co., Ltd., and the map is shown in Figure 1 As shown in A.

[0031] The experimental data of this example are expressed as mean ± standard deviation. The data were compared and analyzed by two-sided Student's-T test (*P<0.05, **P<0.01, ***P<0.001 were statistically significant).

[0032] 1. Test methods

[0033] A549 cells and H460 cells were revived, subcultured, inoculated and transfected. The specific steps are as follows:

[0034] 1. Cell recovery and culture:

[0035] (1) After removing the cells from liquid nitrogen, quickly place them in a 37°C constant temperature water bath to thaw;

[0036] (2) Place the fully thawed cells in a desktop centrifuge and centrifuge at 800 rpm for 5 min;

[0037] (3) Discard the old culture medium and add 1 mL of pre-warmed complete culture medium (containing 10% FBS and 100 units / mL penicillin + streptomycin) to resuspend the cells;

[0038] (4) Transfer the cells to a 10 cm cell culture dish (10 × 10 cm) with a culture volume of 7 mL and gently shake to mix;

[0039] (5) The cells were cultured in a cell culture incubator (37°C, 5% CO2). Fresh complete culture medium was replaced the next day. The cells were passaged when the cell density reached 90%.

[0040] 2. Cell passaging:

[0041] (1) Preheat complete culture medium, 0.25% Trypsin-EDTA, and PBS buffer in advance;

[0042] (2) Take the cells from step 1, discard the old culture medium, and add 2 mL of PBS along the side wall to rinse;

[0043] (3) Discard PBS, add 2 mL of 0.25% Trypsin-EDTA to the dish, and place in a 37°C incubator to digest the cells;

[0044] (4) After the cells are completely digested, add 2 mL of complete medium to terminate the digestion, transfer the cells to a 10 mL centrifuge tube using a micropipette, and centrifuge at 800 rpm in a desktop centrifuge for 5 min;

[0045] (5) Discard the supernatant, resuspend the cells in the corresponding fresh complete medium, and transfer to a 10 cm culture dish and shake gently;

[0046] (6) The cells were cultured in a cell culture incubator (37°C, 5% CO2) and subcultured every other day.

[0047] 3. Cell inoculation:

[0048] (1) Take cells in the logarithmic growth phase, discard the old culture medium, rinse with 2 mL of PBS, add 2 mL of 0.25% Trypsin-EDTA and fully digest in a 37°C incubator;

[0049] (2) Add 2 mL of complete culture medium to neutralize 0.25% Trypsin-EDTA, transfer the cells to a 10 mL centrifuge tube using a micropipette, and centrifuge at 800 rpm in a desktop centrifuge for 5 min;

[0050] (3) Discard the supernatant, resuspend the cells in an appropriate amount of complete culture medium, and count the cells under an inverted microscope using a hemocytometer;

[0051] (4) Calculate the required volume of cell suspension and the volume of culture medium required for the corresponding culture system according to the number of cells to be inoculated in the experiment (6-well plate: 2 mL; 12-well plate: 1 mL; 24-well plate: 500 μL);

[0052] (5) The cells were cultured in a cell culture incubator (37°C, containing 5% CO2).

[0053] 4. Overexpression plasmid transfection:

[0054] Transfection was performed according to the instructions of LIP2000 transfection reagent.

[0055] The transfection steps are as follows:

[0056] (1) Plating: Inoculate 2.5×10 cells in a 6-well plate. 5 cells / well;

[0057] (2) Observation after 24 hours: The cells are growing well, with a confluence of about 60-70%, and can be transfected;

[0058] (3) Change the medium of the cell culture plate inoculated the day before before transfection. Replace 2 mL of complete culture medium with 1.25 mL of serum-free culture medium and perform transfection 1 hour after the medium change.

[0059] (4) Prepare the transfection mixture: add 2 μL of plasmid to each well and add 2 μL of lip2000. The diluent is Optime medium (antibody-free and serum-free). First, dilute lip2000 to 52 μL (50 μL of anti-body-free and serum-free Optime medium + 2 μL of lip2000), and place at room temperature for 5 minutes; then dilute the plasmid to 52 μL, add the diluted lip2000 dropwise to the diluted plasmid, mix gently, and place at room temperature for 15 minutes; add 104 μL / well of the mixture to a 6-well plate and shake gently. After culturing in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 8 hours, remove the complex and replace the culture medium. After incubation in the cell culture incubator for 48 hours, perform subsequent functional experiments.

[0060] 2. Testing methods and results

[0061] 1. qRT-PCR detection

[0062] In step 1, 4, after transfecting human lung cancer cells with the full-length overexpression plasmid of lncRNA DDB2-AS1 for 48 hours, qRT-PCR was used to detect the expression of lnc DDB2-AS1 RNA levels in human lung cancer cells before transfection (lvNC) and after transfection (lv DDB2-AS1). The qRT-PCR experimental steps are as follows:

[0063] (1) RNA extraction: Add 1 mL Trizol to the cells and operate on ice; collect into 1.5 mL enzyme-free EP tubes; add 200 μL chloroform, vortex mix for 30 seconds, and stand on ice for 5 minutes; centrifuge at 12000g, 15 minutes, and 4°C; aspirate the supernatant into a new enzyme-free EP tube; add an equal volume of isopropanol, mix, and stand for 10 minutes; centrifuge at 12000g, 10 minutes, and 4°C; discard the isopropanol, add 1 mL of anhydrous ethanol, mix; centrifuge at 7500g, 5 minutes, and 4°C; repeat the above two steps twice; discard the ethanol, and dry the EP tube at room temperature for 5-10 minutes; add 20 μL DEPC water to each tube, resuspend and mix thoroughly; measure the RNA concentration and purity using the NaNODrop machine. RNA samples that meet the purity requirements will be included in subsequent experiments.

[0064] (2) Reverse transcription: cDNA was synthesized by reverse transcription using III1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) reagent. The reaction was carried out on ice and the product was stored at -20°C. The primer sequences required for reverse transcription are shown in Table 1.

[0065] Table 1

[0066]

[0067] (3) qPCR: The PCR amplification system was prepared using qPCR SYBR Green MasterMix. The reaction program was set as follows: pre-denaturation (95°C, 5 min)*1 cycle, [denaturation (95°C, 10 s), annealing / extension (60°C, 30 s)]*40 cycles, and the machine defaulted to the melting curve stage.

[0068] The experimental results are as follows Figure 1 As shown in B, the results showed that the RNA expression level of lnc DDB2-AS1 in human lung cancer cells was significantly increased, indicating that the lncRNA DDB2-AS1 overexpression plasmid was successfully constructed and can be used for subsequent applications.

[0069] 2. Cell clone formation experiment:

[0070] A plate cloning experiment was performed to verify the human lung cancer cells before and after transfection with the lncRNA DDB2-AS1 overexpression plasmid to detect the effect of the lncRNA DDB2-AS1 overexpression plasmid on the proliferation of human lung cancer cells before (lvNC) and after transfection (lv DDB2-AS1). The specific steps are as follows:

[0071] (1) Inoculation clone:

[0072] ① 48 hours after plasmid transfection, cells were re-seeded in 6-well plates, 5000 cells / well, and three replicate wells were set up for each group;

[0073] ② Place the cells in a cell culture incubator and culture for two weeks until the clones grow to an appropriate size, replacing fresh complete culture medium every 3-5 days;

[0074] (2) Cell fixation and staining:

[0075] ①Discard the old culture medium and add 1mL tissue fixative to fix for 30 minutes;

[0076] ②Discard the tissue fixative and add 1 mL of crystal violet stain for 1 hour;

[0077] ③ Recover the crystal violet, slowly rinse the remaining dye in the well, and dry it naturally;

[0078] ④Manually count the clones and statistically analyze them, and use a scanner to scan and save the original images.

[0079] The results are as follows Figure 2 As shown, the results showed that overexpression of lncRNA DDB2-AS1 significantly inhibited the proliferation of human lung cancer cells.

[0080] 3. Cell apoptosis detection experiment:

[0081] The effects of lncRNA DDB2-AS1 overexpression plasmid on apoptosis of human lung cancer cells before transfection (lv NC) and 48h after transfection (lv DDB2-AS1) were detected. The specific steps are as follows:

[0082] (1) Add 100 μL of 1× Annexin V Binding Solution in the apoptosis detection kit to each cell sample and mix gently;

[0083] (2) Add 5 μL Annexin V / FITC conjugate to each sample and mix gently, then add 5 μL PISolution and mix gently, then incubate at room temperature in the dark for 15 min;

[0084] (3) Add 300 μL 1× Annexin V Binding Solution and mix well before loading;

[0085] (4) On-machine detection: In addition to the experimental samples to be analyzed, a blank control group and single-stained cell groups (Annexin V-FITC and PI groups) were set up. The cells in the blank group and the single-stained group were treated with H2O2 to induce cell apoptosis, which was used to adjust the fluorescence compensation during flow cytometric analysis.

[0086] The results are as follows Figure 3 As shown in A, the results showed that overexpression of lncRNA DDB2-AS1 significantly promoted apoptosis in human lung cancer cells.

[0087] 4. Cell cycle detection test:

[0088] The effects of lncRNA DDB2-AS1 overexpression plasmid on the cell cycle of human lung cancer cells before transfection (lv NC) and 48h after transfection (lv DDB2-AS1) were detected. The specific steps are as follows:

[0089] (1) Carefully collect the transfected cell culture medium into a centrifuge tube for later use. Digest the cells with trypsin until the cells can be gently blown off with a pipette or gun tip, then add the collected cell culture medium, blow off all the adherent cells, and gently blow off the cells. Collect them again into a centrifuge tube. Centrifuge at 800g for 5 minutes to precipitate the cells. Carefully remove the supernatant, add 1mL of ice-cold PBS, resuspend the cells, and transfer to a 1.5mL centrifuge tube. Centrifuge again to precipitate the cells, carefully remove the supernatant, and collect the cells.

[0090] (2) Cell fixation: Add 1 mL of ice-cold 70% ethanol, gently pipette to mix, and fix at 4°C for 30 min. Centrifuge at 800 g for 5 min to pellet the cells. Carefully remove the supernatant, add 1 mL of ice-cold PBS, and resuspend the cells. Centrifuge again to pellet the cells, carefully remove the supernatant, and collect the cells.

[0091] (3) Preparation of propidium iodide staining solution: Prepare an appropriate amount of propidium iodide staining solution according to the number of samples to be tested.

[0092] (4) Staining: Add 0.5 mL of propidium iodide staining solution to each tube of cell sample, slowly and fully resuspend the cell pellet, and incubate at 37°C in the dark for 30 min. Then complete the flow cytometry detection.

[0093] (5) Flow cytometric detection and analysis: Flow cytometry was used to detect red fluorescence at an excitation wavelength of 488 nm and light scattering was detected simultaneously. Analysis software was used to analyze the cell DNA content and light scattering.

[0094] The results are as follows Figure 3 As shown in B, the results showed that overexpression of lncRNA DDB2-AS1 effectively blocked the cell cycle of human lung cancer cells.

[0095] The above results show that the lncRNA DDB2-AS1 overexpression plasmid of the present invention can significantly inhibit the proliferation of human lung cancer cells, promote cell apoptosis and effectively block their cell cycle, indicating that overexpression of lncRNA DDB2-AS1 can effectively kill lung cancer cells.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of lncRNA DDB2-AS1 as a target in the preparation of a drug for treating lung cancer, characterized in that: The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The lncRNA DDB2-AS1 is used to treat lung cancer by overexpression.

3. Use of a lncRNA DDB2-AS1 overexpression reagent in the preparation of a drug for treating lung cancer, characterized in that: The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.

1.

4. Use of a lncRNA DDB2-AS1 overexpression reagent in the preparation of a drug for inhibiting lung cancer cell proliferation, characterized in that: The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.

1.

5. Use of a lncRNA DDB2-AS1 overexpression reagent in the preparation of a drug for promoting apoptosis of lung cancer cells, characterized in that: The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.

1.

6. Use of a lncRNA DDB2-AS1 overexpression reagent in the preparation of a drug for blocking lung cancer cell cycle, characterized in that: The nucleotide sequence of the lncRNA DDB2-AS1 is shown in SEQ ID NO.

1.

7. The use according to any one of claims 3 to 6, characterized in that: The lncRNA DDB2-AS1 overexpression reagent includes a lncRNA DDB2-AS1 overexpression plasmid.

8. The use according to claim 7, characterized in that: The plasmid vector of the lncRNADDB2-AS1 overexpression plasmid includes PCDNA3.

1.

9. A lncRNADDB2-AS1 overexpression plasmid, characterized in that: The lncRNADDB2-AS1 was inserted into the transient overexpression plasmid PCDNA3.1, and the insertion position was the BamHI and XhoI restriction sites between the T7 sequence and the bGH sequence; the nucleotide sequence of the lncRNADDB2-AS1 is shown in SEQ ID NO.

1.

10. Use of the lncRNA DDB2-AS1 overexpression plasmid according to claim 9 in the preparation of a drug for treating lung cancer.

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