Construction method and application of stably transfected cell strain for knocking down and overexpressing Dyrk1B gene

Abstract

The invention relates to a method for efficiently establishing a stably transfected cell strain for knocking down and overexpressing a dyrk1b gene. Comprising the following steps: designing a specific RNA (Ribonucleic Acid) sequence to knock down a dyrk1b gene, constructing a DNA (Deoxyribose Nucleic Acid) sequence for highly expressing dyrk1b, and carrying out virus packaging and predetermined cell infection by utilizing a three-generation lentivirus system. A positive clone cell strain is obtained through Puromycin screening, OVCAR3 and 293T cells are infected by adopting a specific plasmid proportion, and knock-down and overexpression efficiency is verified. According to the method, the function of the dyrk1b gene is evaluated by observing the morphological change of OVCAR3 and COV362 cells, such as cell body elongation and antenna growth. The innovation point of the invention is that an efficient virus packaging system and precise gene expression regulation and control ensure the stability of the cell strain and the reliability of the experiment. In addition, the invention also relates to application of the stably transfected cell strains in comprehensive treatment of ovarian cancer magnetic hyperthermia, small molecule inhibitor and chemotherapy, and provides a new research tool and strategy for cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a stable cell line for knocking down and overexpressing a TZAP gene and its application. Background Art

[0002] Dyrk1B is a member of the Minibrain / dyrk kinase family and is a dual-specific tyrosine protein kinase. Dyrk1B is involved in regulating tumorigenesis, cell growth and development, energy metabolism and other processes. Studies have also found that Dyrk1B is more expressed in human tumor tissues, and it may play a key role in the occurrence and development of malignant tumors such as ovarian cancer. Dyrk1B is expected to become a new target for the treatment of ovarian cancer. When the expression of Dyrk1B in tumor cells is silenced, it is found that it can induce different degrees of apoptosis in tumor cells. When tumor cells are under various stress conditions such as hypoxia, they can activate Dyrk1B, which is a cell cycle checkpoint kinase, so that damaged tumor cells enter the G0 phase to complete self-repair. At the same time, Dyrk1B can significantly reduce the production of ROS and promote tumor cell survival. The Dyrk1B inhibitor AZ191 can efficiently and selectively inhibit the activity of Dyrk1B serine / threonine protein kinase.

[0003] Ovarian cancer is one of the most deadly gynecological diseases in the world. Currently, ovarian cancer is mainly treated with tumor cell reduction surgery, supplemented by chemotherapy, but many patients will develop platinum resistance. Chemotherapy resistance is an important reason for the recurrence of ovarian cancer and ineffective treatment. Therefore, it is very necessary to find a new and effective treatment method. Targeted therapy has also been proposed and gradually applied to a variety of diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a method for establishing a stable cell line for knocking down and overexpressing the dyrk1b gene, and the application of using the cell line in biological research and development of treatment methods.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first purpose of the present invention is to provide a method for constructing a stable cell line for knocking down the dyrk1b gene, comprising the following steps:

[0007] 1) Design RNA sequence to knock down dyrk1b

[0008] 2) The plasmid obtained in step 1) was packaged into virus using the third-generation lentiviral packaging system to obtain a lentivirus that knocked down the dyrk1b gene

[0009] 3) Using the dyrk1b gene knockdown lentivirus obtained in step 2), the ovarian cancer cell line was transfected, and the cell line with dyrk1b gene knockdown was obtained by resistance screening.

[0010] The second purpose of the present invention is to provide a method for constructing a stable cell line overexpressing the dyrk1b gene, comprising the following steps:

[0011] 1) Design of DNA sequence for high expression of dyrk1b

[0012] 2) The plasmid obtained in step 1) was packaged into virus using the third-generation lentiviral packaging system to obtain a lentivirus that knocked down the dyrk1b gene

[0013] 3) Using the dyrk1b gene knockdown lentivirus obtained in step 2), the ovarian cancer cell line was transfected, and the cell line with dyrk1b gene knockdown was obtained by resistance screening.

[0014] The construction method of the present invention is efficient and can obtain stable cell lines with knockdown and overexpression of the dyrk1b gene; the construction method of the gene-modified cell line provided by the present invention is more efficient than the prior art method.

[0015] The method for constructing ovarian cancer cells with knocked-down dyrk1b gene of the present invention can reduce the expression of dyrk1b gene in ovarian cancer cells, thereby reducing the cell proliferation ability, clone formation ability and migration ability of ovarian cancer cells.

[0016] As a preferred embodiment of the method for constructing a cell line for knocking down and overexpressing the dyrk1b gene of the present invention, the ovarian cancer cells include OVCAR3, SK-OV-3, SNU119, and COV362 cell lines.

[0017] The present invention uses a third-generation lentiviral packaging system to improve the packaging efficiency and infectivity of the virus. This system optimizes the process of lentiviral particles entering host cells by using a new helper virus, such as HIV-1, and significantly improves the transfection efficiency. At the same time, the system adjusts the lentiviral structure through genetic engineering, reduces the immune response in the body, further improves efficiency and enhances safety. In addition, the third-generation lentiviral packaging system is multifunctional, not only for gene transfer, but also for precise control of gene expression by adding different regulatory elements. The system can also finely regulate gene expression by adjusting the type and proportion of helper viruses, providing a safe and effective tool for clinical applications.

[0018] For the third purpose, the present invention provides cell lines for knocking down and overexpressing the dyrk1b gene obtained by the above construction method.

[0019] The fourth purpose of the present invention is to provide the use of the above-mentioned cell lines with knockdown and overexpression of dyrk1b gene in the preparation of ovarian cancer drugs.

[0020] The cell line obtained by the above construction method is used to construct a disease model, and the disease model is used for mechanism research of tumor diseases, etc., and development or screening of targeted drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The construction method of the present invention is efficient and can obtain a stable cell line that knocks down and overexpresses the dyrk1b gene; the construction method of the gene-modified cell line provided by the present invention is more efficient than the prior art method. The method of constructing an ovarian cancer cell that knocks down the dyrk1b gene of the present invention can reduce the expression of the dyrk1b gene in ovarian cancer cells, thereby reducing the cell proliferation ability, clone formation ability and migration ability of ovarian cancer cells. The present invention knocks down the dyrk1b gene of the OVCAR3 cell line or the COV362 cell line, and the inventor unexpectedly found that knocking down the dyrk1b gene can reduce the proliferation ability, clone formation ability and migration ability of OVCAR3 cells or COV362 cells. The present invention uses a third-generation lentiviral packaging system to package the virus, and the third-generation lentiviral packaging system is an efficient, stable, multifunctional and safe gene therapy tool. Compared with the traditional first-generation and second-generation lentiviral packaging systems, the third-generation lentiviral packaging system has higher transfection efficiency, lower immune response risk and stronger versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of fluorescence staining of cells stably transfected with knockdown and overexpression of dyrk1b gene;

[0024] Figure 2 is the histogram of Dyrk1B expression in different ovarian cancer cell lines;

[0025] Figure 3 Design map for knockdown of dyrk1b gene expression plasmid;

[0026] Figure 4 Design map for dyrk1b gene overexpression plasmid;

[0027] Figure 5 Schematic diagram of the lentivirus structure;

[0028] Figure 6 This is a schematic diagram summarizing the three-generation lentiviral system;

[0029] Figure 7 To screen the optimal concentration of the susceptibility enhancer Polybrene;

[0030] Figure 8 The expression of dyrk1b protein in 293T cells and the statistical results of the gray value average intensity analysis of Dyrk1B protein bands using Image J software;

[0031] Fig. 9 To observe the cell morphology of cells with stable knockdown of dyrk1b gene under a microscope;

[0032] Fig.10 Bright field and fluorescence images of COV362 stably transfected cells under light microscope, objective lens 10x, scale 100um;

[0033] Fig.11 The results of WB analysis of Dyrk1B gene expression and the statistical results of the gray value average intensity analysis of Dyrk1B protein bands using Image J software;

[0034] Fig.12 The bright field and fluorescence images of SNU119 stably transfected cells under light microscope, objective lens 20x, scale 50um;

[0035] Fig.13 To analyze the expression results of Dyrk1B gene by WB, Image J software was used to analyze the statistical results of the average intensity of the gray value of Dyrk1B protein bands.

[0036] Specific implementation method In order to better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0037] Examples 2-3: Design of RNA sequences for knocking down dyrk1b and DNA sequences for overexpressing dyrk1b

[0038] Examples 4-6: Packaging viruses using a third-generation lentiviral packaging system

[0039] 1. 293T cell plating.

[0040] Take 293T cells in the logarithmic growth phase, digest them with 0.05% trypsin, and count them. If it is a 10cm large dish, it is recommended to evenly spread 293T cells at a number of 10-12×106 per dish; if it is a 6-well plate, it is recommended to evenly spread 293T cells at a number of 2×106 per well.

[0041] 2. The next day: within 24 hours, when the confluence of 293T cells is between 90% and 95%, DNA-liposome complex is added thereto. The preparation method of DNA-liposome complex is as follows:

[0042] 1) Gently mix LipoMax, add the corresponding amount according to the instructions to 500 μl Opti-MEM serum-free medium, mix well and place at room temperature for 5 minutes.

[0043] 2) Dilute the DNA in 500 μl Opti-MEM serum-free medium to a total mass of 15 μg and add DNA according to the ratio of vector plasmid.

[0044] 3) Gently mix the diluted LipoMax and diluted DNA and let stand at room temperature for 20 minutes to form a DNA-LipoMax complex.

[0045] 3. Gently add the DNA-LipoMax complex to the cell culture dish, gently shake the dish to mix, and place it in the cell culture incubator for culture.

[0046] 4. Virus collection

[0047] Concentrated virus: 48 hours after adding DNA-LipoMax complex, collect the virus supernatant. Store the collected virus supernatant in a 4°C refrigerator; collect the 72-hour virus supernatant and mix it with the 48-hour virus supernatant. Cool the centrifuge temperature to 4°C, centrifuge at 600g for 5 minutes to remove cell debris, filter the supernatant through a 0.45μm filter, add the virus concentrate, place the concentrated virus on a shaker in a 4°C refrigerator, and rotate overnight. The next day, centrifuge at 4 degrees, 3000-4000g for 15 minutes. Discard the supernatant and resuspend in PBS or culture medium.

[0048] 5. Infect the intended cells.

[0049] Example 7: Screening of the optimal concentration of the induction-enhancing agent Polybrene

[0050] 1. Take out the virus solution from the -80℃ refrigerator and thaw it on ice

[0051] 2. When the target cells grow to the logarithmic phase, add polybrene to the virus solution until the final concentration of polybrene reaches 5 μg / mL after adding to the culture dish.

[0052] 3. Add virus solution to the target cell culture dish, shake well and place in the incubator for culture.

[0053] 4. Pay attention to the cell status after infection and use the second tube of virus solution for secondary infection.

[0054] Puromysin drug puromycin screening positive clone cell line

[0055] 1. Determine the optimal puromycin concentration: Each cell line has different sensitivity to puromycin. Before starting the experiment, determine the optimal puromycin concentration for the cell line.

[0056] Day 1: Prepare a six-well plate or a 24-well plate, plate the target cells, and the cell confluence on the second day should be 80%-90%

[0057] Day 2: Add puromycin to make the final concentration of puromycin in each well between 1 μg / ml and 10 μg / ml Day 3: Check the cells every day and replace the culture medium with fresh puromycin-containing culture medium every other day. Select the lowest concentration of puromycin that causes complete cell death after three to five days.

[0058] 2. Screening of positive mixed clones and stable transfectants

[0059] Day 1: Infected cells and uninfected control cells were plated and the confluence reached 70%-80% on the next day.

[0060] Day 2: Add the optimal puromycin concentration and screen for 48-72 hours.

[0061] Day 5: When all cells in the blank cell plus puro group died, the remaining cells in the virus-infected group can basically be considered to be stably transfected cells, and the infection efficiency can be observed under a fluorescence microscope.

[0062] Example 8: Verification of the efficiency of viral knockdown and overexpression

[0063] 1. Plasmid preparation and virus packaging: Prepare the required plasmids, including the plasmid expressing the dyrk1b gene, VSVG, pLP2, and pLP1, and mix the plasmids at a mass ratio of 11.25:2.8:7.3:3.9 (as shown in Table 1) to ensure the appropriate ratio of each component during the virus packaging process.

[0064] 2. Cell culture and plating: Select OVCAR3 and 293T cells and culture them to the logarithmic growth phase. Plate 293T cells and adjust the cell density according to the cell counting results to ensure that the cell confluence is appropriate at the beginning of the experiment.

[0065] 3. Virus infection: The mixed plasmid and virus packaging system are co-transfected into 293T cells to produce virus particles. After 48 hours, the supernatant containing the virus is collected and appropriately concentrated.

[0066] 4. Verification of infection efficiency: The collected virus particles were used to infect OVCAR3 and 293T cells, and the adsorption and internalization of the virus by the cells were observed. The fluorescent labeling of the infected cells was examined by fluorescence microscopy to evaluate the efficiency of virus knockdown and overexpression of the dyrk1b gene.

[0067] 5. Protein expression analysis: After virus infection, 293T cell samples were collected for protein extraction. The expression of dyrk1b protein in 293T cells was detected using Western Blot technology.

[0068] 6. Gray value analysis: Use Image J software to digitally analyze the Western Blot bands and calculate the gray value of the Dyrk1B protein band. The gray values ​​were averaged and statistically analyzed to quantitatively evaluate the changes in the expression level of the dyrk1b protein.

[0069] 7. Data recording and analysis: Record all parameters during the experiment, including cell density, infection time, virus concentration, etc. Analyze the experimental data and compare the differences between cells with knockdown and overexpression of the dyrk1b gene and the control group.

[0070] Table 1

[0071]

[0072] Example 9: Observation of morphology and proliferation changes of OVCAR3 cells after dyrk1b gene knockdown

[0073] 1. Cell culture: Culture the OVCAR3 cell line with stable knockdown of dyrk1b gene in a suitable culture medium and keep the cells growing under suitable temperature, humidity and CO2 concentration.

[0074] 2. Cell morphology observation: Observe the cell morphology under a microscope. Use phase contrast microscopy to observe the morphological characteristics of cells more clearly.

[0075] 3. Record cell morphology changes: Record and compare the morphological changes of cells after knocking down the dyrk1b gene. Pay special attention to the changes in the length of the cell body and the extension of the antennae.

[0076] 4. Evaluation of the proliferation ability of stably transfected cells: Observe the proliferation ability of stably transfected OVCAR3 cells and primary cells, and record cell growth curves and cell counting data.

[0077] 5. Monitoring of cell status after passaging: After 5-6 generations of cell passaging, continue to monitor the cell status, including cell density, viability and morphology.

[0078] 6. Observation of floating cells and cell rupture: Observe and record the number of floating cells and cell rupture every 24 hours to evaluate the health of the cells.

[0079] 7. Chromatin status analysis: Observe and record the changes in chromatin after cell rupture, such as the phenomenon of chromatin clumping.

[0080] Examples 10-13. Analysis of Dyrk1B expression using microscopy and WB, quantification using ImageJ software, and comparison of knockdown and overexpression effects 1. Cell imaging

[0081] 1) Cell culture and transfection:

[0082] COV362 and SNU119 cells were cultured to appropriate density and stably transfected.

[0083] 2) Bright field and fluorescence imaging:

[0084] a. Use a 10X objective lens to perform bright field imaging of the COV362 stable cell line and set the scale to 100μm.

[0085] b. Use a 20X objective lens to perform bright field and fluorescence imaging of the SNU119 stably transfected cell line and set the scale to 50μm.

[0086] Fluorescent protein detection:

[0087] 3) Observe the expression of reporter gene in transfected cells under a fluorescence microscope to evaluate the transfection efficiency.

[0088] 2. Western Blot (WB) analysis

[0089] 1) Protein extraction: COV362 and SNU119 stable cell lines were collected for protein extraction.

[0090] 2) Protein quantification: The extracted protein was quantified using the BCA or Bradford method.

[0091] 3) Gel electrophoresis: Load the protein sample into SDS-PAGE gel for electrophoretic separation.

[0092] 4) Membrane transfer: Transfer the separated proteins to PVDF or nitrocellulose membrane.

[0093] 5) Blocking and primary antibody incubation: Use blocking solution to block the part of the membrane not occupied by protein, and then incubate with a specific primary antibody against Dyrk1B.

[0094] 6) Secondary antibody incubation and color development: Incubate with labeled secondary antibody, followed by color development or luminescence detection.

[0095] 3. Protein band gray value analysis

[0096] 1) Image acquisition: Use a gel imaging system to acquire images of the WB membrane.

[0097] 2) Gray value analysis: Image J software was used to analyze the gray value of the Dyrk1B protein band.

[0098] 3) Statistical comparison:

[0099] a. The Dyrk1B protein expression in COV362 cell line was compared between the lentivirus group infected with shRNA1 targeting dyrk1b mRNA knockdown sequence and the shRNA group infected with the empty virus vector (NC).

[0100] b. The protein expression of Dyrk1B (NC) in the SNU119 cell line infected with the lentivirus containing the full-length dyrk1b gene was compared with that in the group infected with the empty virus vector.

[0101] 4) Significance test: Compare the gray values ​​of the experimental group and the control group, and use appropriate statistical methods to perform a significance test. Indicates the significance of the results: ns means not significant, ****P<0.0001 means very significant.

[0102] 5) Result recording: record and organize experimental data and images, and prepare experimental reports.

Claims

1. A method for establishing a stable cell line with knockdown of dyrk1b gene expression. It is characterized in that The following steps are involved: 1) Design and synthesize short hairpin RNA (shRNA) sequences targeting the dyrk1b gene; 2) inserting the shRNA sequence into an expression vector to construct an expression vector for knocking down the dyrk1b gene; 3) using a third-generation lentiviral packaging system to package the RNA interference sequence into lentiviral particles; 4) using the lentiviral particles to infect predetermined host cells; 5) Through Puromycin drug screening, positive clone cell lines that stably express RNA interference sequences are obtained; 6) Verify the efficiency of virus-mediated knockdown; 7) Using cell culture technology, a stable cell line was established from the host cells in which the expression of the dyrk1b gene was successfully knocked down.

2. A method for establishing a stable cell line overexpressing the dyrk1b gene, Features The following steps are involved: 1) Design a DNA sequence for high expression of dyrk1b; 2) inserting the full-length coding sequence of the dyrk1b gene into an expression vector to construct an expression vector that overexpresses the dyrk1b gene; 3) packaging the DNA sequence into lentiviral particles using a third-generation lentiviral packaging system; 4) using the lentiviral particles to infect predetermined host cells; 5) Obtain a stable positive clone cell line through Puromycin drug screening; 6) Verify the efficiency of virus-mediated overexpression; 7) Establishing a stable cell line from the host cells that successfully overexpress the dyrk1b gene by cell culture technology.

3. The method of claim 1, 2, wherein the host cell is selected from one of the following cell lines: 1) OVCAR3 cell line; 2) SK-OV-3 cell line; 3) SNU119 cell line; 4) COV362 cell line.

4. The method of claim 1, 2, wherein the screening step comprises using antibiotic screening, and the drug selected is Puromysin purine toxin.

5. The construction method as claimed in claims 1 and 2, Features In the step 3), the plasmid obtained in step 1) is packaged using a third-generation lentiviral packaging system to obtain lentivirus for knockdown and overexpression of the dyrk1b gene.

6. The construction method as claimed in claim 5, Features , the lentivirus collection utilizes a method for concentrating viruses.

7. The construction method as claimed in claims 1 and 2, Features In step 6), the plasmid:VSVg:pLP2:pLP1 ratio of 11.25:2.8:7.3:3.9 was used to infect ovcar3 and 293T cells respectively to verify the efficiency of virus knockdown and overexpression. 8 . The cell line for knocking down or overexpressing the dyrk1b gene obtained by the construction method according to any one of claims 1 to 7 .

9. Use of the stable cell line knocking down and overexpressing the dyrk1b gene as claimed in claim 8 in the combined treatment of ovarian cancer with magnetic hyperthermia-small molecule inhibitor-chemotherapy.

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