Method for researching effect and mechanism of GOT1 in lung adenocarcinoma cells

Abstract

The invention discloses an effect and mechanism research method of GOT1 in lung adenocarcinoma cells, and relates to the technical field of biomedical research. Comprising the following steps: S1, cell culture and protein extraction: culturing Bas-2b, A549, H1573 and Calu-3 cells, cleaning the cells with PBS, performing pyrolysis treatment, collecting supernate as a total protein solution, and determining the protein concentration per tube by BCA; s2, carrying out Western Blot experiment, taking 10 micrograms of protein as a loading amount, carrying out electrophoresis, transferring a film, sealing, incubating a primary antibody and a secondary antibody, exposing and developing, and storing a picture; the experiment is repeated for multiple times, and Image J is used for analyzing the strip exposure diagram. According to the method disclosed by the invention, effective knock-down of GOT1 in A549 and H1573 cells is successfully realized through the steps of screening proper cell screening concentration by puromycin, exploring an MOI value, transfecting lentivirus, screening puromycin and the like; the invention provides an important tool and method for follow-up research on the function of GOT1 in lung adenocarcinoma cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical research technology, and in particular to a method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells. Background Art

[0002] As one of the malignant tumors with the highest morbidity and mortality worldwide, the treatment and diagnosis of lung cancer has always been the focus of medical research.

[0003] Tumor cells exhibit the Warburg effect, meaning they consume glucose and produce lactate regardless of oxygen availability, whereas normal cells typically utilize oxidative phosphorylation (OXPHOS). To maintain normal mitochondrial function, tumor cells utilize a process called anaplerosis to replenish metabolites from the TCA cycle.

[0004] Aspartate aminotransferase 1 (GOT1) is a unique isoenzyme of aspartate aminotransferase in eukaryotic cells, located in the cytoplasm. GOT1 consumes aspartate to generate oxaloacetate (OAA) and glutamate (Glu), which further replenishes TCA to provide energy for the cell.

[0005] After searching, the application scheme of Chinese patent application number CN201310653721.8 discloses a method for studying the effect of furin inhibitor on the growth and metastasis of lung adenocarcinoma cells, which specifically includes the following steps: (1) MTT assay for cell proliferation; (2) A549 cell clone formation ability assay; (3) Hochest33342 / PI double staining assay for cell apoptosis; (4) monolayer cell migration assay (wound healing); (5) Transwell invasion assay;

[0006] (6) Western blot detection of cell migration-related protein expression levels; (7) enzyme-linked immunosorbent assay; (8) statistical analysis. The research methods in the above patents have the following deficiencies: Although the research also involves cancer cells, it does not disclose the role and influence of aspartate aminotransferase 1 on lung adenocarcinoma cells and its research methods. Summary of the Invention

[0007] The purpose of the present invention is to address the shortcomings of the prior art and to propose a method for studying the role and mechanism of GOT1 in lung adenocarcinoma cells.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for studying the role and mechanism of GOT1 in lung adenocarcinoma cells comprises the following steps:

[0010] S1: Cell culture and protein extraction: Beas-2b, A549, H1573, and Calu-3 cells were cultured, washed with PBS, lysed, and the supernatant was collected as the total protein solution. The protein concentration in each tube was determined by BCA.

[0011] S2: Western Blot experiment, using 10 μg protein as the sample amount, electrophoresis, transfer, blocking, incubation with primary and secondary antibodies, exposure, development and storage of images; the experiment was repeated multiple times, and the band exposure images were analyzed using Image J;

[0012] S3: GOT1 knockdown experiment, puromycin screening of A549 and H1573 cells, determination of the appropriate puromycin screening concentration, determination of the MOI values ​​for A549 and H1573, lentiviral transfection of A549 and H1573 cells, observation of the transfection efficiency of each well using an upright fluorescent microscope 48 and 72 hours later, and screening of the transfected cells using puromycin;

[0013] S4: Functional assay.

[0014] Preferably, in S1, cell culture and protein extraction specifically include the following steps:

[0015] S11: Culture Beas-2b, A549, H1573, and Calu-3 cells respectively. If the cells are growing well, place the cell culture dishes on the operating table and wash them 2-3 times with PBS.

[0016] S12: Mix RIPA lysis buffer with protease inhibitors, then add RIPA to each culture dish and lyse on ice;

[0017] S13: Use a cell scraper to scrape off the cells and reagents, collect them into a 1.5 ml centrifuge tube, place on ice, and shake evenly;

[0018] S14: Lysis using ultrasonic cell disruptor;

[0019] S15: Use a low-temperature high-speed centrifuge at 12,000 rpm and 4°C, centrifuge for 15 minutes after balancing, and collect the supernatant into a new EP tube to obtain the total protein solution;

[0020] S16: BCA assay for protein concentration per tube;

[0021] S17: Add 5× SDS-PAGE loading buffer and denature the protein in a 100°C metal bath for 10 minutes.

[0022] Preferably, in S12, RIPA lysis buffer and protease inhibitor are mixed at a ratio of 100:1, and then 300 ul of RIPA is added to each 10 cm culture dish, and the cells are lysed on ice for 3-5 minutes.

[0023] Preferably, in S2, the steps of the Western Blot experiment are as follows:

[0024] S21: Taking 10 μg protein as the loading amount, calculate the loading amount of each protein sample;

[0025] S22: Start running the gel at a voltage of 80V. After 30 minutes, adjust the voltage to 120V and continue running until the blot reaches the bottom of the glass plate.

[0026] S23: Activate the PDVF membrane and perform electroporation at 220 mA for 60 min to transfer the protein to the PDVF membrane;

[0027] S24: The PDVF membrane was washed with TBS-T and then blocked in 5% milk blocking solution at room temperature for 2 h;

[0028] S25: Use Tublin and GOT 1 primary antibodies in a preset ratio;

[0029] S26: Wash the blocked strips and place them in the corresponding primary antibody tubes, incubate on a shaker at 4°C overnight;

[0030] S27: After the bands were removed from the primary antibody and washed, the GOT1 band was placed in the rabbit secondary antibody, and the Tublin band was placed in the mouse secondary antibody, and incubated on a shaker at room temperature for 60 min;

[0031] S28: Exposure and development: After cleaning the strips, expose and develop and save the image.

[0032] Preferably, in S21, samples are loaded in the order of marker, Beas-2b, A549, H1573, and Calu-3.

[0033] Preferably, in S3, the GOT1 knockdown experiment is as follows:

[0034] S31: Puromycin screening: Use puromycin to determine the screening concentration of A549 and H1573 cells respectively;

[0035] S32: lentiviral transfection of A549 and H1573 cells;

[0036] S33: Puromycin selection: Use puromycin to select the transfected cells.

[0037] Preferably, in S32, the lentiviral transfection of A549 and H1573 is as follows:

[0038] S321: Cell technology and cell plating, so that the cell density is between 30%-50% on the next day;

[0039] S323: On the second day, select the cell plate, discard the old culture medium, add 1 / 2 volume of fresh culture medium, take the measured MOI value for virus transfection, and add polybrene to assist in dyeing;

[0040] S324: Place in a cell culture incubator for incubation;

[0041] S325: After 4 h, add 1 / 2 volume of fresh culture medium and continue culturing in the incubator;

[0042] S326: 24 h after transfection, the culture medium containing lentivirus was removed, washed with PBS, and replaced with fresh 10% complete culture medium;

[0043] S327: Observe the transfection efficiency of cells in each well using an upright fluorescent microscope 48h and 72h after transfection.

[0044] Preferably, in S4, the functional assay includes a CCK8 assay, specifically as follows:

[0045] ① Cells in the logarithmic growth phase were cultured at 5×10 3 Cells were seeded / well in a 96-well culture plate, 100 μL of complete medium was added to each well, and the cells were pre-cultured in an incubator for 24 hours to allow the cells to adhere. An equal volume of blank medium was additionally added, and 3 to 6 replicate wells were set up for each group.

[0046] ② Remove the supernatant from the culture plate and add fresh culture medium containing 10% CCK-8 reagent to each well; shake gently to mix, and continue culturing; use a microplate reader to measure the absorbance of each well at a wavelength of 450 nm;

[0047] ③The experiment was repeated and statistical analysis was performed using GraphPad Prism software.

[0048] Preferably, in S4, the functional assay further comprises a scratch test, specifically as follows:

[0049] ① On the bottom of the six-hole plate, use a marker to draw three horizontal lines for each hole along the ruler;

[0050] ② The day before, seed well-grown A549 GOT1-sh and shnc into a six-well plate so that the cell density in the six-well plate reaches 100% on the second day;

[0051] ③ After overnight incubation, use a ruler to draw a straight line with a 200 μl pipette tip perpendicular to the well plate and the marked line. The intersection is used as the fixed detection point.

[0052] ④Discard the old culture medium and wash with PBS 2-3 times to rinse the cells clean;

[0053] ⑤Add serum-free culture medium;

[0054] ⑥ Remove cells at set time points after scratching, take photos of the same scratch position under a microscope with white light and 4x magnification, and save the photos;

[0055] ⑦Analyze the scratch test results using Image J;

[0056] ⑧Statistical analysis was performed using GraphPad Prism software.

[0057] Preferably, the set time nodes are specifically: 0h, 6h, 12h, 24h, 48h.

[0058] The beneficial effects of the present invention are:

[0059] 1. The present invention successfully achieved effective knockdown of GOT1 in A549 and H1573 cells by screening suitable cell screening concentrations with puromycin, exploring MOI values, and performing lentiviral transfection and puromycin screening. This provides important tools and methods for subsequent studies on the function of GOT1 in lung adenocarcinoma cells.

[0060] 2. The present invention uses BCA to determine the protein concentration of each tube, ensuring the accurate loading amount of protein samples and improving the accuracy and repeatability of the experiment. At the same time, the detailed steps of the Western Blot experiment and the design of multiple repeated experiments further enhance the reliability and credibility of the experimental results.

[0061] 3. This study comprehensively evaluated the effects of GOT1 knockdown on the proliferation and migration of lung adenocarcinoma cells using two different functional assays: the CCK8 assay and the wound wound healing assay. Band exposure images were analyzed using Image J, and statistical analysis was performed using GraphPad Prism software to ensure the scientific nature and accuracy of the experimental data.

[0062] 4. This invention covers multiple steps from cell culture, protein extraction, Western Blot experiments to GOT1 knockdown experiments and functional assays, forming a complete and systematic research process, which helps to further explore the role and mechanism of GOT1 in lung adenocarcinoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Schematic diagram of WB demonstrating high expression of GOT1 in lung adenocarcinoma cell lines in the present invention;

[0064] Figure 2 Schematic diagram of WB verification of GOT1 knockdown in the A549 cell line in the present invention. DETAILED DESCRIPTION

[0065] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0066] Example 1:

[0067] A method for studying the role and mechanism of GOT1 in lung adenocarcinoma cells, comprising:

[0068] S1: Cell culture and protein extraction: Beas-2b, A549, H1573, and Calu-3 cells were cultured, washed with PBS, lysed, and the supernatant was collected as the total protein solution. The protein concentration in each tube was determined by BCA.

[0069] S2: Western Blot experiment, using 10 μg protein as the sample amount, electrophoresis, transfer, blocking, incubation with primary and secondary antibodies, exposure, development and storage of images; the experiment was repeated multiple times, and the band exposure images were analyzed using Image J;

[0070] S3: GOT1 knockdown experiment, puromycin screening of A549 and H1573 cells, determination of the appropriate puromycin screening concentration, determination of the MOI values ​​for A549 and H1573, lentiviral transfection of A549 and H1573 cells, observation of the transfection efficiency of each well using an upright fluorescent microscope 48 and 72 hours later, and screening of the transfected cells using puromycin;

[0071] S4: Functional assay.

[0072] Wherein, in said S1, cell culture and protein extraction specifically include the following steps:

[0073] S11: Culture Beas-2b, A549, H1573, and Calu-3 cells respectively. If the cells are growing well, place the cell culture dishes on the operating table and wash them 2-3 times with PBS.

[0074] S12: Mix RIPA lysis buffer with protease inhibitors at a ratio of 100:1, then add 300 μl of RIPA to each 10 cm culture dish and lyse on ice for 3-5 minutes;

[0075] S13: Use a cell scraper to scrape off the cells and reagents, collect them into a 1.5 ml centrifuge tube, place on ice, and shake evenly;

[0076] S14: Lysis using ultrasonic cell disruptor;

[0077] S15: Use a low-temperature high-speed centrifuge at 12,000 rpm and 4°C, centrifuge for 15 minutes after balancing, and collect the supernatant into a new EP tube to obtain the total protein solution.

[0078] S16: BCA assay for protein concentration per tube;

[0079] S17: Add 5× SDS-PAGE loading buffer and denature the protein in a 100°C metal bath for 10 minutes.

[0080] Among them, the steps of the Western Blot experiment in S2 are as follows:

[0081] S21: Calculate the loading amount of each protein sample, taking 10 μg of protein as the loading amount; load the samples in the order of marker, Beas-2b, A549, H1573, and Calu-3;

[0082] S22: Start running the gel at a voltage of 80V. After about 30 minutes, adjust the voltage to 120V and continue running the gel until the blot reaches the bottom of the glass plate.

[0083] S23: Activate the PDVF membrane and perform electroporation at 220 mA for 60 min to transfer the protein to the PDVF membrane;

[0084] S24: The PDVF membrane was washed with TBS-T and then blocked in 5% milk blocking solution at room temperature for 2 h;

[0085] S25: Tublin and GOT 1 primary antibodies from Mitaka Bio were used, and the proportions were prepared according to the instructions;

[0086] S26: Wash the blocked strips and place them in the corresponding primary antibody tubes, incubate on a shaker at 4°C overnight;

[0087] S27: After the bands were removed from the primary antibody and washed, the GOT1 band was placed in the rabbit secondary antibody, and the Tublin band was placed in the mouse secondary antibody, and incubated on a shaker at room temperature for 60 min;

[0088] S28: Exposure and development: After cleaning the strips, expose and develop and save the image.

[0089] Among them, in S3, the GOT1 knockdown experiment is as follows:

[0090] S31: Puromycin screening: Use puromycin to determine the screening concentration of A549 and H1573 cells respectively;

[0091] S32: lentiviral transfection of A549 and H1573 cells;

[0092] S33: Puromycin selection: Use puromycin to select the transfected cells.

[0093] Among them, in said S32, the lentiviral transfection of A549 and H1573 is specifically as follows:

[0094] S321: Cell technology and cell plating, so that the cell density is between 30%-50% on the next day;

[0095] S323: On the second day, select a cell plate with an appropriate density, discard the old culture medium, add 1 / 2 volume of fresh culture medium, take the measured MOI value for virus transfection, and add polybrene to assist in dyeing;

[0096] S324: Place in a cell culture incubator for incubation;

[0097] S325: After 4 h, add 1 / 2 volume of fresh culture medium and continue culturing in the incubator;

[0098] S326: 24 h after transfection, the culture medium containing lentivirus was removed, washed with PBS, and replaced with fresh 10% complete culture medium;

[0099] S327: Observe the transfection efficiency of cells in each well using an upright fluorescent microscope 48h and 72h after transfection.

[0100] like Figure 1 、 Figure 2 As shown;

[0101] Among them, in said S4, the functional assay includes CCK8 assay, specifically as follows:

[0102] ① Cells in the logarithmic growth phase (A549GOT1 knockdown group and control group) were cultured at 5×10 3 Cells were seeded / well in a 96-well culture plate, 100 μL of complete medium was added to each well, and the cells were pre-cultured in an incubator for 24 hours to allow the cells to adhere. An equal volume of blank medium was additionally added, and 3 to 6 replicate wells were set up for each group.

[0103] ② Remove the supernatant from the culture plate and add fresh culture medium containing 10% CCK-8 reagent to each well; after gentle vortexing to mix, continue incubation (protect from light); measure the absorbance (OD value) of each well at a wavelength of 450 nm using a microplate reader;

[0104] ③The experiment was repeated and statistical analysis was performed using GraphPad Prism software.

[0105] Among them, in said S4, the functional assay also includes a scratch test, which is as follows:

[0106] ① On the bottom of the six-hole plate, use a marker to draw three horizontal lines for each hole along the ruler;

[0107] ② The day before, seed well-grown A549 GOT1-sh and shnc into a six-well plate so that the cell density in the six-well plate reaches 100% on the second day;

[0108] ③ After overnight incubation, use a ruler to draw a straight line with a 200ul pipette tip perpendicular to the well plate and the marked line. The intersection can be used as a fixed detection point;

[0109] ④Discard the old culture medium and wash with PBS 2-3 times to rinse the cells clean;

[0110] ⑤Add serum-free culture medium;

[0111] ⑥ Take out the cells at 0h, 6h, 12h, 24h, and 48h after scratching, take pictures of the same scratch position under a microscope with white light and 4x magnification, and save the pictures;

[0112] ⑦Analyze the scratch test results using Image J;

[0113] ⑧Statistical analysis was performed using GraphPad Prism software.

[0114] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for studying the role and mechanism of GOT1 in lung adenocarcinoma cells, characterized in that: The steps include: S1: Cell culture and protein extraction: Beas-2b, A549, H1573, and Calu-3 cells were cultured, washed with PBS, lysed, and the supernatant was collected as the total protein solution. The protein concentration in each tube was determined by BCA. S2: Western Blot experiment, using 10 μg protein as the sample amount, electrophoresis, transfer, blocking, incubation with primary and secondary antibodies, exposure, development and storage of images; the experiment was repeated multiple times, and the band exposure images were analyzed using Image J; S3: GOT1 knockdown experiment, puromycin screening of A549 and H1573 cells, determination of the appropriate puromycin screening concentration, determination of the MOI values ​​for A549 and H1573, lentiviral transfection of A549 and H1573 cells, observation of the transfection efficiency of each well using an upright fluorescent microscope 48 and 72 hours later, and screening of the transfected cells using puromycin; S4: Functional assay.

2. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 1, characterized in that: In S1, cell culture and protein extraction specifically include the following steps: S11: Culture Beas-2b, A549, H1573, and Calu-3 cells respectively. If the cells are growing well, place the cell culture dishes on the operating table and wash them 2-3 times with PBS. S12: Mix RIPA lysis buffer with protease inhibitors, then add RIPA to each culture dish and lyse on ice; S13: Use a cell scraper to scrape off the cells and reagents, collect them into a 1.5 ml centrifuge tube, place on ice, and shake evenly; S14: Lysis using ultrasonic cell disruptor; S15: Use a low-temperature high-speed centrifuge at 12,000 rpm and 4°C, centrifuge for 15 minutes after balancing, and collect the supernatant into a new EP tube to obtain the total protein solution; S16: BCA assay for protein concentration per tube; S17: Add 5× SDS-PAGE loading buffer and denature the protein in a 100°C metal bath for 10 minutes.

3. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 2, characterized in that: In the above S12, RIPA lysis buffer and protease inhibitor were mixed at a ratio of 100:1, and then 300 ul of RIPA was added to each 10 cm culture dish and lysed on ice for 3-5 minutes.

4. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 1, characterized in that: In S2, the steps of the Western Blot experiment are as follows: S21: Taking 10 μg protein as the loading amount, calculate the loading amount of each protein sample; S22: Start running the gel at a voltage of 80V. After 30 minutes, adjust the voltage to 120V and continue running until the blot reaches the bottom of the glass plate. S23: Activate the PDVF membrane and perform electroporation at 220 mA for 60 min to transfer the protein to the PDVF membrane; S24: The PDVF membrane was washed with TBS-T and then blocked in 5% milk blocking solution at room temperature for 2 h; S25: Use Tublin and GOT 1 primary antibodies in a preset ratio; S26: Wash the blocked strips and place them in the corresponding primary antibody tubes, incubate on a shaker at 4°C overnight; S27: After the bands were removed from the primary antibody and washed, the GOT1 band was placed in the rabbit secondary antibody, and the Tublin band was placed in the mouse secondary antibody, and incubated on a shaker at room temperature for 60 min; S28: Exposure and development: After cleaning the strips, expose and develop and save the image.

5. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 4, characterized in that: In the above-mentioned S21, samples were loaded in the order of marker, Beas-2b, A549, H1573, and Calu-3.

6. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 1, characterized in that: In S3, the GOT1 knockdown experiment is as follows: S31: Puromycin screening: Use puromycin to determine the screening concentration of A549 and H1573 cells respectively; S32: lentiviral transfection of A549 and H1573 cells; S33: Puromycin selection: Use puromycin to select the transfected cells.

7. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 6, characterized in that: In S32, the lentiviral transfection of A549 and H1573 cells is as follows: S321: Cell technology and cell plating, so that the cell density is between 30%-50% on the next day; S323: On the second day, select the cell plate, discard the old culture medium, add 1 / 2 volume of fresh culture medium, take the measured MOI value for virus transfection, and add polybrene to assist in dyeing; S324: Place in a cell culture incubator for incubation; S325: After 4 h, add 1 / 2 volume of fresh culture medium and continue culturing in the incubator; S326: 24 h after transfection, the culture medium containing lentivirus was removed, washed with PBS, and replaced with fresh 10% complete culture medium; S327: Observe the transfection efficiency of cells in each well using an upright fluorescent microscope 48h and 72h after transfection.

8. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 7, characterized in that: In S4, the functional assay includes CCK8 assay, as follows: ① Cells in the logarithmic growth phase were cultured at 5×10 3 Cells were seeded / well in a 96-well culture plate, 100 μL of complete medium was added to each well, and the cells were pre-cultured in an incubator for 24 hours to allow the cells to adhere. An equal volume of blank medium was additionally added, and 3 to 6 replicate wells were set up for each group. ② Remove the supernatant from the culture plate and add fresh culture medium containing 10% CCK-8 reagent to each well; shake gently to mix, and continue culturing; use a microplate reader to measure the absorbance of each well at a wavelength of 450 nm; ③Repeat the experiment and perform statistical analysis.

9. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 8, characterized in that: In S4, the functional assay also includes a scratch test, as follows: ① On the bottom of the six-hole plate, use a marker to draw three horizontal lines for each hole along the ruler; ② The day before, seed well-grown A549 GOT1-sh and shnc into a six-well plate so that the cell density in the six-well plate reaches 100% on the second day; ③ After overnight incubation, use a ruler to draw a straight line with a 200 μl pipette tip perpendicular to the well plate and the marked line. The intersection is used as the fixed detection point. ④Discard the old culture medium and wash with PBS 2-3 times to rinse the cells clean; ⑤Add serum-free culture medium; ⑥ Remove cells at set time points after scratching, take photos of the same scratch position under a microscope with white light and 4x magnification, and save the photos; ⑦Analyze the scratch test results using Image J; ⑧Perform statistical analysis.

10. The method for studying the effect and mechanism of GOT1 in lung adenocarcinoma cells according to claim 9, characterized in that: The set time nodes are specifically: 0h, 6h, 12h, 24h, 48h.

Images