Method for promoting proliferation and migration of liver cancer cells through hypoxia induction of ACO2 nucleation

Abstract

The invention discloses a method for promoting proliferation and migration of liver cancer cells through hypoxia induction of ACO2 nucleation, and relates to the field of cells. Comprising the following steps: cell culture and passage: placing cells in a complete medium of a DMEM (dulbecco modified eagle medium), culturing under culture conditions, then performing passage, selecting hepatoma carcinoma cell line PLC5 cells, and culturing the hepatoma carcinoma cell line PLC5 cells in a culture medium containing 10% of fetal calf serum and 1% of streptomyces penicillium; constructing plasmids: selecting the plasmids and constructing the plasmids; transfecting the cells, namely transfecting the cultured and subcultured cells; detecting the condition that the plasmids enter the cells through an EdU experiment; detecting the expression of related proteins through immunofluorescent staining; detecting cell migration through a scratch experiment; cell invasion is detected through a Transwell experiment; and counting results. The action mechanism of the ACO2 for nuclear metastasis in liver cancer cells and the influence of the ACO2 on HCC can be accurately determined, so that the application has important clinical significance on a new treatment direction.

Description

Technical Field

[0001] The present invention relates to the field of cells, and in particular to a method for promoting proliferation and migration of liver cancer cells by inducing ACO2 into the nucleus due to hypoxia. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. According to the latest statistics from the International Agency for Research on Cancer, HCC ranks sixth in global incidence and is the third leading cause of cancer-related deaths worldwide. Although there are clear risk factors for HCC, most patients are already in the advanced stage at the time of diagnosis, and the prognosis of patients with HCC is poor, with a 3-year survival rate of only 12.7%. Therefore, exploring the potential mechanisms of occurrence, development and metastasis of HCC and finding effective molecular targets are of great clinical significance in providing new treatment directions.

[0003] The tricarboxylic acid cycle (TCA cycle) is the central hub of cellular metabolism and energy production and is essential for cellular homeostasis. It has been confirmed that abnormalities in TCA cycle enzymes are closely related to the occurrence and development of tumors. The latest studies have found that TCA cycle-related enzymes also exist in the cell nucleus, including citrate synthase (CS), aconitase 2 (ACO2) and isocitrate dehydrogenase 3 (IDH3), and confirmed that the biochemical reactions catalyzed by these nuclear enzymes are similar to the corresponding biochemical reactions catalyzed by these enzymes in mitochondria, and constitute an incomplete TCA cycle in the cell nucleus (nuclear TCA cycle, nTCA cycle).

[0004] ACO2 is a key enzyme in the TCA cycle. It can catalyze the mutual conversion between citrate and isocitrate. It is also an essential enzyme connecting the TCA cycle and lipid metabolism. Studies have found that ACO2 is highly expressed in a variety of tumor types, and ACO2 can be used as a biomarker for tumor diagnosis and prediction. It has been confirmed that ACO2 is significantly upregulated in the cytoplasm of liver cancer cells compared with the cytoplasm of normal liver cells, and can promote the proliferation, migration and invasion of liver cancer cells; knocking down ACO2 can significantly inhibit the proliferation, migration and invasion of liver cancer cells, and high ACO2 expression is negatively correlated with the prognosis of hepatocellular carcinoma.

[0005] However, the mechanism of nuclear translocation of ACO2 in liver cancer cells and its impact on HCC are also of particular importance.

[0006] To this end, the present invention proposes a method for promoting the proliferation and migration of liver cancer cells by inducing ACO2 into the nucleus due to hypoxia Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for promoting the proliferation and migration of liver cancer cells by inducing ACO2 into the nucleus under hypoxia.

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

[0009] A method for promoting the proliferation and migration of liver cancer cells by inducing ACO2 into the nucleus through hypoxia, comprising the following steps:

[0010] S1: Cell culture and passaging: cells are placed in complete medium of DMEM, cultured under culture conditions, and then passaged;

[0011] S2: plasmid construction, plasmid selection and construction;

[0012] S3: Cell transfection: take the cultured and subcultured cells for transfection;

[0013] S4: EdU assay to detect plasmid entry into cells;

[0014] S5: Immunofluorescence staining to detect the expression of related proteins;

[0015] S6: Scratch assay to detect cell migration;

[0016] S7: Transwell assay to detect cell invasion;

[0017] S8: Statistical results.

[0018] Preferably: the step S1 includes the following steps:

[0019] S11: selecting liver cancer cell line PLC5 cells, culturing them in a culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, at a carbon dioxide concentration of 5% and a temperature of 37 degrees Celsius;

[0020] S12: When the cell density reaches 50%, the cells are immediately placed in an incubator maintained under hypoxic conditions of 94% nitrogen, 5% carbon dioxide, and 1% oxygen for culture;

[0021] S13: Subculture was performed after the cell density reached 70%.

[0022] Preferably: in the step S2, the plasmid is a wild-type ACO2 plasmid: pCMV-ACO2 (human)-3×FLAG-Neo, and an ACO2 plasmid lacking a nuclear localization sequence is constructed, and the upstream primer sequence of NLS is 5'-CAGTGGGACAGGGAGAAGAACAC-3'; the downstream primer sequence is 3'-GTGTTCTTCTCCCTGTCCCACTG-5'.

[0023] Preferably: the step S3 includes the following steps:

[0024] S31: PLC5 liver cancer cells in the logarithmic growth phase were collected and counted under a microscope;

[0025] S32: 1.5 × 10 cells / well were seeded in 500 μL of culture medium. 4 cells;

[0026] S33: After culturing the cells in an incubator for 24 h, TM According to the requirements of transfection reagent, wild-type ACO2 plasmid and ACO2 plasmid with nuclear localization sequence deleted were transfected into PLC5 cells respectively;

[0027] S34: Replace the culture medium with fresh one 12 hours after transfection and perform the test 48 hours later.

[0028] Preferably: the step S4 comprises the following steps:

[0029] S41: PLC5 cells in the logarithmic growth phase were taken and counted under a microscope. 1.5×10 cells were inoculated into each well of a 24-well plate in 500 μL of culture medium. 4 After culturing for 24 hours, cells were transfected.

[0030] S42: The two groups of cells transfected with ACO2WT plasmid and transfected with ACO2ΔNLS plasmid were cultured in an incubator containing 5% CO2, 37°C and in an incubator under hypoxic conditions containing 94% N2, 5% CO2, 1% O2 for 48 h respectively;

[0031] S43: The cells were stained, and the results were observed and photographed under an inverted fluorescence microscope. This was repeated three times.

[0032] Preferably: the step S5 comprises the following steps:

[0033] S51: First, the effect of hypoxia on ACO2 nuclear entry was examined;

[0034] S52: Next, the effect of the deletion of the nuclear import sequence on the nuclear import of ACO2 was detected.

[0035] Preferably: the step S6 comprises the following steps:

[0036] S61: The cell system was divided into four groups, namely ACO2WT+Control group, ACO2WT+1%O2 group, ACO2ΔNLS+Control group, and ACO2ΔNLS+1%O2 group;

[0037] S62: Take PLC5 cells in the logarithmic growth phase and add 5×10 cells per well in 2 ml of culture medium. 4 The cells were uniformly seeded in a 6-well plate.

[0038] S63: When the cell density grows to 50%, ACO2WT plasmid and ACO2ΔNLS plasmid are transfected into the cells respectively;

[0039] S64: the ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator with 5% CO2 and 37°C, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator with 9% N2, 5% CO2, and 1% O2;

[0040] S65: After the cells have grown completely, use a sterilized 200 μL pipette tip to make a straight scratch in the center of the cell monolayer, wash off the scratched cells with PBS, add DMEM complete medium, and then observe and take pictures under a microscope;

[0041] S66: The ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator with 5% CO2 and 37°C, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator under hypoxic conditions. After 24 hours of culture, the cells were observed under a microscope. Three fields of view were randomly selected from each group and photographed and analyzed using ImageJ software.

[0042] Preferably: the step S7 comprises the following steps:

[0043] S71: The cells were divided into four groups, namely, ACO2WT+Control group, ACO2WT+1%O2 group, ACO2ΔNLS+Control group, and ACO2ΔNLS+1%O2 group;

[0044] S72: When the cell density in the culture dish grows to 50%, the ACO2WT plasmid and the ACO2ΔNLS plasmid are transfected into the cells respectively;

[0045] S73: The ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator at 37°C and 5% CO2, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator at 9% N2, 5% CO2, and 1% O2, respectively;

[0046] S74: Dilute Matrigel with serum-free DEME medium at a ratio of 1:8 and mix thoroughly. Spread 100 μL of the mixture on the Transwell chamber membrane in each well and place at 37°C. After solidification for 6 hours, add 100 μL of serum-free DEME medium and hydrate for 1 hour.

[0047] S75: Take four groups of PLC5 cells in logarithmic growth phase after transfection, resuspend them in serum-free medium, and add 5×10 4 The cells were inoculated in the upper chamber of the Transwell chamber, and 500 μL of LDMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber of the chamber;

[0048] S76: After 24 hours of culture, wipe off the non-invaded cells in the upper chamber with a cotton swab, fix with 4% paraformaldehyde at room temperature for 30 minutes; rinse twice with PBS; stain with crystal violet for 15 minutes; observe under a microscope, and randomly select 3 fields of view to take pictures.

[0049] Preferably: In the step S8, GraphPad Prism 9 statistical software is used for analysis, and all data are express.

[0050] The beneficial effects of the present invention are:

[0051] 1. The present invention can accurately determine the mechanism of nuclear transfer of ACO2 in liver cancer cells and its effect on HCC, thus having important clinical significance for new treatment directions. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A method proposed by the present invention for inducing ACO2 to enter the nucleus due to hypoxia to promote the proliferation and migration of liver cancer cells. A schematic diagram of the expression of ACO2 in normal liver cells verified by the HPA database;

[0053] Figure 2 A method proposed by the present invention for inducing ACO2 to enter the nucleus due to hypoxia to promote the proliferation and migration of liver cancer cells. A schematic diagram of the expression of ACO2 in liver cancer cells verified by the HPA database;

[0054] Figure 3A schematic diagram of the method proposed by the present invention for inducing ACO2 to enter the nucleus and promote the proliferation and migration of liver cancer cells by immunofluorescence (x400) detection of the effect of hypoxia on the nuclear localization of ACO2 in PLC5 cells;

[0055] Figure 4 A schematic diagram of the method proposed by the present invention for inducing ACO2 nuclear translocation into hypoxia to promote liver cancer cell proliferation and migration, using immunofluorescence to detect the effect of NLS on ACO2 nuclear translocation;

[0056] Figure 5 Schematic diagram of the method proposed by the present invention for inducing ACO2 into the nucleus to promote the proliferation and migration of liver cancer cells by using EDU experiment (x100) to detect the effect of nuclear ACO2 on the proliferation of PLC5 cells;

[0057] Figure 6 Schematic diagram of a method proposed by the present invention for inducing ACO2 to enter the nucleus and promote the proliferation and migration of liver cancer cells by using a scratch assay to detect the effect of nuclear ACO2 on the migration of PLC5 cells;

[0058] Figure 7 This is a schematic diagram of a method proposed in the present invention that hypoxia induces ACO2 to enter the nucleus to promote the proliferation and migration of liver cancer cells, using a Transwell experiment to detect the effect of nuclear ACO2 on the invasion of PLC5 cells. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0060] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected or set, or detachably connected or set, or integrally connected or set. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0061] Embodiment 1:

[0062] A method for promoting the proliferation and migration of liver cancer cells by inducing ACO2 into the nucleus through hypoxia, comprising the following steps:

[0063] S1: Cell culture and passaging: cells are placed in complete medium of DMEM, cultured under culture conditions, and then passaged;

[0064] S2: plasmid construction, plasmid selection and construction;

[0065] S3: Cell transfection: take the cultured and subcultured cells for transfection;

[0066] S4: EdU assay to detect plasmid entry into cells;

[0067] S5: Immunofluorescence staining to detect the expression of related proteins;

[0068] S6: scratch assay to detect cell migration;

[0069] S7: Transwell assay to detect cell invasion;

[0070] S8: Statistical results.

[0071] The step S1 includes the following steps:

[0072] S11: selecting liver cancer cell line PLC5 cells, culturing them in a culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, at a carbon dioxide concentration of 5% and a temperature of 37 degrees Celsius;

[0073] S12: When the cell density reaches 50%, the cells are immediately placed in an incubator maintained under hypoxic conditions of 94% nitrogen, 5% carbon dioxide, and 1% oxygen for culture;

[0074] S13: Subculture was performed after the cell density reached 70%.

[0075] In the S2 step, the plasmid is a wild-type ACO2 plasmid: pCMV-ACO2 (human)-3×FLAG-Neo (P43542) purchased from Miaoling Biotechnology Co., Ltd., and an ACO2 plasmid lacking a nuclear localization sequence is constructed. The upstream primer sequence of NLS is 5'-CAGTGGGACAGGGAGAAGAACAC-3'; the downstream primer sequence is 3'-GTGTTCTTCTCCCTGTCCCACTG-5'.

[0076] The step S3 includes the following steps:

[0077] S31: PLC5 liver cancer cells in the logarithmic growth phase were collected and counted under a microscope;

[0078] S32: 1.5 × 10 cells / well were seeded in 500 μL of culture medium. 4 cells;

[0079] S33: After culturing the cells in an incubator for 24 h, TM According to the requirements of transfection reagent, wild-type ACO2 plasmid and ACO2 plasmid with nuclear localization sequence deleted were transfected into PLC5 cells respectively;

[0080] S34: Replace the culture medium with fresh one 12 hours after transfection and perform the test 48 hours later.

[0081] The S4 step comprises the following steps:

[0082] S41: PLC5 cells in the logarithmic growth phase were taken and counted under a microscope. 1.5×10 cells were inoculated into each well of a 24-well plate in 500 μL of culture medium. 4 After culturing for 24 hours, cells were transfected.

[0083] S42: The two groups of cells transfected with ACO2WT plasmid and transfected with ACO2ΔNLS plasmid were cultured in an incubator containing 5% CO2, 37°C and in an incubator under hypoxic conditions containing 94% N2, 5% CO2, 1% O2 for 48 h respectively;

[0084] S43: The cells were stained, and the results were observed and photographed under an inverted fluorescence microscope. This was repeated three times.

[0085] The step S5 comprises the following steps:

[0086] S51: First, the effect of hypoxia on ACO2 nuclear entry was detected: the experiment was set up in two groups: control group and hypoxia group, with 3 replicates in each group. PLC5 cells (logarithmic growth phase) were taken and 500uL DMEM complete medium containing 1.5×10 4 The cells were evenly inoculated in a 24-well plate at a density of 100 cells / well. After culturing for 24 hours, the control group was placed in an incubator containing 5% CO2 and 37°C, and the hypoxic group was placed in an incubator under hypoxic conditions of 94% N2, 5% CO2, and 1% O2 for 24 hours. The culture medium was discarded and the cells were washed twice with PBS buffer for 5 minutes each. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed three times with PBS for 5 minutes each. The cells were added with strong immunostaining permeabilization solution for 30 minutes and washed three times with PBS for 5 minutes each. After the immunostaining blocking solution was used for 30 minutes, 200 μL of primary antibody was added to each well and incubated overnight at 4°C. The cells were washed three times with PBS for 5 minutes each. The fluorescent secondary antibody was added and incubated at 37°C in the dark for 2 hours. The cells were washed three times with PBS for 5 minutes each. DAPI was added to stain the nucleus in the dark for 5 minutes. The cells were washed three times with PBS for 5 minutes each. After adding anti-fluorescence quencher in the dark, the results were observed and photographed using an inverted fluorescence microscope. The experiment was repeated three times.

[0087] S52: Then, the effect of nuclear import sequence deletion on ACO2 nuclear import was detected: PLC5 cells (logarithmic growth phase) were evenly seeded in a 24-well plate with a density of 1.5×104 cells per well in 500uL DMEM complete medium. After culturing for 24 hours, the medium was discarded. The experiment was set up with four groups: ACO2WT+Control group (after transfection with ACO2WT plasmid, the cells were placed in a 5% CO2, 37°C incubator), ACO2WT+1% O2 group (after transfection with ACO2WT plasmid, the cells were placed in an incubator under hypoxic conditions of 9% N2, 5% CO2, and 1% O2), ACO2ΔNLS+Control (after transfection with ACO2ΔNLS plasmid, the cells were placed in a 5% CO2, 37°C incubator), and ACO2ΔNLS+1% O2 (after transfection with ACO2ΔNLS plasmid, the cells were placed in an incubator under hypoxic conditions) group, with 3 replicate wells in each group, and each group was cultured for 48 hours; the culture medium was discarded, the above-mentioned immunofluorescence staining steps were repeated, and finally the results were photographed and recorded using an inverted fluorescence microscope. The experiment was repeated 3 times.

[0088] The step S6 comprises the following steps:

[0089] S61: The cell system was divided into four groups, namely ACO2WT+Control group, ACO2WT+1%O2 group, ACO2ΔNLS+Control group, and ACO2ΔNLS+1%O2 group;

[0090] S62: Take PLC5 cells in the logarithmic growth phase and add 5×10 cells per well in 2 ml of culture medium. 4 The cells were uniformly seeded in a 6-well plate.

[0091] S63: When the cell density grows to 50%, ACO2WT plasmid and ACO2ΔNLS plasmid are transfected into the cells respectively;

[0092] S64: the ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator with 5% CO2 and 37°C, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator with 9% N2, 5% CO2, and 1% O2;

[0093] S65: After the cells have grown completely, use a sterilized 200 μL pipette tip to make a straight scratch in the center of the cell monolayer, wash off the scratched cells with PBS, add DMEM complete medium, and then observe and take pictures under a microscope;

[0094] S66: The ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator with 5% CO2 and 37°C, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator under hypoxic conditions. After 24 hours of culture, the cells were observed under a microscope. Three fields of view were randomly selected from each group and photographed and analyzed using ImageJ software.

[0095] The step S7 comprises the following steps:

[0096] S71: The cells were divided into four groups, namely, ACO2WT+Control group, ACO2WT+1%O2 group, ACO2ΔNLS+Control group, and ACO2ΔNLS+1%O2 group;

[0097] S72: When the cell density in the culture dish grows to 50%, the ACO2WT plasmid and the ACO2ΔNLS plasmid are transfected into the cells respectively;

[0098] S73: The ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator at 37°C and 5% CO2, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator at 9% N2, 5% CO2, and 1% O2, respectively;

[0099] S74: Dilute Matrigel with serum-free DEME medium at a ratio of 1:8 and mix thoroughly. Spread 100 μL of the mixture on the Transwell chamber membrane in each well and place at 37°C. After solidification for 6 hours, add 100 μL of serum-free DEME medium and hydrate for 1 hour.

[0100] S75: Take four groups of PLC5 cells in logarithmic growth phase after transfection, resuspend them in serum-free medium, and add 5×10 4 The cells were inoculated in the upper chamber of the Transwell chamber, and 500 μL of LDMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber of the chamber;

[0101] S76: After 24 hours of culture, wipe off the non-invaded cells in the upper chamber with a cotton swab, fix with 4% paraformaldehyde at room temperature for 30 minutes; rinse twice with PBS; stain with crystal violet for 15 minutes; observe under a microscope, and randomly select 3 fields of view to take pictures.

[0102] In step S8, GraphPad Prism 9 statistical software was used for analysis, and all data were expressed as x±s.

[0103] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for promoting the proliferation and migration of liver cancer cells by inducing ACO2 into the nucleus through hypoxia, characterized in that: The following steps are involved: S1: Cell culture and passaging: cells are placed in complete medium of DMEM, cultured under culture conditions, and then passaged; S2: plasmid construction, plasmid selection and construction; S3: Cell transfection, taking cultured and subcultured cells for transfection; S4: EdU assay to detect plasmid entry into cells; S5: Immunofluorescence staining to detect the expression of related proteins; S6: Scratch assay to detect cell migration; S7: Transwell assay to detect cell invasion; S8: Statistical results.

2. The method according to claim 1, wherein the hypoxia-induced ACO2 nuclear entry promotes the proliferation and migration of liver cancer cells, The step S1 includes the following steps: S11: selecting liver cancer cell line PLC5 cells, culturing them in a culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, at a carbon dioxide concentration of 5% and a temperature of 37 degrees Celsius; S12: When the cell density reaches 50%, the cells are immediately placed in an incubator maintained under hypoxic conditions of 94% nitrogen, 5% carbon dioxide, and 1% oxygen for culture; S13: Subculture was performed after the cell density reached 70%.

3. The method according to claim 1, wherein: In the S2 step, the plasmid is a wild-type ACO2 plasmid: pCMV-ACO2 (human)-3×FLAG-Neo, and an ACO2 plasmid lacking a nuclear localization sequence is constructed. The upstream primer sequence of NLS is 5'-CAGTGGGACAGGGAGAAGAACAC-3'; and the downstream primer sequence is 3'-GTGTTCTTCTCCCTGTCCCACTG-5'.

4. The method of claim 1, wherein the method comprises: The step S3 includes the following steps: S31: PLC5 liver cancer cells in the logarithmic growth phase were collected and counted under a microscope; S32: 1.5 × 10 cells / well were seeded in 500 μL of culture medium. 4 cells; S33: After culturing the cells in an incubator for 24 h, TM According to the requirements of transfection reagent, wild-type ACO2 plasmid and ACO2 plasmid with nuclear localization sequence deleted were transfected into PLC5 cells respectively; S34: Replace the culture medium with fresh one 12 hours after transfection and perform the test 48 hours later.

5. The method of claim 1, wherein the hypoxia-induced ACO2 nuclear entry promotes the proliferation and migration of liver cancer cells, The S4 step comprises the following steps: S41: PLC5 cells in the logarithmic growth phase were taken and counted under a microscope. 1.5×10 cells were inoculated into each well of a 24-well plate in 500 μL of culture medium. 4 After culturing for 24 hours, cells were transfected. S42: The two groups of cells transfected with ACO2WT plasmid and transfected with ACO2ΔNLS plasmid were cultured in an incubator containing 5% CO2, 37°C and in an incubator under hypoxic conditions containing 94% N2, 5% CO2, 1% O2 for 48 h respectively; S43: The cells were stained, and the results were observed and photographed under an inverted fluorescence microscope. This was repeated three times.

6. The method of claim 1, wherein: The step S5 comprises the following steps: S51: First, the effect of hypoxia on ACO2 nuclear entry was examined; S52: Next, the effect of the deletion of the nuclear import sequence on the nuclear import of ACO2 was detected.

7. The method of claim 1, wherein the method comprises: The step S6 comprises the following steps: S61: The cell system was divided into four groups, namely ACO2WT+Control group, ACO2WT+1%O2 group, ACO2ΔNLS+Control group, and ACO2ΔNLS+1%O2 group; S62: Take PLC5 cells in the logarithmic growth phase and add 5×10 cells per well in 2 ml of culture medium. 4 The cells were uniformly seeded in a 6-well plate. S63: When the cell density grows to 50%, ACO2WT plasmid and ACO2ΔNLS plasmid are transfected into the cells respectively; S64: the ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator with 5% CO2 and 37°C, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator with 9% N2, 5% CO2, and 1% O2; S65: After the cells have grown completely, use a sterilized 200 μL pipette tip to make a straight scratch in the center of the cell monolayer, wash off the scratched cells with PBS, add DMEM complete medium, and then observe and take pictures under a microscope; S66: The ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator with 5% CO2 and 37°C, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator under hypoxic conditions. After 24 hours of culture, the cells were observed under a microscope. Three fields of view were randomly selected from each group and photographed and analyzed using ImageJ software.

8. The method of claim 1, wherein the hypoxia-induced ACO2 nuclear entry promotes the proliferation and migration of liver cancer cells, The step S7 comprises the following steps: S71: The cells were divided into four groups, namely, ACO2WT+Control group, ACO2WT+1%O2 group, ACO2ΔNLS+Control group, and ACO2ΔNLS+1%O2 group; S72: When the cell density in the culture dish grows to 50%, the ACO2WT plasmid and the ACO2ΔNLS plasmid are transfected into the cells respectively; S73: The ACO2WT+Control group and the ACO2ΔNLS+Control group were placed in an incubator at 37°C and 5% CO2, and the ACO2WT+1% O2 group and the ACO2ΔNLS+1% O2 group were placed in an incubator at 9% N2, 5% CO2, and 1% O2, respectively; S74: Dilute Matrigel with serum-free DEME medium at a ratio of 1:8 and mix thoroughly. Spread 100 μL of the mixture on the Transwell chamber membrane in each well and place at 37°C. After solidification for 6 hours, add 100 μL of serum-free DEME medium and hydrate for 1 hour. S75: Take four groups of PLC5 cells in logarithmic growth phase after transfection, resuspend them in serum-free medium, and add 5×10 4 The cells were inoculated in the upper chamber of the Transwell chamber, and 500 μL of LDMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber of the chamber; S76: After 24 hours of culture, wipe off the non-invaded cells in the upper chamber with a cotton swab, fix with 4% paraformaldehyde at room temperature for 30 minutes; rinse twice with PBS; stain with crystal violet for 15 minutes; observe under a microscope, and randomly select 3 fields of view to take pictures.

9. The method of claim 1, wherein: In step S8, GraphPad Prism 9 statistical software was used for analysis, and all data were expressed as x±s.

Images