Pazopanib drug-resistant cell strain for renal clear cell carcinoma as well as construction method and application of pazopanib drug-resistant cell strain

Abstract

The invention provides a renal clear cell carcinoma pazopanib drug-resistant cell strain as well as a construction method and application thereof, the preservation number of the renal clear cell carcinoma drug-resistant cell strain is GDMCC NO: 65811, and the renal clear cell carcinoma drug-resistant cell strain is a pazopanib drug-resistant cell strain of 786-O. The Pazopanib is constructed in a manner of inducing the proliferation of kidney cancer cells 786-O in the Pazopanib by adopting a method of gradually increasing the concentration, and the drug resistance molecular mechanism of the Pazopanib is analyzed and read comprehensively through transcriptome, so that a new treatment idea is provided for clinically coping with the drug resistance of the Pazopanib.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pazopanib-resistant cell line of clear cell renal cell carcinoma, a construction method thereof, and an application thereof. Background Art

[0002] Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer subspecies, accounting for 70 - 80% of all renal cancer cases. Among them, 30% of clear cell renal cell carcinoma patients have metastases at the time of initial diagnosis. Systemic treatment for patients with metastatic renal cell carcinoma with clear cell histological components has shifted from cytokines to drugs targeting angiogenesis.

[0003] Pazopanib is a tyrosine kinase inhibitor mainly used for the treatment of advanced renal cell carcinoma. By inhibiting a variety of tyrosine kinases, it blocks tumor angiogenesis and tumor cell proliferation, and has been approved by the US Food and Drug Administration (FDA) for the treatment of clear cell metastatic renal cell carcinoma. Among tyrosine kinase inhibitors, pazopanib is a first-line treatment option. However, patients receiving pazopanib treatment are prone to develop drug resistance, leading to tumor recurrence. Therefore, there is an urgent clinical need to develop effective salvage therapies to address this acquired drug resistance. Summary of the Invention

[0004] One advantage of the present invention is to provide a pazopanib-resistant cell line of clear cell renal cell carcinoma, a construction method thereof, and an application thereof. By constructing a pazopanib-resistant cell line of clear cell renal cell carcinoma 786 - O, through comprehensive transcriptome analysis, its drug resistance molecular mechanism is interpreted, providing new treatment ideas for clinical response to its drug resistance.

[0005] Another advantage of the present invention is to provide a pazopanib-resistant cell line of clear cell renal cell carcinoma, a construction method thereof, and an application thereof. By gradually increasing the drug concentration, a drug-resistant cell system of clear cell renal cell carcinoma is established, which is more in line with the actual drug treatment process, enabling cells to gradually adapt and select cells with higher drug tolerance under the gradually increasing drug concentration.

[0006] Another advantage of the present invention is to provide a pazopanib-resistant cell line of clear cell renal cell carcinoma, a construction method thereof, and an application thereof. The pazopanib-resistant cell line of clear cell renal cell carcinoma 786 - O constructed by the present invention can provide new treatment targets for the treatment of clinical drug resistance, and is of great significance for screening other anti-tumor drugs, discovering tumor drug resistance markers, and evaluating new anti-tumor drugs.

[0007] Another advantage of the present invention is to provide a pazopanib-resistant cell line of clear cell renal carcinoma, a construction method and an application thereof. Through testing, it is found that the invasion ability and cloning ability of the resistant cells are significantly enhanced compared with the parental cells.

[0008] According to one aspect of the present invention, the present invention provides a pazopanib-resistant cell line of clear cell renal carcinoma, with the preservation number: GDMCC NO: 65811; the pazopanib-resistant cell line of clear cell renal carcinoma is a pazopanib-resistant cell line of renal carcinoma 786-O.

[0009] The pazopanib-resistant cell line of renal carcinoma 786-O is constructed by inducing the renal carcinoma cells 786-O to proliferate in pazopanib by a method of gradually increasing the concentration.

[0010] According to another aspect of the present invention, the present invention provides a construction method of a pazopanib-resistant cell line of clear cell renal carcinoma, comprising the following steps: (S10) Take clear cell renal carcinoma 786-O cells and culture them in 1640 complete medium containing 10% serum; (S20) Gradually increase the concentration of pazopanib to induce the stable proliferation of renal carcinoma cells; and (S30) After the final concentration reaches 50 μM, culture stably for 4 weeks to obtain a pazopanib-resistant cell line of clear cell renal carcinoma 786-O.

[0011] In the step (S20), the stable proliferation of renal carcinoma cells is induced by cyclically increasing the concentration of pazopanib by 5-10 μM.

[0012] The construction method of the pazopanib-resistant cell line of clear cell renal carcinoma, wherein the step (S20) comprises the following steps: (S201) On the basis of the step (S10), when the cell culture reaches a confluence of 60%-70%, add 5 μM of pazopanib to the culture medium; (S202) Continue to culture the cells, change the culture medium every 3 days during the culture process, remove the dead cells, and continue to culture the remaining cells until the cultured cells can stably proliferate in 5 μM pazopanib; (S203) Add 10 μM of pazopanib and repeat the above steps; (S204) Cyclically increase the concentration by 5 μM and repeat the above steps; (S205) After reaching a concentration of 50 μM, culture stably for 4 weeks to obtain a pazopanib-resistant cell line of clear cell renal carcinoma 786-O.

[0013] Among them, in the step (S10), 786-O cells are cultured in a complete 1640 medium containing 10% serum in a constant temperature culture phase at 37°C and containing 5% carbon dioxide for 786-O cells.

[0014] Among them, the half-lethal concentration of the parental cells and the drug-resistant cells is tested by culturing the renal cancer parental cells in the logarithmic growth phase and the 786-O pazopanib-resistant cells of renal clear cell carcinoma and adding different concentrations of pazopanib and CCK-8 reagent to evaluate the construction of the 786-O pazopanib-resistant cell line of renal clear cell carcinoma.

[0015] Among them, in the step (S204), the concentration is increased cyclically by 5 µM until the final concentration reaches 50 µM, and the above steps are not repeated thereafter.

[0016] According to another aspect of the present invention, the present invention provides an application of a 786-O pazopanib-resistant cell line of renal clear cell carcinoma, The 786-O pazopanib-resistant cell line of renal clear cell carcinoma is the 786-O pazopanib-resistant cell line of renal cancer, and the preservation number is: GDMCC NO: 65811, which is suitable for application in the research of anti-cancer drugs. Brief Description of the Drawings

[0017] Figure 1 It is a schematic diagram of the comparison result of the sensitivity of the parental cells (786-O) and the drug-resistant cells (RT-786-O) to pazopanib in the present invention.

[0018] Figure 2 It is a schematic diagram of the comparison result of the invasion ability of the drug-resistant cells (RT-786-O) and the parental cells (786-O).

[0019] Figure 3 It is a schematic diagram of the comparison result of the colony formation ability of the drug-resistant cells (RT-786-O) and the parental cells (786-O).

[0020] Figure 4 It is a schematic diagram of the volcano plot of the differential genes between the parental cells (786-O) and the drug-resistant cells (RT-786-O).

[0021] Figure 5 It is a schematic diagram of the grouped clustering of the differential genes between the parental cells (786-O) and the drug-resistant cells (RT-786-O).

[0022] Figure 6 It is a schematic diagram of the main biological functions enriched by the GO analysis of the differential genes between the parental cells and the drug-resistant cells.

[0023] Figure 7 It is a schematic diagram of the main signaling pathways enriched by the KEGG analysis of the differential genes between the parental cells and the drug-resistant cells.

[0024] Figure 8A and Figure 8B are both schematic diagrams of the distribution of the differential transcription factor family.

[0025] Figure 9 is a schematic diagram showing the distribution of the target genes of the transcription factor family among the differential genes.

[0026] Figure 10 is a schematic diagram of the protein - protein interaction network of the differential genes. Detailed implementation manners

[0027] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only examples, and those skilled in the art can think of other obvious variants. The basic principles defined in the following description can be applied to other implementation schemes, variant schemes, improvement schemes, equivalent schemes, and other technical schemes that do not deviate from the spirit and scope of the present invention.

[0028] One advantage of the present invention is to provide a pazopanib - resistant cell line of renal clear cell carcinoma and its construction method. By constructing a pazopanib - resistant cell line of renal clear cell carcinoma 786 - O, and through comprehensive transcriptome analysis, its drug - resistant molecular mechanism is interpreted, providing new treatment ideas for clinical response to its drug resistance.

[0029] The preservation information of the pazopanib - resistant cell line of renal clear cell carcinoma 786 - O is shown in Table 1.

[0030] Table 1 Preservation information table of the pazopanib - resistant cell line of renal clear cell carcinoma 786 - O NO. Item Information 1 Preservation Center Guangdong Microbial Culture Collection Center 2 Name of Culture Human Renal Clear Cell Adenocarcinoma Cell 786-O - pazopanib-resistant 3 Preservation Number GDMCC NO: 65811 4 Preservation Date January 16, 2025 5 Address of Preservation Unit 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou The construction method of the pazopanib - resistant cell line of renal clear cell carcinoma 786 - O includes the following steps: (S10) Take renal clear cell carcinoma 786 - O cells and culture them in a 1640 complete medium containing 10% serum; (S20) Gradually increase the concentration of pazopanib to induce the stable proliferation of renal cancer cells; and (S30) After the final concentration reaches 50 µM, culture stably for 4 weeks to obtain the pazopanib - resistant cell line of renal clear cell carcinoma 786 - O.

[0031] Among them, in the step (S10), the 786 - O cells are cultured in a 1640 complete medium containing 10% serum in a constant temperature culture phase at 37°C and containing 5% carbon dioxide.

[0032] In the step (S20), the stable proliferation of renal cancer cells is induced by cyclically increasing the concentration of pazopanib by 5 - 10 µM.

[0033] Among them, the step (S20) includes the following steps: (S201) On the basis of the step (S10), when the cell confluence reaches 60%-70%, add 5 µM pazopanib to the culture medium; (S202) Continue to culture the cells, change the culture medium every 3 days during the culture process, remove the dead cells, and continue to culture the remaining cells until the cultured cells can stably proliferate in 5 µM pazopanib; (S203) Add 10 µM pazopanib and repeat the above steps; (S204) Increase the concentration in a cyclic increment of 5 µM until the final concentration reaches 50 µM; (S205) After reaching the concentration of 50 µM, culture stably for 4 weeks to obtain the pazopanib-resistant cell line of renal clear cell carcinoma 786-O.

[0034] Determine the median lethal concentration of the parental cells (786-O) and the resistant cells (RT-786-O).

[0035] Seed the 786-O parental cells and the resistant cells in the logarithmic growth phase in a 96-well plate, with 10,000 cells per well and five replicates for each concentration. After the cells adhere, add 0, 10, 20, 40, 80, 160, 320, 640 µM pazopanib respectively, and add CCK-8 reagent after culturing in a constant temperature incubator for 72 h to detect the cell viability. The detection results are as shown in the appendix Figure 1 shown, where the appendix Figure 1 is the comparison result graph of the sensitivity of the parental cells (786-O) and the resistant cells (RT-786-O) to pazopanib after the final concentration of human renal cancer cell 786-O resistance reaches 50 µM.

[0036] From Figure 1 it can be found that the median lethal concentration IC50 of the parental cells is 69.35 µM, while the median lethal concentration IC50 of the resistant cells is 386.0 µM. From this result, it can be seen that the tolerance of the resistant cells to pazopanib is significantly increased, proving that the resistant cells constructed by the present invention are successfully constructed.

[0037] Comparison of the invasion ability between the resistant cells (RT-786-O) and the parental cells (786-O).

[0038] The invasion ability of cells is an important index for evaluating the metastasis of tumor cells. The present invention evaluates the invasion ability of the resistant cells and the parental cells through the transwell invasion experiment. The specific method is as follows.

[0039] (1) Required materials Transwell chambers (12 µm), 24-well plates, Matrigel (Corning), basic materials required for cell experiments.

[0040] (2) Operating procedures ① Basement membrane hydration: Before using the Transwell chamber, its basement membrane needs to be hydrated. The specific operation is to add 50 µL of serum-free culture medium to each well and then incubate at 37 °C for 30 minutes. ② Cell preparation: Starve the cells under serum-free conditions for 12 - 24 hours to remove the influence of serum. Subsequently, wash the cells with PBS and resuspend them in serum-free medium containing BSA, and adjust the cell density to 5×10 5 cells / mL.

[0041] ③ Cell seeding: Add 100 µL of cell suspension to the upper chamber of the Transwell chamber, and add 600 µL of medium containing 10% FBS to the lower chamber. Culture the cells for 12 - 48 hours, and the specific time depends on the migration ability of the cells.

[0042] ④ Result statistics: After the culture is completed, take out the Transwell chamber, fix and stain it, and then observe and count the cells that have passed through the membrane under the microscope. Usually, stain with 0.1% crystal violet for 20 minutes, and then remove it with a cotton swab. The test results are as Figure 2 shown, where Figure 2 is a schematic diagram of the comparison of invasion abilities between drug-resistant cells (RT-786-O) and parental cells (786-O).

[0043] As can be seen from the appendix Figure 2 the invasion ability of drug-resistant cells (RT-786-O) is significantly enhanced compared to parental cells (786-O).

[0044] The comparison test of colony formation ability between drug-resistant cells (RT-786-O) and parental cells (786-O) is as follows: The colony formation ability of cells is an important indicator for evaluating tumor proliferation. In this invention, the tumor proliferation and formation abilities of drug-resistant cells (RT-786-O) and parental cells (786-O) are evaluated through colony formation experiments. The specific experimental methods are as follows.

[0045] The plate colony formation experiment includes the following steps.

[0046] (1) Required materials Basic culture medium, fetal bovine serum, trypsin, PBS, 6-well plates (or culture dishes), 4% paraformaldehyde fixative, crystal violet staining solution, sterile pipettes and tips, cell counting plates and other cell culture consumables.

[0047] (2)Operation steps ① Cell preparation: Collect the cells or pre-treated cells after normal digestion with trypsin, resuspend them in complete medium to form a single-cell suspension, and count the cells; ② Cell seeding: Adjust the cell suspension concentration to 1000 cells / mL, and seed 1000 cells per well in a 6-well plate (the appropriate number of seeded cells varies for different cells and should be adjusted according to the actual situation); ③ Cell clone culture: Continue to culture in a cell incubator for about 1 - 3 weeks. During this period, change the medium every 3 days and observe the cell status; ④ Fixation and staining: After the culture is completed, wash the cells with PBS, add 1 mL of 4% paraformaldehyde to each well to fix the cells for 30 - 60 min. After washing again, add 1 mL of crystal violet staining solution to each well and stain for 10 - 20 min; ⑤ Counting: Count the number of clones formed under a microscope, or take pictures of the entire 6-well plate and each well.

[0048] The test results of the clone formation ability of drug-resistant cells (RT-786-O) and parental cells (786-O) are shown in the appendix Figure 3 as follows, where the appendix Figure 3 is a schematic diagram of the comparison results of the clone formation ability of drug-resistant cells (RT-786-O) and parental cells (786-O).

[0049] As can be seen from the appendix Figure 3 the clone formation ability of drug-resistant cells is significantly enhanced.

[0050] In addition to the differences in cell function phenotypes observed above, to further compare the differences between parental cells (786-O) and drug-resistant cells (RT-786-O) at the molecular level, we extracted the RNA of the above cells for transcriptome sequencing.

[0051] The specific experimental method for RNA extraction is as follows.

[0052] Steps for extracting cell RNA: ① Add 1 mL of Trizol to each well of cells in the 6-well plate, place on ice for 5 min, and pipette with a pipette tip.

[0053] ② Aspirate the lysate from each well into a 1.5 mL EP tube, add 0.2 mL of chloroform per tube, shake vigorously for 15 s, incubate at 15 - 30 °C for 2 - 3 min, and centrifuge (4 °C, 12000 g, 15 min).

[0054] ③ After centrifugation, the liquid is divided into three layers (the upper layer - colorless aqueous layer is RNA, the middle layer is white DNA, and the bottom layer is red protein). Carefully aspirate the upper colorless liquid and transfer it to a new EP tube.

[0055] ④ Add an equal volume of isopropanol, mix well with 0.4 - 0.5 mL, incubate at 15 - 30 °C for 10 - 30 min, and centrifuge (4 °C, 12000 g, 10 min). If after adding an equal volume of isopropanol, place it on a test tube rack, seal it with PE gloves, and put it in a 4 °C refrigerator, the precipitation effect is better for 30 min.

[0056] ⑤ Discard the supernatant, add 1 mL of 75% ethanol to the precipitate, vortex for 30 s, and centrifuge (4 °C, 7500 g, 5 min).

[0057] ⑥ Carefully discard the supernatant, and let the precipitate in the tube air-dry in a laminar flow hood for 3 - 5 min. It is best to aspirate the supernatant with a pipette tip to remove as much as possible.

[0058] ⑦ Add 20 μL of DEPC water to dissolve, aliquot 5 μL / tube, and send it to the sequencing platform for sequencing.

[0059] The sequencing results are as attached Figure 4 shown, attached Figure 4 shown is the volcano plot of differential genes between parental cells (786 - O) and drug-resistant cells (RT - 786 - O). The differential situations generated by the comparison are reflected in the volcano plot. Gray represents genes with non-significant differences, and red and blue represent genes with significant differences; the horizontal axis is log 2 fold change, and the vertical axis is -log 10 q value, where RT - 786 - O - vs - 786 - O: q value < 0.05, and the absolute value of log 2 fold change is greater than 1.

[0060] It can be learned from the attachment Figure 4 that compared with the parental cells (786 - O), there are 1693 significantly down-regulated genes and 2197 significantly up-regulated genes in the drug-resistant cells (RT - 786 - O). This significant gene difference indicates that the drug-resistant cells (RT - 786 - O) and the parental cells (786 - O) have been completely differentiated. The results are as attached Figure 5 shown, attached Figure 5 shown is the grouped clustering plot of differential genes between parental cells (786 - O) and drug-resistant cells (RT - 786 - O), where red in the figure represents relatively highly expressed protein-coding genes, and blue represents relatively lowly expressed protein-coding genes, where RT - 786 - O - vs - 786 - O: q value < 0.05, and the absolute value of log 2 fold change is greater than 1.

[0061] From the attachment Figure 5It can be seen that, compared with the parental cells (786-O), the top ten significantly downregulated genes are PPL, CHD11, GALNT6, GSTP1, UGT1A8, SERPINB9, GLUL, PBDC1, SOD3, and CCDC8. The top ten significantly upregulated genes are CPVL, ADGRL3, RNF150, NOX5, OR13A1, PUPRL, ACSS3, LIN7A, MIR548XAG, and TMPRSS15.

[0062] To better understand the differences in biological functions between parental cells and drug-resistant cells, after obtaining the overall differential genes in the present invention, GO enrichment analysis was performed on the genes to describe their corresponding functions. Figure 6 The top 10 most significant information enriched by the differential genes was shown from three levels: biological process, cellular component, and molecular function.

[0063] Appendix Figure 6 The main biological function schematic diagram of GO analysis of differential gene enrichment between parental cells and drug-resistant cells is shown in the appendix. The vertical axis is the name of the GO term, and the horizontal axis is -log 10 p-value, showing the top 30 GO functional terms of RT-786-O-vs-786-O (total).

[0064] Meanwhile, to better understand the changes in signaling pathways between parental cells and drug-resistant cells, KEGG analysis was performed in the present invention. Figure 7 It shows the most significantly changed signaling pathways enriched by the differential genes, RT-786-O-vs-786-O: KEGG pathway classification. Specifically, the appendix Figure 7 is the main pathway schematic diagram of KEGG analysis of differential gene enrichment between parental cells and drug-resistant cells. The horizontal axis is the ratio (%) of the upregulated (downregulated) differentially expressed genes annotated to each pathway to the total number of upregulated (downregulated) differentially expressed genes annotated to all KEGG pathways, and the vertical axis represents the name of the signaling pathway. The number on the right side of the column represents the number of upregulated (downregulated) differentially expressed genes annotated to this pathway. It can be seen from the appendix Figure 7 KEGG pathway analysis that the differential genes are mainly enriched in which cellular pathways. These significantly changed genes and the enriched signaling pathways may play important roles in the process of cell drug resistance and can provide new therapeutic targets for the treatment of clinical drug resistance.

[0065] In addition, transcription factors are a group of protein molecules that can specifically bind to specific sequences upstream of the 5` end of genes, thus ensuring the expression of target genes at a specific intensity in a specific time and space. In Figure 8A and Figure 8BAmong them, by comparing the distribution of transcription factors of all genes and differential genes (up-regulated and down-regulated), transcription factors with obvious proportional differences between the parent and drug-resistant cells can be found. Next, differential target genes corresponding to the differential transcription factors can be extracted according to the relationship list between transcription factors and target genes, and a statistical chart of target genes of the differential transcription factor family can be drawn, as shown in the appendix Figure 9 as shown.

[0066] Among them, the appendix Figure 8A and Figure 8B are both distribution diagrams of the differential transcription factor family. The abscissa is the transcription factor family; the ordinate is the number of genes; dark blue represents all genes regulated by the transcription factor family; yellow represents the differential transcription factors regulated by the transcription factor family; pink represents the differential transcription factors with an up-regulated trend regulated by the transcription factor family; light blue represents the differential transcription factors with a down-regulated trend regulated by the transcription factor family. Among them Figure 8B is Figure 8A a partial enlarged schematic diagram of the ordinate gene number from 0 to 50 in

[0067] The appendix Figure 9 is a distribution display of target genes of the transcription factor family in differential genes, that is, a statistical chart of (transcription factor regulation of differential genes RT-786-O-vs-786-O). The abscissa is the transcription factor family; the ordinate is the number of target genes; pink represents that the target genes show an up-regulated trend in this differential grouping; light blue represents that the target genes show a down-regulated trend in this differential grouping; the value represents the number of up / down-regulated genes.

[0068] To further understand the protein interaction of differential genes between the parent and drug-resistant genes, we annotated the species information in the STRING database or aligned the species gene sequences with the protein sequences of this species in the STRING database (e-value < 10 -10 ), obtained the interaction relationship of genes, sorted them according to the interaction combined score from high to low, and screened a circular diagram of the interaction relationship results of the top 30 differential genes, as shown in the appendix Figure 10 as shown.

[0069] Through the above analysis, the present invention comprehensively analyzes the differences between the parent cells and drug-resistant cells at the transcriptome level. These significantly different genes play important roles in the process of pazopanib resistance in renal cancer and can provide new treatment targets for the treatment of clinically drug-resistant patients.

[0070] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the accompanying drawings are only examples and do not limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functions and structural principles of the present invention have been demonstrated and explained in the embodiments, and without departing from the said principles, any variations or modifications can be made to the embodiments of the present invention.

Claims

1. A renal clear cell carcinoma pazopanib-resistant cell line, characterized in that: The deposit number is: GDMCC NO: 65811; the renal clear cell carcinoma pazopanib-resistant cell line is the renal carcinoma 786-O pazopanib-resistant cell line.

2. The renal clear cell carcinoma pazopanib-resistant cell line according to claim 1, wherein the renal cancer 786-O pazopanib-resistant cell line is constructed by inducing the proliferation of renal cancer cells 786-O in pazopanib by gradually increasing the concentration.

3. A method for constructing a pazopanib-resistant renal clear cell carcinoma cell line, characterized in that: The following steps are involved: (S10) Renal clear cell carcinoma 786-O cells were cultured in a serum-containing medium; (S20) gradually increasing the concentration of pazopanib to induce stable proliferation of renal cancer cells; and (S30) After the final concentration reaches a predetermined concentration, the cell line is cultured to obtain a renal clear cell carcinoma 786-O pazopanib-resistant cell line.

4. The method for constructing a pazopanib-resistant renal clear cell carcinoma cell line according to claim 3, wherein in the step (S20), the stable proliferation of renal cancer cells is induced by cyclically increasing the concentration of pazopanib by 5-10 µM.

5. The method for constructing a pazopanib-resistant renal clear cell carcinoma cell line according to claim 4, wherein the step (S20) comprises the following steps: (S201) Based on the step (S10), when the cells are cultured to a confluence of 60%-70%, 5 µM pazopanib is added to the culture medium; (S202) continuing to culture the cells, changing the culture medium every 3 days during the culture process, removing dead cells, and continuing to culture the remaining cells until the cultured cells can stably proliferate in 5 µM pazopanib; (S203) Add 10 µM pazopanib and repeat the above steps; (S204) Repeat the above steps with the concentration increasing by 5 µM; (S205) When the concentration reached 50 µM, the cell line was stably cultured for 4 weeks to obtain the renal clear cell carcinoma 786-O pazopanib-resistant cell line.

6. The method for constructing a pazopanib-resistant renal clear cell carcinoma cell line according to any one of claims 3 to 5, wherein in the step (S10), 786-O cells are cultured in a constant temperature culture phase at 37°C and containing 5% carbon dioxide using 1640 complete medium containing 10% serum.

7. The method for constructing a pazopanib-resistant renal clear cell carcinoma cell line according to claim 6, wherein the renal cancer parental cells in the logarithmic growth phase and the renal clear cell carcinoma 786-O pazopanib-resistant cells are cultured and different concentrations of pazopanib and CCK-8 reagent are added to test the half lethal concentration of the parental cells and the resistant cells to judge the construction of the renal clear cell carcinoma 786-O pazopanib-resistant cell line.

8. The method for constructing a pazopanib-resistant renal clear cell carcinoma cell line according to claim 5, wherein in the step (S204), the concentration is increased by 5 µM in a cycle until the final concentration reaches 50 µM.

9. A use of a pazopanib-resistant renal clear cell carcinoma cell line, characterized in that: The renal clear cell carcinoma pazopanib-resistant cell line is a renal carcinoma 786-O pazopanib-resistant cell line, with a deposit number of GDMCC NO: 65811, and is suitable for use in research on anticancer drugs.

10. Use of the renal clear cell carcinoma pazopanib-resistant cell line according to claim 9, wherein the renal cancer 786-0 pazopanib-resistant cell line is prepared according to the construction method of the renal clear cell carcinoma pazopanib-resistant cell line according to any one of claims 3 to 9.

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