A pazopanib-resistant renal clear cell carcinoma cell line and its construction method and application

Abstract

The present invention provides a pazopanib-resistant renal clear cell carcinoma cell line, a construction method, and an application thereof. The deposit number of the renal clear cell carcinoma cell line is GDMCC NO: 65811. The renal clear cell carcinoma cell line is a 786-O pazopanib-resistant cell line. The cell line is constructed by inducing the proliferation of renal cancer cells 786-O in the presence of pazopanib by gradually increasing the concentration of pazopanib. Through comprehensive transcriptome analysis, the molecular mechanism of drug resistance is interpreted, providing a new treatment strategy for clinical treatment of drug resistance.

Description

Technical Field

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

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

[0003] Pazopanib is a tyrosine kinase inhibitor primarily used to treat advanced renal cell carcinoma. It inhibits multiple tyrosine kinases, thereby blocking tumor angiogenesis and tumor cell proliferation. It 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 are prone to developing drug resistance, leading to tumor recurrence. Therefore, there is an urgent need to develop effective salvage therapies to address this acquired resistance. Summary of the Invention

[0004] One advantage of the present invention is that it provides a pazopanib-resistant renal clear cell carcinoma cell line, a construction method, and an application. By constructing a pazopanib-resistant renal clear cell carcinoma 786-O cell line and comprehensively analyzing the transcriptome, the molecular mechanism of its resistance is interpreted, providing a new treatment strategy for clinical treatment of its resistance.

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

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

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

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

[0009] The renal cancer 786-O pazopanib-resistant cell line is constructed by inducing the proliferation of renal cancer cell 786-O in pazopanib using a method of gradually increasing the concentration.

[0010] According to another aspect of the present invention, the present invention provides a method for constructing a pazopanib-resistant renal clear cell carcinoma cell line, comprising the following steps:

[0011] (S10) Renal clear cell carcinoma 786-O cells were cultured in 1640 complete medium containing 10% serum;

[0012] (S20) Gradually increasing the concentration of pazopanib to induce stable proliferation of renal cancer cells; and

[0013] (S30) After the final concentration reached 50 μM, the cells were stably cultured for 4 weeks to obtain the renal clear cell carcinoma 786-O pazopanib-resistant cell line.

[0014] In the step (S20), the stable proliferation of renal cancer cells is induced by cyclically increasing the concentration of pazopanib by 5-10 μM.

[0015] The method for constructing a pazopanib-resistant renal clear cell carcinoma cell line, wherein the step (S20) comprises the following steps:

[0016] (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;

[0017] (S202) continuing to culture the cells, changing the culture medium every 3 days during the culture process to remove dead cells, and continuing to culture the remaining cells until the cultured cells can stably proliferate in the presence of 5 µM pazopanib;

[0018] (S203) Add 10 µM pazopanib and repeat the above steps;

[0019] (S204) Repeat the above steps with the concentration increasing by 5 μM;

[0020] (S205) When the concentration reached 50µM, the cells were stably cultured for 4 weeks to obtain the renal clear cell carcinoma 786-O pazopanib-resistant cell line.

[0021] In the step (S10), 786-O cells are cultured in a 1640 complete culture medium containing 10% serum at 37° C. in a constant temperature culture phase containing 5% carbon dioxide.

[0022] The construction of the renal clear cell carcinoma 786-O pazopanib-resistant cell line was evaluated by culturing renal cancer parental cells in the logarithmic growth phase and adding different concentrations of pazopanib and CCK-8 reagent to test the half-lethal concentration of the parental cells and the resistant cells.

[0023] In the step (S204), the concentration is increased by 5 μM in a cycle until the final concentration reaches 50 μM, and the above steps are not repeated.

[0024] According to another aspect of the present invention, the present invention provides a use of a pazopanib-resistant renal clear cell carcinoma cell line.

[0025] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram showing the comparison of the sensitivity of the parental cells (786-O) and the drug-resistant cells (RT-786-O) to pazopanib in the present invention.

[0027] Figure 2 Schematic diagram of the comparison of the invasive ability of drug-resistant cells (RT-786-O) and parental cells (786-O).

[0028] Figure 3 Schematic diagram of the comparison of the clone-forming ability of drug-resistant cells (RT-786-O) and parental cells (786-O).

[0029] Figure 4 Schematic diagram of the volcano plot of differentially expressed genes between parental cells (786-O) and drug-resistant cells (RT-786-O).

[0030] Figure 5 Schematic diagram of grouping and clustering of differentially expressed genes between parental cells (786-O) and drug-resistant cells (RT-786-O).

[0031] Figure 6 Schematic diagram of the main biological functions of differentially enriched genes between parental cells and drug-resistant cells according to GO analysis.

[0032] Figure 7 Schematic diagram of the main signaling pathways of differentially enriched genes between parental cells and drug-resistant cells analyzed by KEGG.

[0033] Figure 8A and Figure 8B All are schematic diagrams of the distribution of differential transcription factor families.

[0034] Figure 9 Schematic diagram showing the distribution of transcription factor family target genes in differentially expressed genes.

[0035] Figure 10 Schematic diagram of the differentially expressed gene-protein interaction network. DETAILED DESCRIPTION

[0036] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are for illustrative purposes only, and those skilled in the art will readily appreciate other obvious variations. The basic principles of the present invention defined in the following description may be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the present invention.

[0037] One advantage of the present invention is that it provides a pazopanib-resistant renal clear cell carcinoma cell line and a construction method thereof. By constructing a pazopanib-resistant renal clear cell carcinoma 786-O cell line and performing comprehensive transcriptome analysis, the molecular mechanism of its resistance is interpreted, providing a new treatment strategy for clinical response to its resistance.

[0038] The deposit information of the renal clear cell carcinoma 786-O pazopanib-resistant cell line is shown in Table 1.

[0039] Table 1 Deposit information of renal clear cell carcinoma 786-O pazopanib-resistant cell line

[0040] NO. item information 1 Collection Center Guangdong Provincial Microbial Culture Collection Center 2 Culture name Human renal clear cell adenocarcinoma cells 786-O-pazopanib-resistant 3 Accession number GDMCC NO:65811 4 Storage time January 16, 2025 5 Address of depository unit 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0041] The method for constructing the renal clear cell carcinoma 786-O pazopanib-resistant cell line comprises the following steps:

[0042] (S10) Renal clear cell carcinoma 786-O cells were cultured in 1640 complete medium containing 10% serum;

[0043] (S20) Gradually increasing the concentration of pazopanib to induce stable proliferation of renal cancer cells; and

[0044] (S30) After the final concentration reached 50 μM, the cells were stably cultured for 4 weeks to obtain the renal clear cell carcinoma 786-O pazopanib-resistant cell line.

[0045] In the step (S10), 786-O cells are cultured in a 1640 complete culture medium containing 10% serum at 37° C. in a constant temperature culture phase containing 5% carbon dioxide.

[0046] In the step (S20), the stable proliferation of renal cancer cells is induced by cyclically increasing the concentration of pazopanib from 5 to 10 μM.

[0047] The step (S20) includes the following steps:

[0048] (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;

[0049] (S202) continuing to culture the cells, changing the culture medium every 3 days during the culture process to remove dead cells, and continuing to culture the remaining cells until the cultured cells can stably proliferate in the presence of 5 µM pazopanib;

[0050] (S203) Add 10 µM pazopanib and repeat the above steps;

[0051] (S204) The concentration was increased by 5 μM in a cycle until the final concentration reached 50 μM;

[0052] After (S205) reached a concentration of 50µM, it was stably cultured for 4 weeks to obtain the renal clear cell carcinoma 786-O pazopanib-resistant cell line.

[0053] The half-lethal concentrations of parental cells (786-O) and drug-resistant cells (RT-786-O) were determined.

[0054] 786-O parent cells and drug-resistant cells in the logarithmic growth phase were plated in 96-well plates, with 10,000 cells per well and five replicates per well for each concentration. After the cells adhered, 0, 10, 20, 40, 80, 160, 320, and 640 µM pazopanib were added, and the cells were cultured in a constant temperature incubator for 72 hours before adding CCK-8 reagent to detect cell viability. The test results are shown in the attached figure. Figure 1 As shown, with Figure 1 The figure shows the comparative results of the sensitivity of the parental cells (786-O) and the resistant cells (RT-786-O) to pazopanib after the final drug-resistant concentration of human renal cancer cell 786-O reached 50µM.

[0055] from Figure 1 It can be found that the half-lethal concentration (IC50) of the parental cells is 69.35 μM, while the half-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 of the present invention were successfully constructed.

[0056] Comparison of the invasive ability of drug-resistant cells (RT-786-O) and parental cells (786-O).

[0057] The invasive ability of cells is an important indicator for evaluating tumor cell metastasis. The present invention uses a transwell invasion assay to evaluate the invasive ability of drug-resistant cells and parental cells. The specific method is as follows.

[0058] (1) Required materials

[0059] Transwell chambers (12µm), 24-well plates, corning, and other basic materials required for cell experiments.

[0060] (2) Operation steps

[0061] ① Basement membrane hydration: Before using the Transwell chamber, the basement membrane needs to be hydrated. The specific operation is to add 50µL of serum-free culture medium to each well and then incubate at 37°C for 30 minutes.

[0062] ② Cell preparation: Starve the cells for 12-24 hours in serum-free conditions to remove the effects of serum. Then, wash the cells with PBS and resuspend them in serum-free medium containing BSA. Adjust the cell density to 5×10 5 pieces / mL.

[0063] ③ Seeding cells: Add 100µL of cell suspension to the upper chamber of a Transwell chamber and 600µL of culture medium containing 10% FBS to the lower chamber. Culture the cells for 12-48 hours, depending on the cell migration ability.

[0064] ④ Result statistics: After the culture is completed, the Transwell chamber is removed, fixed and stained, and the cells that have passed through the membrane are observed and counted under a microscope. Usually 0.1% crystal violet is used for staining for 20 minutes, and then removed with a cotton swab. The test results are as follows: Figure 2 As shown, Figure 2 Schematic diagram of the comparison of the invasive ability of drug-resistant cells (RT-786-O) and parental cells (786-O).

[0065] From the attached Figure 2 It can be seen that the invasive ability of drug-resistant cells (RT-786-O) is significantly enhanced compared with the parental cells (786-O).

[0066] The comparative test of the clone formation ability of drug-resistant cells (RT-786-O) and parental cells (786-O) is as follows:

[0067] The ability of cells to form colonies is an important indicator for evaluating tumor proliferation. This study used colony formation assays to assess the tumor proliferation and formation abilities of drug-resistant cells (RT-786-O) and parental cells (786-O). The specific experimental methods are as follows.

[0068] The plate colony formation experiment includes the following steps.

[0069] (1) Required materials

[0070] Basal 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 chambers and other cell culture consumables.

[0071] (2) Operation steps

[0072] ① Cell preparation: Collect cells or pre-treated cells after normal digestion with trypsin, resuspend them in complete culture medium to form a single cell suspension, and count them;

[0073] 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 seeding cell number varies for different cell types, so proceed according to the actual situation);

[0074] ③ Cell clone culture: Continue to culture in a cell culture incubator for approximately 1-3 weeks, changing the medium every 3 days and observing the cell status;

[0075] ④ Fixation and staining: After the culture is completed, wash the cells with PBS, add 1 mL of 4% paraformaldehyde to each well and fix the cells for 30-60 minutes. After washing again, add 1 mL of crystal violet stain to each well and stain for 10-20 minutes.

[0076] ⑤Counting: Count the number of clones formed under a microscope, or take pictures of the entire 6-well plate and each well.

[0077] The results of the clone formation ability test of drug-resistant cells (RT-786-O) and parental cells (786-O) are shown in the attached Figure 3 As shown, with Figure 3 Schematic diagram of the comparison of the clone-forming ability of drug-resistant cells (RT-786-O) and parental cells (786-O).

[0078] From the attached Figure 3 It can be seen that the clone-forming ability of drug-resistant cells is significantly enhanced.

[0079] In addition to the differences in cell functional phenotypes observed above, in order to further compare the differences between parental cells (786-O) and drug-resistant cells (RT-786-O) at the molecular level, we extracted RNA from the above cells for transcriptome sequencing.

[0080] The specific experimental method for RNA extraction is as follows.

[0081] Steps for extracting cellular RNA:

[0082] ① Add 1 mL of Trizol to each well of a six-well plate, place on ice for 5 minutes, and pipette through the cells.

[0083] ② Pipette the lysate from each well into a 1.5 mL EP tube, add 0.2 mL of chloroform per tube, shake vigorously for 15 seconds, incubate at 15-30°C for 2-3 minutes, and centrifuge (4°C, 12,000 g, 15 minutes).

[0084] ③ After centrifugation, the liquid is divided into three layers (the upper layer - colorless water layer is RNA, the middle white layer is DNA, and the bottom red layer is protein). Carefully aspirate the upper colorless liquid and transfer it to a new EP tube.

[0085] ④ Add an equal volume of isopropanol (0.4-0.5 mL) and mix well. Incubate at 15-30°C for 10-30 min and centrifuge (4°C, 12,000 g, 10 min). It is better to add an equal volume of isopropanol, place the tube in a test tube rack, seal it with PE gloves, and place it in a 4°C refrigerator to precipitate for 30 min.

[0086] ⑤ Remove the supernatant, add 1 mL of 75% ethanol to the precipitate, vortex for 30 seconds, and centrifuge (4°C, 7500g, 5 minutes).

[0087] ⑥ Carefully remove the supernatant and place the pellet in the tube in a clean bench with air to dry for 3-5 minutes. It is best to use a pipette to absorb the supernatant and remove as much as possible.

[0088] ⑦ Dissolve the sample in 20µL of DEPC water, aliquot 5µL into each tube, and send to the sequencing platform for sequencing.

[0089] The sequencing results are as attached Figure 4 As shown, attached Figure 4 The volcano plot shows the differentially expressed genes between the parental cells (786-O) and the drug-resistant cells (RT-786-O). The differences generated by the comparison are reflected in the volcano plot. Gray represents genes with non-significant differences, while red and blue represent genes with significant differences. The horizontal axis is the log2 fold change, and the vertical axis is the -log 10 q value, among which RT-786-O-vs-786-O: q value < 0.05, and the absolute value of the log2 fold change is greater than 1.

[0090] Can be obtained from the attached Figure 4It was found that compared with the parental cells (786-O), there were 1693 genes significantly downregulated and 2197 genes significantly upregulated 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 shown in the attached figure. Figure 5 As shown, attached Figure 5 Shown is a grouping cluster diagram of differentially expressed genes between parental cells (786-O) and drug-resistant cells (RT-786-O), where red indicates relatively highly expressed protein-coding genes and blue indicates relatively lowly expressed protein-coding genes. RT-786-O-vs-786-O: q value < 0.05, and the absolute value of the log2 fold change is greater than 1.

[0091] From the attached Figure 5 It can be seen that compared with the parental cells (786-O), the top ten genes significantly downregulated are PPL, CHD11, GALNT6, GSTP1, UGT1A8, SERPINB9, GLUL, PBDC1, SOD3 and CCDC8. The top ten genes significantly upregulated are CPVL, ADGRL3, RNF150, NOX5, OR13A1, PUPRL, ACSS3, LIN7A, MIR548XAG and TMPRSS15.

[0092] In order to better understand the differences in biological functions between parental cells and drug-resistant cells, the present invention performed GO enrichment analysis on the genes after obtaining the overall differentially expressed genes, and described their corresponding functions. Figure 6 The top 10 most significant enriched information of differentially expressed genes were displayed from three levels: biological process, cellular composition, and molecular function.

[0093] Attachment Figure 6 Schematic diagram of the main biological functions of differentially enriched genes between parental cells and drug-resistant cells in GO analysis, where the vertical axis is the GO entry name and the horizontal axis is -log 10 p-values showing the top 30 GO function entries for RT-786-O-vs-786-O (total).

[0094] At the same time, in order to better understand the changes in signal pathways between parental cells and drug-resistant cells, the present invention conducted KEGG analysis. Figure 7 The most significantly altered signaling pathways of differential gene enrichment were shown, RT-786-O-vs-786-O: KEGG pathway classification. Figure 7This is a schematic diagram of the main pathways of differential gene enrichment between parental cells and drug-resistant cells analyzed by KEGG. The horizontal axis is the ratio (%) of up-regulated (down-regulated) differentially expressed genes annotated to each pathway to the total number of up-regulated (down-regulated) differentially expressed genes annotated to all KEGG pathways. The vertical axis represents the name of the signal pathway, and the numbers on the right side of the column represent the number of up-regulated (down-regulated) differentially expressed genes annotated to the pathway. Figure 7 KEGG pathway analysis reveals which cellular pathways differentially expressed genes are primarily enriched in. These significantly altered genes and enriched signaling pathways may play an important role in cellular drug resistance and could provide new therapeutic targets for the treatment of clinical drug resistance.

[0095] In addition, transcription factors are a group of protein molecules that can specifically bind to specific sequences upstream of the 5' end of a gene, thereby ensuring that the target gene is expressed at a specific intensity at a specific time and space. Figure 8A and Figure 8B By comparing the distribution of transcription factors of all genes and differentially regulated genes (up-regulated and down-regulated), we can find transcription factors with significant differences in proportion between parental and drug-resistant cells. Next, we can extract the differential target genes corresponding to the differential transcription factors based on the relationship list between transcription factors and target genes, and draw a statistical graph of target genes of the differential transcription factor family, as shown in the attached figure. Figure 9 shown.

[0096] Attached Figure 8A and Figure 8B These are all distribution maps of differential transcription factor families. The horizontal axis is the transcription factor family; the vertical axis is the number of genes; dark blue represents all genes regulated by the transcription factor family; yellow represents differential transcription factors regulated by the transcription factor family; pink represents differential transcription factors regulated by the transcription factor family with an up-regulated trend; light blue represents differential transcription factors regulated by the transcription factor family with a down-regulated trend. Figure 8B for Figure 8A The middle vertical axis is a locally enlarged schematic diagram with the number of genes ranging from 0 to 50.

[0097] Attachment Figure 9 This figure shows the distribution of transcription factor family target genes among differentially expressed genes (RT-786-O vs. -786-O). The horizontal axis represents the transcription factor family; the vertical axis represents the number of target genes. Pink indicates a target gene that is upregulated in that differentially expressed group; light blue indicates a target gene that is downregulated in that differentially expressed group. The numerical value represents the number of upregulated / downregulated genes.

[0098] To further understand the protein interactions between the parents and the differentially expressed genes in the drug-resistant genes, we annotated the species information in the STRING database or compared the species gene sequence with the protein sequence of the species in the STRING database (e-value < 10 -10 ), obtain the relationship between genes, sort them from high to low according to the combined interaction score, and screen the interaction relationship results of the top 30 differentially expressed genes in a circle diagram, as shown in the attached figure. Figure 10 shown.

[0099] Through the above analysis, the present invention comprehensively analyzes the differences between parental cells and resistant cells at the transcriptome level. These significantly different genes play an important role in the pazopanib resistance process of renal cancer and can provide new therapeutic targets for the treatment of clinical resistant patients.

[0100] Those skilled in the art will appreciate that the embodiments of the present invention described above and shown in the accompanying drawings are intended to be illustrative only and are not intended to limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments. Any variations or modifications may be made to the embodiments of the present invention without departing from the principles described.

Claims

1. A pazopanib-resistant renal clear cell carcinoma cell line, characterized in that: The deposit number is: GDMCC NO: 65811; the renal clear cell carcinoma pazopanib-resistant cell line is the renal cancer 786-O pazopanib-resistant cell line, wherein a cyclic increase of 5µM concentration is adopted until the final concentration reaches 50µM, and the renal clear cell carcinoma 786-O pazopanib-resistant cell line is obtained after stable culture. Compared with the parental cell 786-O, the top ten genes significantly downregulated are PPL, CHD11, GALNT6, GSTP1, UGT1A8, SERPINB9, GLUL, PBDC1, SOD3 and CCDC8, and the top ten genes significantly upregulated are CPVL, ADGRL3, RNF150, NOX5, OR13A1, PUPRL, ACSS3, LIN7A, MIR548XAG and TMPRSS15.

2. The use of the pazopanib-resistant renal clear cell carcinoma cell line according to claim 1, characterized in that: The renal clear cell carcinoma pazopanib-resistant cell line is a renal cancer 786-O pazopanib-resistant cell line, and is used for screening anti-renal cancer drugs.

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