Application of chromatin remodeling factor BPTF as a diagnostic marker for sunitinib resistance in renal cell carcinoma

Abstract

The present invention discloses the use of a chromatin remodeling factor BPTF as a diagnostic marker for sunitinib resistance in renal cancer. The present invention discloses the use of an active ingredient that downregulates or blocks the expression of BPTF molecules in renal cancer cells in the preparation of relevant kits and drugs for diagnosing and treating sunitinib resistance in renal cancer. The present invention provides a new diagnostic and therapeutic means for targeted drug resistance in renal cancer.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and tumor prevention and treatment technology, and in particular to the use of a chromatin remodeling factor BPTF as a diagnostic marker for sunitinib resistance in renal cancer. Background Art

[0002] Metastatic renal cell carcinoma (mRCC) is insensitive to both radiotherapy and chemotherapy and has a poor prognosis, with a 5-year survival rate typically below 5%. In recent years, targeted therapy has become a hot topic in renal cancer research and treatment, with several molecular targeted drugs introduced and used, achieving remarkable therapeutic effects in the treatment of mRCC. Among them, sunitinib is the second multi-target kinase inhibitor approved for the treatment of advanced renal cancer. The National Comprehensive Cancer Network (NCCN) guidelines, the European Renal Cancer Guidelines, and the CSCO Renal Cancer Diagnosis and Treatment Guidelines all recommend sunitinib as a targeted treatment for advanced renal cancer as Class 1 evidence.

[0003] However, long-term use of sunitinib can induce tumor resistance, and this, combined with the innate resistance of some patients, limits the application of this drug. The initial efficacy response rate of sunitinib is close to 30%-40%, but after 6-15 months of use, sunitinib resistance begins to appear, leading to the progression and metastasis of renal cancer. In recent years, the molecular mechanism of sunitinib resistance in renal cancer cells has been studied, and it has been found that sunitinib resistance is not caused by a single factor, but is a complex process involving multiple factors. In order to reverse sunitinib resistance in renal cancer and improve the efficacy of treatment, this field urgently needs to use multiple methods to study the mechanism of sunitinib resistance in renal cancer, the key gene targets of renal cancer resistance, and design new drugs to treat renal cancer targeted drug resistance based on these targets, thereby improving the efficacy of targeted treatment for renal cancer.

[0004] Bromodomain PHD-finger Transcription Factor (BPTF) is the largest subunit of the Nucleosome Remodeling Factor (NURF) complex. It recruits other NURF subunits to gene promoters or enhancer regions, regulating nucleosome sliding and promoting gene transcription. BPTF has recently been found to influence cancer progression, particularly melanoma survival, by directly activating oncogenic signaling or through synergistic interactions with other key protein factors. BPTF also significantly impacts prognosis in gliomas, lung cancer, colon cancer, liver cancer, and esophageal squamous cell carcinoma. Studies have shown that BPTF is closely associated with chemotherapy resistance and sensitization. Overexpression of BPTF in hepatocellular carcinoma (HCC) promotes liver cancer progression and poor prognosis. BPTF knockdown significantly sensitizes the anti-liver cancer activity of 5-fluorouracil / cisplatin. NSUN2-methylated lncRNA (NMR) promotes esophageal squamous cell carcinoma progression and induces cisplatin chemotherapy resistance by binding to BPTF and regulating the expression of matrix metalloproteinases (MMPs). Overexpression of BPTF in melanoma promotes metastasis and poor prognosis. Furthermore, BPTF induces resistance to the melanoma-targeted drugs vemurafenib and dabrafenib. Furthermore, our previous studies have shown that the BPTF-specific antagonist AU1 significantly inhibits renal cell carcinoma cell migration and invasion. Furthermore, AU1 (13.2 mg / kg) significantly inhibits lung metastasis and prolongs survival in mice with renal cell carcinoma.

[0005] These studies indicate that the chromatin remodeling factor BPTF may be used as a diagnostic marker for clinical resistance to targeted drugs in renal cancer, and that active ingredients that downregulate or block the expression of the chromatin remodeling factor BPTF molecule in renal cancer may be used as clinically effective drugs to combat drug-resistant metastasis of renal cancer. Summary of the Invention

[0006] The present invention aims to provide a method for the use of the chromatin remodeling factor BPTF as a diagnostic marker for sunitinib resistance in renal cancer. By studying the clinical significance of BPTF in renal cancer and the molecular pathways regulating renal cell proliferation and migration, the results demonstrate that BPTF may serve as a new therapeutic target for sunitinib resistance in renal cancer, providing a practical basis for the treatment of patients with renal cancer resistance and metastasis. The present invention also provides an active ingredient that downregulates or blocks BPTF expression in renal cancer for the preparation of drugs to combat renal cancer resistance. Furthermore, the present invention provides a highly sensitive method and kit for diagnosing drug resistance in renal cancer patients, providing a practical basis for treatment.

[0007] To achieve this goal, the inventors investigated the protein expression of BPTF in renal cancer tissue from patients with renal cancer. Fluorescence quantitative PCR and Western blot experiments showed that BPTF expression in renal cancer tissue from patients with sunitinib-resistant renal cancer was significantly higher than that in patients with non-resistant renal cancer, suggesting that this may be a new biomarker.

[0008] By gradually increasing the concentration of sunitinib (2.5, 5, 7.5, and 10 μmol / L) in renal cancer cell lines 786-O and ACHN in vitro, researchers successfully established sunitinib-resistant renal cancer cell lines 786-OR and ACHN-R. CCK-8 assays revealed that the half-maximal inhibitory concentration (IC50) of sunitinib in 786-OR and ACHN-R cells was significantly increased compared to the parental cells. These cells can serve as in vitro cell models for subsequent renal cancer drug resistance studies.

[0009] Subsequent studies have shown that treatment of drug-resistant renal cancer cells (786-OR and ACHN-R cells) with shRNA targeting BPTF significantly downregulated their activity in response to sunitinib, promoting sensitization to sunitinib. This invention provides a new approach to treating sunitinib resistance in renal cancer. This invention has significant implications for selecting treatment options for renal cancer patients, reducing recurrence, and improving patient survival.

[0010] The present invention provides the use of BPTF in the preparation of an agent for diagnosing or treating renal cancer drug resistance. BPTF is a member of the chromatin remodeling factor family. The NCBI accession number for human BPTF is NM_182641.4, and the protein is NP_872579.2. Nucleosome-remodeling factor subunit BPTF isoform 1 [Homo sapiens] is available. BPTF can be used as a diagnostic marker for sunitinib resistance in renal cancer. The active ingredient of the agent is a formulation that downregulates or blocks the expression of the BPTF molecule in renal cancer.

[0011] The present invention also provides an anti-renal cancer drug resistance drug, wherein the active ingredient of the anti-renal cancer drug resistance drug is a preparation of an active ingredient that downregulates or blocks the expression of BPTF molecules in renal cancer cells; that is, the use of the active ingredient that downregulates or blocks the expression of BPTF molecules in renal cancer in the preparation of an anti-renal cancer drug resistance drug; the active ingredient that downregulates or blocks the expression of BPTF molecules in renal cancer is a BPTF antagonist, a BPTF antibody, an RNA interference molecule or an antisense oligonucleotide targeting the BPTF coding sequence.

[0012] The present invention also provides a kit for detecting sunitinib resistance in renal cancer. The kit contains a reagent for detecting BPTF expression. Using the kit to detect BPTF expression further provides evidence for the prognosis or progression of renal cancer patients, and also provides a new intervention strategy for renal cancer patients after recurrence (surgery + targeted therapy). Furthermore, the kit also contains a standard sample or molecular weight marker of BPTF. More preferably, the kit contains amplification primers or binding polypeptides for detecting BPTF.

[0013] The present invention relates to the field of tumor treatment, and the invention discloses the use of BPTF as a target for the treatment of drug-resistant renal cancer. Utilizing this target can effectively prevent and treat renal cancer sunitinib resistance, improve efficacy while reducing toxic side effects, and is of great significance for the development of targeted treatments for renal cancer and even the development of new individualized comprehensive treatments. BPTF's current uses involve serving as a therapeutic target for a variety of tumors (but not renal cancer). The use of BPTF in the preparation of drugs for treating drug-resistant renal cancer, as disclosed herein, is the first disclosure. Since BPTF is highly expressed in drug-resistant renal cancer, and downregulating BPTF significantly inhibits the cell activity of drug-resistant renal cancer cells (786-OR and ACHN-R cells) in sunitinib, promotes sunitinib sensitization, and has outstanding substantive characteristics, the application of BPTF in the prevention and treatment of renal cancer sunitinib resistance represents a groundbreaking advancement. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A The expression of BPTF mRNA in renal cancer tissues of sunitinib-sensitive and -resistant renal cancer patients in the examples;

[0015] Figure 1B The expression of BPTF protein in renal cancer tissues of sunitinib-sensitive and -resistant renal cancer patients in the examples;

[0016] Figure 2A The relationship between BPTF protein expression level and patient survival time (OS) in the examples is shown;

[0017] Figure 2B The relationship between BPTF protein expression level and patient progression-free survival (PFS) is shown in the examples;

[0018] Figure 3A The results show that BPTF shRNA down-regulates BPTF in the embodiment, significantly down-regulating the cell activity of drug-resistant renal cancer cells (786-OR) in sunitinib, and promoting sunitinib sensitization.

[0019] Figure 3BThe results show that BPTF shRNA down-regulates BPTF in the embodiment, significantly down-regulating the cell activity of drug-resistant renal cancer cells (ACHN-R) in sunitinib and promoting sunitinib sensitization.

[0020] Figure 4 The results show that BPTF shRNA down-regulates BPTF in the examples, significantly inhibits the migration of drug-resistant renal cancer cells (ACHN-R or 786-OR) in sunitinib, and promotes sunitinib sensitization.

[0021] Figure 5 The results show that the BPTF-specific antagonist AU1 in the examples significantly inhibited glucose uptake and lactate production in drug-resistant renal cancer cells (786-OR and ACHN-R), reduced the glycolysis level of drug-resistant renal cancer cells, and promoted sunitinib sensitization. DETAILED DESCRIPTION

[0022] The present invention will be described in detail and specifically below through specific examples to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.

[0023] The following details various biological research contents on BPTF conducted using the present invention.

[0024] Example 1

[0025] Fluorescence quantitative PCR experiments were used to test the expression level of BPTF mRNA in renal cancer tissues of sunitinib-resistant and non-resistant renal cancer patients.

[0026] 1. Clinical Sample Collection: Fresh renal cancer tissue samples were collected from patients with sunitinib-resistant and non-resistant renal cancer. The samples were rinsed with physiological saline, frozen with liquid nitrogen, and stored in a -80°C refrigerator.

[0027] 2. RNA extraction from renal cancer tissue:

[0028] 1) Remove the postoperative renal cancer tissue samples stored in liquid nitrogen, cut 50 mg of tissue and place it in a mortar and pestle with liquid nitrogen;

[0029] 2) Grind the tissue into powder and add liquid nitrogen as needed during the grinding process;

[0030] 3) Transfer the powder into a 2 mL enzyme-free centrifuge tube on ice, add 1.5 mL of TRNzol, and shake to lyse.

[0031] 4) Add approximately 0.3 mL of chloroform, shake to mix, and let stand on ice for 15 minutes;

[0032] 5) Precool the centrifuge to 4°C, balance, and centrifuge at 12,000 rpm for 15 minutes.

[0033] 6) Use a pipette to transfer the upper aqueous phase to another 2 mL enzyme-free centrifuge tube;

[0034] 7) Add an equal volume of isopropanol to the centrifuge tube, mix well, and let stand at room temperature for 10 minutes;

[0035] 8) Place the centrifuge tube in a 4°C centrifuge, balance, and centrifuge at 12,000 rpm for 10 minutes.

[0036] 9) Discard the supernatant. A trace amount of white RNA precipitate will be seen at the bottom of the tube. Add 1 mL of 75% ethanol (prepared with DEPC water) and resuspend.

[0037] 10) Precool the centrifuge to 4°C, balance the tubes, and centrifuge at 8000 rpm for 5 minutes.

[0038] 11) Discard the supernatant and dry at room temperature for 7 minutes;

[0039] 12) Add 50 μL of DEPC water to dissolve;

[0040] 13) Pipette 2 μL of sample into a 200 μL enzyme-free centrifuge tube;

[0041] 14) Dilute to 100 μL with DEPC water;

[0042] 15) Measure OD260 and OD280 using a spectrophotometer. (Total RNA concentration was determined using a spectrophotometer. RNA concentration was calculated as: OD260 × dilution factor × 40 μg / mL. Purity was determined by the OD260 / OD280 ratio: OD260 / OD280 = 1.8-2.0 indicates good purity.)

[0043] Note: All instruments used in the experiment were free of RNAase contamination.

[0044] 3. Real-time PCR was used to detect BPTF mRNA expression in renal cancer tissues of patients with sunitinib-resistant and non-resistant renal cancer.

[0045] 1) Take out the total RNA of the two groups of patients from the -80℃ freezer;

[0046] 2) Thaw on ice;

[0047] 3) Place 0.2 mL enzyme-free centrifuge tubes on ice and add the mixed solution components of each group in Table 1 to each tube (three parallel samples form one group).

[0048] 4) Briefly centrifuge and mix;

[0049] 5) Place on a reverse transcription instrument at 65°C for 10 minutes;

[0050] 6) Immediately remove the tubes and cool on ice. Then, add the reaction solution components in Table 2 to the cooled centrifuge tubes.

[0051] 7) After mixing, continue incubating the tube at 37°C for 60 minutes and 85°C for 5 minutes;

[0052] 8) After finishing, cool on ice;

[0053] 9) Add the components in Table 4 to each PCR tube;

[0054] 10) Mix thoroughly after adding the sample;

[0055] 11) Set the reaction conditions and add a melting curve on the PCR instrument, and complete the amplification. The primer sequences, reaction system, and amplification program for PCR amplification are shown in Tables 3-5.

[0056] Table 1: Reverse transcription mix components

[0057] Table 1: Components of reverse transcription fluid

[0058]

[0059] Table 2: Reverse transcription reaction components

[0060] Table 2: Components of reverse transcription reaction fluid

[0061]

[0062] Table 3: Primer sequences

[0063] Table 3: Primer sequences

[0064]

[0065] Table 4: PCR amplification reaction system

[0066] Table 4:PCR amplification reaction system

[0067]

[0068]

[0069] Table 5: PCR amplification program

[0070] Table 5:PCR amplification procedure

[0071]

[0072] This example compares the expression of BPTF mRNA in renal cancer tissues of two groups of renal cancer patients who are resistant to sunitinib and those who are sensitive to sunitinib. The experimental results show that the expression of BPTF mRNA in renal cancer tissues of resistant renal cancer patients is abundant and significantly higher than that of renal cancer tissues of sunitinib-sensitive patients (P<0.01). Figure 1A This indicates that BPTF can be a diagnostic marker for sunitinib resistance in renal cell carcinoma.

[0073] Example 2

[0074] Western Blot assay was used to detect the expression of BPTF protein in renal cancer tissues of sunitinib-resistant and non-resistant renal cancer patients

[0075] 1. Protein extraction from renal cancer tissue:

[0076] 1) Prepare cell lysis buffer: Add 15 mL of RIPA lysis buffer to 150 μL of PMSF, mix well, and pre-chill in an ice box.

[0077] 2) Take out the frozen postoperative tissue samples from liquid nitrogen, cut about 1g of tissue, and then cut into about 1mm 3 Put the large and small pieces into a mortar pre-cooled with liquid nitrogen;

[0078] 3) Grind into powder, adding liquid nitrogen to keep the temperature low during the grinding process;

[0079] 4) Transfer the powder to a 15 mL enzyme-free centrifuge tube, add the prepared lysis buffer, and place on ice;

[0080] 5) Continue lysis for 30 minutes, vortexing every 5 minutes;

[0081] 6) Precool the centrifuge to 4°C, balance, and centrifuge at 12,000 rpm for 10 minutes.

[0082] 7) Transfer the supernatant into new enzyme-free centrifuge tubes and freeze at -80°C.

[0083] 2. Total protein concentration detection and adjustment

[0084] The protein concentration of each sample was determined using the BCA protein assay and adjusted to 3 μg / μL. After adding 5× protein loading buffer, the sample was denatured by boiling in 100°C water for 5 minutes and stored in aliquots at -80°C to avoid repeated freeze-thaw cycles.

[0085] 3. Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE)

[0086] 1) Prepare 12% SDS-PAGE gel:

[0087] Using the Bio-rad gel preparation tool, align two glass plates, clamp them with clips, and secure them to the gel preparation rack. First, prepare 12% separating gel according to the recipe and sequence in Table 6:

[0088] Table 6: Preparation of separation gel

[0089] Table 6:Production of separating glue

[0090]

[0091] After repeatedly blowing and mixing the mixture with a pipette, slowly inject the mixture into the interlayer between the two glass plates mentioned above with a pipette, taking care to avoid bubbles in the process of adding the separation gel mixture. Add about 3.5-4mL of separation gel mixture to each set of glass plates, so that the liquid level of the separation gel mixture is about 2.5cm from the top of the glass plate. Then slowly add deionized water to the upper layer of the glue for sealing, and let the glass plate with the separation gel stand at room temperature for 30min to allow the separation gel to solidify. After the separation gel solidifies, slowly pour out the deionized water used for sealing the upper layer, and use filter paper to absorb the residual deionized water between the glass plates. Then prepare the upper 5% concentrated gel according to the formula and sequence of Table 7:

[0092] Table 7: Preparation of stacking gel

[0093] Table 7:Confection of concentrated glue

[0094]

[0095]

[0096] Use a pipette to mix the stacking gel mixture thoroughly and slowly add it to the solidified separating gel layer, adding approximately 2 mL of stacking gel. Finally, bring the gel level to the same level as the glass plate. Quickly and gently insert the comb into the wells and remove any excess gel with filter paper. Be careful not to create bubbles during the addition of the stacking gel mixture and the insertion of the comb. Let the stacking gel solidify at room temperature for 30 minutes. Once the stacking gel solidifies, gently remove the comb to avoid deforming the wells.

[0097] 2) Protein sample loading and electrophoresis

[0098] Fix the prepared 12% SDS-PAGE gel in the Bio-rad electrophoresis tank and pour the prepared 1× SDS-PAGE running buffer into the electrophoresis tank to the corresponding scale line. Take 10μL of the protein sample that has been mixed with 5× protein loading buffer and denatured by boiling. After cooling on ice, use a pipette to gently mix the protein sample before loading. Then add it to the sample wells, with 5μL of protein sample per well. After all samples are loaded, place the electrophoresis tank in a basin covered with crushed ice to prevent the electrophoresis solution from being too hot and affecting the electrophoresis. After turning on the power, start electrophoresis. Electrophoresis conditions: voltage 80V for about 30 minutes, change to 130V voltage for about 2 hours after the sample enters the separation gel. The electrophoresis conditions should be adjusted appropriately according to the size of the target protein. When the bromophenol blue electrophoresis reaches the edge of the separation gel, immediately turn off the power switch and stop the electrophoresis.

[0099] 4. Transfer

[0100] Use the marker bands on the gel to determine the position of the target protein on the gel, then use a gel cutting board to cut the gel in the required range for the target protein, and gently and slowly place the cut gel into the membrane transfer buffer to soak. Use a ruler to measure the size of the cut gel and cut 6 layers of filter paper (three layers + three layers) and PVDF membrane according to the size of the cut gel. The size of the membrane should be slightly larger than the gel, and the size of the filter paper should be smaller than or equal to the size of the gel. This can prevent the two layers of filter paper sandwiched between the outermost layers of the gel from contacting and causing a short circuit. After cutting the PVDF membrane, place the cut PVDF membrane in methanol so that the methanol just covers the PVDF membrane, soak for 5-10 seconds, and soak until the membrane is completely transparent. Then place the PVDF membrane and the six layers of filter paper into the transfer buffer and soak for about 2-3 minutes. Use flat-tipped tweezers to create a transfer "sandwich" structure: from top to bottom, three layers of filter paper, glue, PVDF membrane, and three layers of filter paper. Each layer of filter paper or PVDF membrane must be thoroughly moistened with transfer solution and pressed with a glass rod to remove air bubbles between layers to prevent transfer efficiency. Once the "sandwich" structure is complete, first drip a small amount of transfer solution onto the anode plate of a semi-dry transfer apparatus. Place the "sandwich" flat on the anode plate of the semi-dry transfer apparatus. Then, drip a small amount of transfer solution around the "sandwich." Then, cover the "sandwich" with the cathode metal plate and outer cover in that order and connect the power supply. Transfer the membrane at a constant current of 100 mA for 30 minutes. After transfer is complete, remove the PVDF membrane and cut off a corner from the top left corner of the front of the PVDF membrane to mark the front and back.

[0101] 5. Closed

[0102] After blocking, remove the membrane and wash it three times with TBST on a shaker for 5 minutes each. Apply the primary antibody (BPTF 1:1000 dilution, β-actin 1:3000 dilution), ensuring the protein-side of the membrane faces downward for full contact with the antibody, and incubate overnight at 4°C. Remove the membrane and wash it three times with TBST at room temperature on a shaker for 10 minutes each. Apply the corresponding HRP-conjugated secondary antibody and incubate on a shaker at room temperature for 2 hours.

[0103] 6. Development, Image Acquisition, and Analysis

[0104] Remove the membrane and wash it three times with TBST on a decolorizing shaker at room temperature for 10 minutes each time. Develop with an ECL chemiluminescence kit. Immerse the membrane in the luminescent solution and incubate for 5 minutes. Remove the PVDF membrane promptly and transfer it to one side of the pre-laid plastic wrap in the X-ray film holder. Absorb the residual liquid and turn the other side over to cover it. Cut the X-ray film of appropriate size and place it on the membrane for pressing. In a darkroom, open the pressing box, take out the X-ray film and quickly perform the development and fixing operation. Adjust the exposure time appropriately according to the development situation to achieve the best effect. After completion, wash the film to remove the residual reagent, dry it and store it. Scan the film in grayscale and save it. Use the image processing system Image J software to digitize the grayscale value of the bands on the image. Use the ratio of the target protein to the internal reference protein as a relative indicator of the target protein expression level for analysis. Repeat the measurement three times for each sample and take the average value.

[0105] This example compares the expression of BPTF protein in renal cancer tissues of two groups of renal cancer patients who are resistant to sunitinib and those who are sensitive to sunitinib. The experimental results show that the expression of BPTF protein in renal cancer tissues of resistant renal cancer patients is abundant and significantly higher than that of renal cancer patients who are sensitive to sunitinib. Figure 1B This indicates that BPTF can be a diagnostic marker for sunitinib resistance in renal cell carcinoma.

[0106] Example 3

[0107] The two groups of patients were followed up by visiting the hospital and following up by phone. Progression-free survival (PFS) is defined as the period from the start of treatment to the observation of disease progression or death due to any cause in patients with renal cancer. The follow-up results were collected and sorted, and the overall survival curve and progression-free survival curve were drawn using GraphpadPrism 8.0.2. The experimental results showed that the overall survival and progression-free survival of patients with renal cancer with low BPTF expression were significantly higher than those of patients with high BPTF expression, see Figure 2A and 2B .

[0108] Example 4

[0109] The CCK-8 proliferation assay was used to determine the effect of BPTF shRNA on the cell viability of drug-resistant renal cancer cells (786-OR, ACHN-R) in sunitinib.

[0110] This example illustrates the effect of the BPTF shRNA of the present invention on the proliferation of drug-resistant renal cancer cells (786-OR and ACHN-R). The results show that shBPTF significantly inhibits the proliferation of renal cancer resistant cells in response to sunitinib and promotes sunitinib sensitization. This demonstrates that the BPTF of the present invention exhibits an extremely important key effect in promoting sunitinib resistance in renal cancer cells.

[0111] The CCK-8 cell proliferation assay is a technique familiar to those skilled in the art.

[0112] 1.786-OR and ACHN-R cell culture and plating in 96-well cell culture plates:

[0113] Take out 786-OR and ACHN-R in good logarithmic growth phase, observe the cell morphology, and start passage when the cells are 80% fused. Discard the old culture medium in the culture dish, wash 3-5 times with 1mL of sterile PBS, add 1mL of warmed trypsin, and shake the culture dish horizontally to evenly distribute the trypsin and fully digest. Place the cells in the incubator for 1-2 minutes, take out the cells, and observe the cell digestion under an inverted microscope. When the intercellular gap becomes larger and the cell protrusions retract and become round, immediately add 10% FBS culture medium to terminate the digestion, and gently blow the cells with a pipette. Repeatedly blow the digested cells gently to detach the wall and disperse them to form a uniform cell suspension and adjust the cell density. With a cell count of 5×10 3 The cell suspension was seeded into 96-well plates at 200 μL per well and cultured in a 37°C, 5% CO2 incubator. After the cells adhered, the original 10% FBS medium was aspirated and discarded. After washing 3-5 times with sterile PBS, the culture medium was replaced with 0.1% FBS and starved for 24 hours to homogenize the cells in the G0 / G1 phase.

[0114] 2. Detection

[0115] After 48 hours of BPTF shRNA intervention in 786-OR and ACHN-R cells, different concentrations of sunitinib were added to each well and cultured for another 24 hours. The old culture medium in the 96-well plate was discarded, and fresh culture medium was added. 10 μL of CCK-8 reagent was added to each well and incubated in a 37°C 5% CO2 incubator for 2 hours. The absorbance value (A) was measured at a single wavelength of 450 nm using an enzyme-labeled analyzer. 450 ). Six replicate wells were set for each concentration, and the average value was taken. The experiment was repeated 3 times.

[0116] In this example, shRNA targeting BPTF was used to treat drug-resistant renal cancer cells (786-OR, ACHN-R) to determine the effect of BPTF shRNA on the cell viability of drug-resistant renal cancer cells (786-OR, ACHN-R) in the presence of sunitinib. The experimental results showed that BPTF shRNA significantly inhibited the proliferation of drug-resistant renal cancer cells in the presence of sunitinib and promoted the sensitization of sunitinib. Figure 3A and 3B This indicates that the BPTF involved in the present invention shows an extremely important key effect in sunitinib resistance in renal cancer.

[0117] Example 5

[0118] The Transwell tumor cell migration assay was used to determine the effect of BPTF shRNA on the migration of drug-resistant renal cancer cells (786-OR, ACHN-R) in sunitinib.

[0119] This example illustrates that BPTF shRNA can effectively inhibit the migration of drug-resistant renal cancer cells in sunitinib, indicating that BPTF plays a crucial role in the migration of drug-resistant renal cancer cells.

[0120] The Transwell tumor cell migration assay is a technique familiar to those skilled in the art.

[0121] 1. Prepare Transwell chamber:

[0122] The upper chamber surface of the bottom membrane of the Transwell chamber was coated with Matrigel 1:8 (50 mg / L) dilution to coat the basement membrane and air-dry at 4°C. After air-drying, the basement membrane was rehydrated, the remaining liquid in the culture plate was aspirated, and 50 μL of serum-free DMEM containing 10 g / L BSA was added to each well. The cells were incubated in a 37°C incubator for 30 minutes.

[0123] 2. Preparation of cell suspension:

[0124] 1) Take cells in good growth condition, wait for the cells to adhere to the wall, and then switch to DMEM cell culture medium with 1% FBS for starvation culture for 12-24 hours;

[0125] 2) Add BPTF shRNA to the culture medium to intervene in 786-OR and ACHN-R cells for 48 hours;

[0126] 3) Digest the cells, centrifuge and discard the culture medium after digestion, wash 1-2 times with PBS, and resuspend in DMEM cell culture medium containing 1% FBS. Adjust the cell density to 1-10×10 5 , and try to ensure that the cell density of the control group and the treatment group is consistent.

[0127] 3. Seeding cells:

[0128] 1) 200 μL of cell suspension was added to a Transwell chamber, and sunitinib was added to the upper chamber of the Transwell chamber;

[0129] 2) Add 500 μL of culture medium containing 10 ng / mL VEGF to the lower chamber of a 24-well plate (be especially careful when seeding the plate; if bubbles appear, lift the chamber, remove the bubbles, and then place the chamber back into the culture plate).

[0130] 3) Incubate the 24-well plate in a 37°C, 5% CO2 incubator for 24 hours.

[0131] 4. Dyeing:

[0132] 1) Discard the culture medium in the wells and fix with 90% alcohol at room temperature for 30 minutes;

[0133] 2) Staining: Use 0.1% crystal violet staining. First, prepare 0.1% crystal violet staining, then stain with 0.1% crystal violet at room temperature for 10 minutes. Rinse with clean water and gently wipe off the upper layer of non-migrated cells with a cotton swab.

[0134] 5. Cell Counting:

[0135] Observe and photograph using a Leica DC 300F upright microscope (200×). Turn the Transwell chamber upside down so that cells attached to the upper and lower sides of the chamber membrane can be clearly seen. Count the cells in five randomly selected fields of view.

[0136] In this example, shRNA targeting BPTF was used to treat drug-resistant renal cancer cells (786-OR, ACHN-R) to determine the effect of BPTF shRNA on the migration of drug-resistant renal cancer cells in sunitinib. The experimental results showed that BPTF shRNA significantly inhibited the invasion and metastasis of drug-resistant renal cancer cells in sunitinib (P<0.01), and promoted sunitinib sensitization. Figure 4 This indicates that the BPTF involved in the present invention shows an extremely important key effect in sunitinib resistance in renal cancer.

[0137] Example 5

[0138] Cell glycolysis level detection (glucose uptake, lactate production experiment)

[0139] 1) Take drug-resistant renal cancer cells (786-OR, ACHN-R) with good growth status, digest the cells and collect them into a 15 mL centrifuge tube. Centrifuge at 2000 rpm for 5 minutes and resuspend them in 1 mL of sterile PBS. Take a small amount of cell suspension and dilute it and count it. Adjust the cell density to 10 6The cells were divided into three groups: one control group and two experimental groups. 5 μM and 10 μM AU1 were added to the experimental groups, respectively. The three groups of cells were placed in culture dishes and cultured for 48 hours.

[0140] 2) Glucose uptake and lactate production were detected according to the instructions of the corresponding detection kits, and replicate wells were set to facilitate error calculation.

[0141] This example measured the effect of BPTF-specific antagonist AU1 (5 μM, 10 μM) on glucose uptake and lactate production in drug-resistant renal cancer cells (786-OR, ACHN-R). The experimental results showed that AU1 could significantly inhibit glucose uptake and lactate production in drug-resistant renal cancer cells (786-OR and ACHN-R). Figure 5 This suggests that inhibiting BPTF can regulate the abnormal glucose metabolism of drug-resistant renal cancer cells, indicating that BPTF involved in the present invention has an extremely important key effect in sunitinib resistance in renal cancer.

[0142] While the specific embodiments of the present invention have been described in detail above, these are merely exemplary and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. Application of chromatin remodeling factor BPTF in the preparation of diagnostic markers for sunitinib resistance in renal cancer.

2. The use of an active ingredient that downregulates or blocks the expression of chromatin remodeling factor BPTF molecules in renal cancer in the preparation of an anti-sunitinib resistant drug for renal cancer, wherein the active ingredient that downregulates or blocks the expression of chromatin remodeling factor BPTF molecules in renal cancer is the BPTF antagonist AU1.

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