New application of GPRC5A

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to novel application of GPRC5A which serves as a novel molecular target point GPRC5A for overcoming pancreatic cancer chemotherapy drug resistance, so that the problems that in the prior art, the chemotherapy effect of pancreatic cancer patients is poor, and chemotherapy drug resistance is likely to be generated are solved, the curative effect of a classical chemotherapy regimen is improved, and the long-term survival rate of the pancreatic cancer patients is increased.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a new use of GPRC5A, in particular to the application of targeting GPRC5A in chemosensitization of pancreatic cancer. Background Art

[0002] Due to the lack of effective screening methods and non-invasive diagnostic techniques, most pancreatic cancer patients are already in the middle and late stages when diagnosed. Currently, the treatments for pancreatic cancer mainly include surgical resection, chemotherapy, and immunotherapy, but the overall efficacy is limited, with a five-year survival rate of only about 9%. For advanced pancreatic cancer, gemcitabine combined with albumin-paclitaxel (GnP) is the main first-line chemotherapy regimen, but its effectiveness is still low. The poor sensitivity of GnP chemotherapy is a key factor leading to the low survival rate of patients with advanced pancreatic cancer. At present, effective methods to improve the sensitivity of GnP chemotherapy are very limited. Therefore, screening and identifying key molecules that regulate GnP chemotherapy resistance in pancreatic cancer and conducting clinical transformation are the key to improving the effectiveness of pancreatic cancer chemotherapy.

[0003] Pancreatic cancer is in urgent need of new treatments to improve the effect of chemotherapy due to its high malignancy and resistance to existing treatment options. At present, pancreatic cancer has limited responsiveness to traditional chemotherapy drugs such as gemcitabine and albumin-paclitaxel, resulting in generally poor prognosis for patients. Pancreatic cancer cells are highly heterogeneous and invasive, which makes them less sensitive to classic chemotherapy drugs such as gemcitabine and albumin-paclitaxel. The microenvironment of pancreatic cancer cells is also an important factor in their drug resistance. There are a large number of stromal cells and dense matrix in the tumor tissue of pancreatic cancer, which inhibit the penetration and action of chemotherapy drugs through various mechanisms. At the same time, the hypoxic state in the tumor microenvironment will also induce metabolic changes in cancer cells, further enhancing their drug resistance. Therefore, target drugs that sensitize pancreatic cancer chemotherapy are an important direction of current tumor treatment. However, target drugs that sensitize pancreatic cancer chemotherapy are yet to be developed. Summary of the invention

[0004] The present invention aims to solve the problem in the prior art that pancreatic cancer patients have poor chemotherapy effects and are prone to chemotherapy resistance, provide a new use of GPRC5A as a molecular target for inducing GnP chemotherapy resistance in pancreatic cancer, and provide new ideas and directions for target drugs for sensitizing pancreatic cancer chemotherapy.

[0005] The first aspect of the present invention provides a molecular target for inducing GnP chemotherapy resistance in pancreatic cancer, namely GPRC5A.

[0006] The specific information of GPRC5A is as follows: the gene information encoding the protein is Gene ID: 9052; the existing aliases are: GPCR5A, PEIG-1, RAI3, RAIG1, TIG1, and the encoding gene is located at 12p13.1.

[0007] G protein-coupled receptors are one of the most diverse receptor superfamilies, and their abnormal expression is closely related to the occurrence and development of diseases. G protein-coupled receptor Class C Group 5Member A (GPRC5A) is a member of the G protein-coupled receptor family, which can be induced by all-trans retinoic acid and encodes a 40kDa protein. The inventors found that GPRC5A is highly expressed in pancreatic cancer and is associated with poor prognosis of patients. Knocking down GPRC5A in pancreatic cancer cell lines can increase their sensitivity to gemcitabine. However, there is currently no technical solution for targeting GPRC5A in pancreatic cancer to improve the efficacy of GnP chemotherapy. The role of GPRC5A in the GnP chemotherapy regimen for pancreatic cancer remains unclear.

[0008] Therefore, the present invention has conducted in-depth research on the mechanism of pancreatic cancer chemotherapy resistance and found that GPRC5A is an important molecular target for inducing pancreatic cancer resistance to gemcitabine combined with albumin-paclitaxel chemotherapy. This molecule can be used as a new therapeutic target to enhance the sensitivity of GnP chemotherapy regimens and improve the survival rate of pancreatic cancer patients by blocking GPRC5A.

[0009] The second aspect of the present invention provides the use of an agent for blocking or inhibiting GPRC5A expression in a drug for treating pancreatic cancer. Furthermore, the application is a combined drug.

[0010] Furthermore, the combination drug is a combination of an agent that blocks or inhibits GPRC5A expression and gemcitabine combined with albumin-paclitaxel.

[0011] Specifically, the agent for blocking or inhibiting GPRC5A expression is GPRC5A small interfering RNA.

[0012] The third aspect of the present invention provides a drug for treating pancreatic cancer, comprising GPRC5A small interfering RNA, which can block the expression of GPRC5A.

[0013] Furthermore, it also includes gemcitabine and albumin paclitaxel, and the GPRC5A small interfering RNA is used in combination with gemcitabine and albumin paclitaxel.

[0014] By implementing the above technical scheme, the present invention has discovered a new molecular target GPRC5A that promotes GnP chemotherapy resistance in pancreatic cancer through technologies such as cytokine array, and applied it to the treatment of pancreatic cancer as a new therapeutic intervention target. The reagent that blocks or inhibits GPRC5A expression is combined with gemcitabine and albumin paclitaxel, in order to solve the problems of poor chemotherapy effect and easy chemotherapy resistance in pancreatic cancer patients in the prior art, increase the efficacy of classic chemotherapy regimens, and improve the long-term survival rate of pancreatic cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of immunofluorescence detection of GPRC5A expression in pancreatic cancer tissues; (A) Immunofluorescence shows GPRC5A expression in pancreatic cancer tissues; (B) Pancreatic cancer patients with high GPRC5A expression have a shorter progression-free survival rate and a poor prognosis.

[0016] Figure 2 Schematic diagram of the results of neutrophils participating in pancreatic cancer chemotherapy resistance; (A) Flow cytometry detected the effect of GnP on immune cells in pancreatic cancer; (B) Among the various groups of immune cells, the proportion of neutrophils increased significantly; (C) ELISA detected neutrophil cytokines, and the level of elastase increased significantly; (D) SYTOX Green fluorescence intensity showed that the level of NETs increased significantly after neutrophils were co-cultured with tumor supernatant;

[0017] Figure 3 Schematic diagram of the results showing that GPRC5A of pancreatic cancer cells participates in regulating the formation of neutrophil NETs and causes chemotherapy resistance; (A) qRT-PCR detection showed that the mRNA expression level of GPRC5A in pancreatic cancer cells increased significantly after GnP treatment; (B) Western Blot detection showed that the protein level of GPRC5A in pancreatic cancer cells increased significantly after GnP treatment; (C) MPO-DNA detection showed that the level of neutrophil NETs decreased significantly after GPRC5A inhibition.

[0018] Figure 4 Schematic diagram of the results showing increased secretion of CXCL8 by pancreatic cancer cells after GnP treatment.

[0019] Figure 5Schematic diagram of the results of NLRP3-mediated GnP / CXCL8 promoting NETs formation; (A) qRT-PCR detection showed that the mRNA expression level of NLRP3 in neutrophils was significantly increased after treatment with GnP-treated pancreatic cancer cell conditioned medium; (B) Western Blot detection showed that the protein level of NLRP3 in neutrophils co-cultured with GnP-treated pancreatic cancer cell culture supernatant was significantly increased; (C) Western Blot detection showed that the expression level of NLRP3 in neutrophils in the co-culture system was significantly decreased after blocking CXCL8.

[0020] Figure 6 Schematic diagram of the results of blocking cfDNA or GPRC5A to improve the efficiency of GnP treatment of pancreatic cancer; (A) Mouse tumors shrank after cfDNA blocking; (B) The survival of pancreatic cancer mice was prolonged after cfDNA blocking. (C) The growth of pancreatic cancer tumors in mice was slowed down after small interfering RNA blocked tumor cell GPRC5A. (D) The tumors in mice shrank after GPRC5A blocking. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in the art. Of course, the adoption of instruments and materials in the embodiment is not limited to the enumeration of this example, but is based on the technical problems that can be solved in the present invention and the corresponding technical effects are realized.

[0022] Unless otherwise specified, the cell lines used in the context of the present invention are cultured according to the prior art, and all cell lines are identified by short tandem repeat analysis of the China Center for Type Culture Collection, and the presence of mycoplasma contamination is verified using a PCR detection kit, and frozen in liquid nitrogen and used for subsequent experiments. All reagents used in the present invention are commercially available. The experimental methods used in the present invention, such as nucleic acid extraction, Western Blot, immunofluorescence, primer design, cell experiments, etc., are conventional methods and techniques in the art. Representative results selected from the biological experiment repetitions are presented in the accompanying drawings below, and the data are displayed as mean±SD or mean±SEM as specified in the figure. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. The data were analyzed using GraphPad Prism 8.0 software. Conventional medical statistical methods such as t-test, chi-square test, and analysis of variance were used to compare the mean differences between two or more groups. P<0.05 was considered a significant difference.

[0023] Example 1

[0024] First, surgical resection specimens of pancreatic cancer patients were selected. The inclusion criteria of patients were as follows: 1) aged 18 years or older. 2) pancreatic ductal adenocarcinoma confirmed by pathology and treated with gemcitabine combined with albumin paclitaxel. 3) paraffin sections prepared from surgical resection specimens were collected. 4) survival period was greater than or equal to 3 months. Then, the GPRC5A level in the tumor tissue was detected. The specific steps are as follows:

[0025] (1) Store the paraffin sections in a 4°C refrigerator.

[0026] (2) Dewax the sections in three portions of xylene, each for 10 minutes.

[0027] (3) The sections were placed in a gradient of alcohol (100%, 95%, 85%, 70%) for hydration for 3-5 minutes each time, and then washed three times with ddH2O.

[0028] (4) Immerse the sections in citrate-EDTA antigen retrieval solution and heat in a water bath at 100°C for 20 minutes for antigen retrieval. After heating, let the sections cool naturally to room temperature in the antigen retrieval solution. (5) Place the sections in a light-proof humidified box, add blocking solution, and block at room temperature for 30 minutes.

[0029] (6) Wash off the blocking solution and directly add the primary antibody and incubate overnight at 4°C.

[0030] (7) Wash three times with TBST, 5 minutes each time.

[0031] (8) Add secondary antibody and incubate at room temperature for 30 min. Wash three times with TBST for 5 min each.

[0032] (9) Add sealing agent and seal the slide with a cover glass.

[0033] (10) Without knowing the patient’s clinical information, researchers randomly photographed three fields of view for each immunofluorescence section of the patient and calculated the average fluorescence intensity of GPRC5A.

[0034] Example 2

[0035] Flow cytometry was used to detect the effect of GnP on immune cells in pancreatic cancer. The specific steps are as follows:

[0036] (1) Aspirate the culture medium from MIA PaCa-2 cells and rinse twice with PBS.

[0037] (2) Digest with trypsin-EDTA for 3 minutes, centrifuge at 1000 rpm for 5 minutes at room temperature and discard the supernatant.

[0038] (3) Resuspend the cell pellet in 1 ml of DMEM medium and count the cells, 5 × 10 per nude mouse. 6The cell concentration was adjusted based on the number of cells (the cell suspension was kept on ice).

[0039] (4) 5×10 6 MIA PaCa-2 cells were injected subcutaneously into the right hind limb of 5-week-old nude mice.

[0040] (5) The mice were randomly divided into two groups: a control group (normal saline) and a GnP group.

[0041] (6) GnP (100 mg / kg gemcitabine and 20 mg / kg albumin paclitaxel) or an equal volume of saline was intraperitoneally injected twice a week according to the group. After 3 weeks of treatment, the mice were killed.

[0042] (7) The mouse subcutaneous tumor was separated and minced, and enzymes such as collagenase and DNase were added for digestion at 37°C in a water bath for 30 minutes. The mixture was ground and sieved, and centrifuged at 1000 rpm and 4°C for 5 minutes.

[0043] (8) Discard the supernatant, add FACS buffer to resuspend the cells, add equal amounts to a U-bottom 96-well plate, and incubate at 1000 rpm.

[0044] Centrifuge at 4°C for 5 minutes. Discard the supernatant, add 100ul of FACS buffer containing FcR antibody to each well, and incubate at 4°C for 15 minutes.

[0045] (9) Prepare the corresponding flow cytometry antibody mixture according to the volume of 70ul per well. Centrifuge the incubated cell suspension at 1000rpm at 4℃ for 5 minutes, discard the supernatant, add the staining mixture and mix well, and stain at 4℃ in the dark for 30 minutes.

[0046] (10) After staining, add 200 μl of FACS buffer and mix well. Centrifuge at 1000 rpm and 4°C for 5 minutes.

[0047] (11) After discarding the supernatant, add 200 μl of PBS and mix well. After filtering again, transfer to a 5 ml flow cytometer and analyze the data using Flowjo software.

[0048] Example 3

[0049] MPO and sytox green staining were used to detect the level of NETs produced by neutrophils.

[0050] (1) Take the neutrophils after tumor co-culture, add PBS, and centrifuge at 1000 rpm for 5 minutes at room temperature. (2) Discard the supernatant and resuspend in 1 ml PBS.

[0051] (3) MPO content was detected using an MPO kit (Cayman).

[0052] (4) cfDNA content was detected using a cfDNA kit (Thermo).

[0053] (5) The samples were stained with Sytox green dye (Thermo) and the fluorescence intensity was measured using a Synergy microplate reader.

[0054] Example 4

[0055] Cytokine array was used to detect the changes of cytokines in the culture supernatant of tumor cells after GnP treatment. (1) When MIAPaCa-2 cells grew to 60%-80% confluence, gemcitabine and albumin-paclitaxel were added to DMEM complete medium to a final concentration of 1uM and 0.68uM, respectively.

[0056] (2) After culturing for 24 hours, the cells were rinsed twice with PBS and replaced with complete medium without GnP.

[0057] (3) After the cells have recovered their normal morphology, the same concentration of GnP is added again for 24 hours.

[0058] (4) Repeat this process until the cells can grow stably in the GnP-treated culture medium.

[0059] (5) Collect the culture supernatant and centrifuge at 2000 rpm for 10 minutes at 4°C.

[0060] (6) Collect the supernatant after centrifugation in a new centrifuge tube

[0061] (7) The supernatant was processed according to the instructions of the cytokine array kit and incubated with the membrane, and the gray value was detected under a chemiluminescence instrument.

[0062] Example 5

[0063] qRT-PCR was used to verify the expression level of GPRC5A mRNA in pancreatic cancer cells. The specific steps are as follows:

[0064] (1) After washing pancreatic cancer cells twice with PBS, trypsin was used to digest the cells, and the cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded to obtain the cell pellet.

[0065] (2) Add 1 ml of Trizol to each EP tube and place it horizontally at room temperature for 5 minutes.

[0066] (3) Add chloroform, shake vigorously to mix, and let stand at room temperature for 5 minutes.

[0067] (4) Centrifuge at 13,000 g for 15 min at 4°C. Carefully aspirate the upper aqueous phase, add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 min.

[0068] (5) Centrifuge at 13000 g for 10 min at 4°C, discard the supernatant, and add 1 ml of ethanol prepared with 75% DEPC water.

[0069] (6) Centrifuge at 7500 g for 5 min at 4°C, discard the supernatant, open the lid and dry the precipitate, add 15 μl of DEPC water to redissolve the RNA and measure the concentration. Use HiScript reverse transcriptase to reverse transcribe the total RNA into cDNA, and calculate the reaction system based on the measured RNA concentration.

[0070] (7) Dilute cDNA as a template and perform qPCR using ChamQ SYBR qPCR Master Mix.

[0071] Example 6

[0072] Western Blot was used to further verify the NLRP3 protein level in neutrophils. The specific steps are as follows:

[0073] (1) First, neutrophils were co-cultured with the supernatant of PANC-1 cells treated with GnP, collected in a 1.5 ml EP tube, centrifuged to remove the supernatant, washed twice with PBS, added with cell lysis buffer containing protease inhibitors and phosphatase inhibitors, and placed on ice for 30 minutes.

[0074] (2) After cell lysis, the cells were collected into 1.5 ml EP tubes and centrifuged at 13,000 rpm and 4°C for 10 minutes.

[0075] (3) After centrifugation, the supernatant is the total cell protein and is transferred to a new 1.5 ml EP tube.

[0076] (4) After protein quantification by the BCA method, add 5× protein loading buffer according to the sample volume, mix well, and place in a metal bath at 100°C for 10 minutes.

[0077] (5) The obtained samples were subjected to protein electrophoresis, with the initial electrophoresis voltage at 80 V and the voltage adjusted to 120 V when the protein samples ran into the separation gel.

[0078] (6) After protein electrophoresis, add an appropriate amount of transfer solution into the container, and then place the sponge, filter paper, protein gel, methanol-activated PVDF membrane, filter paper, and sponge in sequence. After removing the bubbles, place the transfer tank into ice water and transfer the membrane at a constant current of 300 mA for 2 hours.

[0079] (7) After transfer, place the PVDF membrane in a 5% BSA solution and incubate at room temperature for 1 hour.

[0080] (8) After blocking, cut the required bands, add NLRP3 and GAPDH primary antibodies respectively, and place on a shaker at 4°C overnight.

[0081] (9) After blocking is completed the next day, wash the PVDF membrane three times with TBST buffer, each time for 10 minutes.

[0082] (10) Depending on the antibody species, incubate with secondary antibody at room temperature for 1 hour. After completion, wash the membrane three times with TBST buffer, each for 10 minutes.

[0083] (11) After washing the membrane, perform chemiluminescence, prepare ECL luminescent solution, and develop using a chemiluminescence instrument.

[0084] Example 7

[0085] A nude mouse subcutaneous tumor model was constructed and treated with GnP±si-GPRC5A to explore the therapeutic value of inhibiting GPRC5A on GnP resistance in animal models.

[0086] (1) MIA PaCa-2 cells were cultured and rinsed twice with PBS

[0087] (2) Digest with trypsin-EDTA for 3 minutes, centrifuge at 1000 rpm for 5 minutes at room temperature and discard the supernatant

[0088] (3) Resuspend the cell pellet in 1 ml of DMEM medium and count the cells, 5 × 10 per nude mouse. 6 The cell concentration was adjusted based on the number of cells (the cell suspension was kept on ice).

[0089] (4) 5×10 6 MIAPaCa-2 cells were injected subcutaneously into the right hind limb of 5-week-old nude mice.

[0090] (5) After tumor formation, the mice were randomly divided into 4 groups and marked by ear clipping: (a) saline, (b) GnP,

[0091] (c) si-GPRC5A, (d) GnP+si-GPRC5A, tumor size was measured twice a week.

[0092] (6) GnP (100 mg / kg gemcitabine and 20 mg / kg albumin paclitaxel) was injected intraperitoneally twice a week according to the group, and si-GPRC5A (5 nmol / mouse) was injected intratumorally twice a week. After 2 weeks of treatment, the mice were killed, and the tumors were collected, weighed, and photographed.

[0093] (7) After the tumor tissue is digested into a single cell suspension, the proportion of each immune cell is detected according to the flow cytometry method in Example 2.

[0094] Example 1 The expression level of GPRC5A in pancreatic cancer cells was detected to determine the chemotherapy resistance and prognosis of pancreatic cancer patients. Inhibition of GPRC5A can effectively improve the sensitivity of pancreatic cancer chemotherapy, providing a scientific basis for the formulation of chemotherapy regimens and prognosis assessment for pancreatic cancer patients. Figure 1 Figure A is a representative result of paraffin section staining, and Figure B shows that pancreatic cancer patients with high expression of GPRC5A have a shorter progression-free survival.

[0095] Example 2 Flow cytometry revealed that the proportion of neutrophils in pancreatic cancer tissue increased after GnP chemotherapy (see results). Figure 2 A and B in Figure 1), and the tumors after GnP chemotherapy were significantly reduced after inhibiting mouse neutrophils, indicating that neutrophils play a certain role in GnP resistance in pancreatic cancer. To explore the specific mechanism of neutrophils in GnP treatment, this study detected the tumor supernatant after co-culture of neutrophils and pancreatic cancer cells and found that the level of elastase was significantly increased (see the results). Figure 2 C). Elastase is an important component of NETs, ​​indicating that neutrophils may secrete NETs to cause GnP chemotherapy resistance in pancreatic cancer.

[0096] Example 3 SYTOX Green staining, cell migration and invasion experiments showed that NETs levels were associated with GnP resistance (see results). Figure 2 D).

[0097] Example 4 Cytokine arrays revealed that the level of CXCL8 in the supernatant of pancreatic cancer cell culture fluid after GnP treatment increased significantly, indicating that pancreatic cancer cells secrete CXCL8 to promote NETs-mediated chemotherapy resistance. Figure 4 , cytokine array showed that CXCL8 level increased significantly. Previous studies have found that GPRC5A is highly expressed in pancreatic cancer tissues, and GnP may affect the expression level of GPRC5A.

[0098] In Example 5 and Example 6, qRT-PCR and Western Blot were used to find that GnP significantly increased the expression of GPRC5A in pancreatic cancer cells. After co-culture with pancreatic cancer cells with GPRC5A knockdown, the results of neutrophil NETs were shown in Figure 3 Furthermore, by knocking out GPRC5A in pancreatic cancer cells treated with GnP, the present invention confirmed that GPRC5A knockout inhibited the secretion of CXCL8 in pancreatic cancer cells after GnP treatment. Since NLRP3 plays a key role in the chemotaxis of neutrophils, this study further explored the role of NLRP3 in the formation of NETs induced by GnP / CXLC8. Through cell co-culture, it was found that blocking CXCL8 could reduce NLRP3 in neutrophils induced by GnP-treated pancreatic cancer cells.

[0099] Example 7 demonstrated at the mouse level that inhibiting GPRC5A expression or inhibiting cfDNA, an important component of NETs, ​​can improve the GnP treatment efficacy of pancreatic cancer. Figure 6 By intratumorally injecting small interfering RNA to inhibit the expression of GPRC5A in pancreatic cancer cells, the growth of pancreatic cancer tumors in mice was slowed down and their size was reduced.

Claims

1. A new use of GPRC5A, characterized in that: As a molecular target for inducing GnP chemotherapy resistance in pancreatic cancer.

2. Application of agents that block or inhibit GPRC5A expression in the treatment of pancreatic cancer.

3. The use according to claim 2, characterized in that: The application is combined medication.

4. The use according to claim 3, characterized in that: The combined drug is a combination of an agent that blocks or inhibits GPRC5A expression and gemcitabine combined with albumin-paclitaxel.

5. The use according to claim 2, 3 or 4, characterized in that: The agent for blocking or inhibiting GPRC5A expression is GPRC5A small interfering RNA.

6. A drug for treating pancreatic cancer, characterized in that: Capable of blocking or inhibiting GPRC5A expression.

7. The drug for treating pancreatic cancer according to claim 5, characterized in that: Including GPRC5A small interfering RNA.

8. The drug for treating pancreatic cancer according to claim 5, characterized in that: The GPRC5A small interfering RNA was used in combination with gemcitabine and nab-paclitaxel.

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