Drugs targeting the interaction between STC2 and Cav1.2 and their applications

Abstract

The present invention provides the use of an agent targeting the interaction between STC2 and Cav1.2 in the preparation of a drug for tumor prevention, treatment, or drug resistance enhancement. The agent targeting the interaction between STC2 and Cav1.2 is selected from an agent that inhibits STC2 expression, an agent that promotes Cav1.2 expression, or a combination of the two. The interaction between STC2 and Cav1.2 discovered in the present invention can be used for targeted therapy of ovarian cancer or other tumor types.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug research and development and application, and specifically relates to drugs targeting the interaction between STC2 and Cav1.2 and their applications in tumor prevention, treatment and drug resistance enhancement. Background Art

[0002] The stanniocalcin (STC) protein family was first discovered in teleosts. They are secreted by type I secretory cells in the kidneys of Stanniocalcin corpuscles, a gland unique to fish. STCs are secretory glycoprotein hormones that regulate calcium and phosphate homeostasis in fish. When serum calcium concentrations rise, STCs are secreted extracellularly, controlling cellular calcium uptake and thereby lowering blood calcium concentrations. In 1998, Ishibashi et al. cloned another homolog of the STC family from a human osteosarcoma cDNA library. Because these proteins differ in structure and physiological function from the originally discovered stanniocalcin, they were named STC1 and STC2, respectively. Human STCs are expressed in various organs, including the ovary, pancreatic α-cells, nerves, and muscles, where they participate in regulating calcium-phosphate homeostasis and inhibiting elevated blood calcium concentrations. Recent studies have revealed that stanniocalcins also play important roles in regulating mineral metabolism, bone cell proliferation and differentiation, and protecting cardiomyocytes and neurons.

[0003] Studies have shown that STC2 expression is closely related to tumors. STC2 is highly expressed in various tumor tissues, such as gastric cancer, lung cancer, and neuroblastoma. Furthermore, STC2 expression levels are correlated with the prognosis of tumor patients; elevated STC2 expression indicates a poor prognosis.

[0004] STC2 is known to promote tumor development and progression through multiple pathways. Law et al. found that under hypoxic conditions, HIF1a upregulates STC2 expression, promoting cell proliferation and aiding cell survival. STC2 can also promote tumor cell metastasis. STC2 overexpression promotes the expression of N-cadherin and Vimentin proteins and reduces E-cadherin levels, thereby promoting epithelial-mesenchymal transition in SKOV3 cells. Increased MMP2 and MMP9 activity also contribute to STC2's promotion of metastasis. In primary human neuroblastoma tissue, STC2 expression promotes cell invasion and increases MMP2 activity, increasing the risk of metastasis and bleeding. In cervical cancer, STC2 expression is associated with lymph node metastasis. Furthermore, patients with high STC2 expression after radiotherapy have shorter overall survival, while patients with low STC2 levels have longer overall survival and progression-free survival, suggesting that STC2 expression may be associated with radioresistance. Therefore, STC2 plays a broad role in tumor development and progression. STC2 can be used as an objective indicator to judge tumor metastasis, recurrence and treatment efficacy. In-depth research on STC2 can open up a new path for tumor diagnosis and treatment.

[0005] Cav1.2 plays a crucial role in the L-type voltage-gated calcium pathway, controlling the influx of extracellular calcium ions into cells. Cav1.2 functions in excitable cells such as cardiomyocytes and skeletal cells. Recent studies have confirmed the presence of Cav1.2 in tumor cells. It is generally believed that tumor cells inhibit apoptosis by inhibiting calcium influx or downregulating the expression of calcium channel proteins, thereby promoting cancer development.

[0006] STC2 is a secreted glycoprotein that regulates target cell function through autocrine or paracrine pathways. As an extracellular signaling molecule, the mechanism by which STC2 transmits signals to cells and exerts regulatory functions remains unknown, and the identification of the STC2 receptor has not yet been reported.

[0007] In summary, it is of great significance to identify the receptors that interact with STC2 and control the occurrence, development and prognosis of tumors by regulating the interaction between STC2 and its receptors. Summary of the Invention

[0008] To solve the above problems, the present invention adds doxycycline to the culture medium of the STC2-inducible expression cell line SKOV3pLVX-STC2 to induce the cells to express STC2, and isolates and identifies the STC2 receptor through experiments such as IP, mass spectrometry, GST Pulldown and immunofluorescence.

[0009] Specifically, the first aspect of the present invention provides the use of an agent targeting the interaction between STC2 and Cav1.2 in the preparation of drugs for tumor prevention, treatment, and drug resistance enhancement.

[0010] In certain embodiments, the agent targeting the interaction between STC2 and Cav1.2 is selected from an STC2 expression inhibitory agent, a Cav1.2 expression promoting agent, or a combination of the two.

[0011] In certain embodiments, the STC2 expression inhibitory agent is selected from siRNA, shRNA, small molecule inhibitors, and STC2 antibodies.

[0012] In certain embodiments, the Cav1.2 expression-promoting agent is selected from vectors or cells carrying Cav1.2 cDNA.

[0013] In certain embodiments, the drug resistance is resistance to a chemotherapy drug; preferably, the chemotherapy drug is a platinum drug; more preferably, the platinum drug is cisplatin.

[0014] In certain embodiments, the tumor is selected from ovarian cancer, cervical cancer, gastric cancer, lung cancer, and neuroblastoma; preferably, the tumor is ovarian cancer.

[0015] The second aspect of the present invention provides the use of Cav1.2 protein peptide segments aa291-380 and / or aa674-728 in the study of the interaction mechanism between STC2 and Cav1.2.

[0016] The third aspect of the present invention provides the use of the STC2 protein peptide segment aa62-144 in studying the interaction mechanism between STC2 and Cav1.2.

[0017] A fourth aspect of the present invention provides the use of an STC2 expression inhibitory agent combined with a Cav1.2 expression promoting agent in the preparation of drugs for tumor prevention, treatment, and drug resistance enhancement.

[0018] In certain embodiments, the STC2 expression inhibition agent is selected from siRNA, shRNA, small molecule inhibitors, and STC2 antibodies.

[0019] In certain embodiments, the Cav1.2 expression-promoting agent is selected from vectors or cells carrying Cav1.2 cDNA.

[0020] In certain embodiments, the drug resistance is resistance to a chemotherapy drug; preferably, the chemotherapy drug is a platinum drug; more preferably, the platinum drug is cisplatin.

[0021] In certain embodiments, the tumor is selected from ovarian cancer, cervical cancer, gastric cancer, lung cancer, and neuroblastoma; preferably, the tumor is ovarian cancer.

[0022] The fifth aspect of the present invention provides a pharmaceutical composition for tumor prevention, treatment, and drug resistance enhancement, which comprises an STC2 expression inhibitory agent and a Cav1.2 expression promoting agent; the STC2 expression inhibitory agent and the Cav1.2 expression promoting agent can be administered sequentially or simultaneously.

[0023] In certain embodiments, the STC2 expression inhibition agent is selected from siRNA, shRNA, small molecule inhibitors, and STC2 antibodies.

[0024] In certain embodiments, the Cav1.2 expression-promoting agent is selected from vectors or cells carrying Cav1.2 cDNA.

[0025] In certain embodiments, the drug resistance is resistance to a chemotherapy drug; preferably, the chemotherapy drug is a platinum drug; more preferably, the platinum drug is cisplatin.

[0026] In certain embodiments, the tumor is selected from ovarian cancer, cervical cancer, gastric cancer, lung cancer, and neuroblastoma; preferably, the tumor is ovarian cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention demonstrates significant binding between the STC2 and Cav1.2 proteins at both the protein and cellular levels through experimental techniques such as IP, mass spectrometry, PLA, and immunofluorescence. The structural regions of Cav1.2 that bind to STC2 are identified as its extracellular peptides aa291-380 and aa674-728, while the STC2 peptide that binds to Cav1.2 is aa62-144. Cav1.2 is an L-type calcium channel and a key channel protein regulating extracellular calcium influx. STC2 has also been found to play a role in regulating calcium homeostasis in the body. The interaction between STC2 and Cav1.2 influences cellular calcium regulation, impacting the development, progression, and chemotherapy resistance of ovarian cancer. Therefore, the STC2 and Cav1.2 molecules discovered in the present invention, as well as their related molecules, can be used for the diagnosis and targeted treatment of ovarian cancer and other tumor types. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0030] Figure 1Identification of STC2-binding proteins. A. Immunofluorescence analysis shows that STC2-associated receptors are localized on the cell membrane; B. Immunofluorescence analysis shows that STC2-associated receptors are localized on the cell membrane after doxycycline induction; C. Coomassie Brilliant Blue staining shows STC2-binding proteins; D. Mass spectrometry analysis of the SKOV3pLVX-STC2-HA protein after doxycycline induction; E. STC2 interacts with Cav1.2 in doxycycline-induced SKOV3pLVX-STC2-HA cells; F. STC2 binds to Cav1.2 in STC2-overexpressing cells; G. Cav1.2 binds to STC2 in STC2-overexpressing cells; H. Immunofluorescence analysis shows the colocalization of STC2 and Cav1.2.

[0031] Figure 2 Figure 2. STC2 binding to Cav fragments. A. In a prokaryotic GST pulldown assay, STC2 interacts with the Ca-1 and Ca-2 fragments. B. In a eukaryotic GST pulldown assay, STC2 interacts with the Ca-1 fragment. C. Identification of the binding fragments in STC2.

[0032] Figure 3 Colocalization of STC2 and Cav1.2. A. Proximity ligation assay analysis of the colocalization of STC2 and Cav1.2 in ovarian cancer; B. IP assay confirms that STC2 N73A mutation prevents binding to Cav1.2; C. GST pulldown assay confirms that STC2 N73A mutation prevents binding to Cav1.2.

[0033] Figure 4 Figure 1 shows that STC2 overexpression and Cav1.2 knockdown promote tumor cell proliferation in animals. A. STC2 knockdown inhibits HEY cell proliferation; B. Tumor weight of STC2 knockdown cells and corresponding control cells (shGFP); C. Tumor growth curves of STC2 knockdown cells and corresponding control cells (shGFP); D. STC2 overexpression promotes SKOV3 cell proliferation; E. Tumor weight of STC2 overexpressing cells and control cells. F. Tumor growth curves of STC2 overexpressing cells and control cells; G. Cav1.2 knockdown inhibits HEY cell proliferation; H. Tumor weight of Cav1.2 knockdown cells and corresponding control cells (shLuc); I. Tumor growth curves of Cav1.2 knockdown cells and corresponding control cells (shLuc).

[0034] Figure 5Figure 3: STC2 overexpression and Cav1.2 knockdown promote cisplatin resistance in ovarian cancer. A. STC2 overexpression promotes cisplatin resistance in SKOV3 cells; B. Cav1.2 knockdown promotes cisplatin resistance in HEY cells; C. STC2 overexpression and apoptosis rate in SKOV3 cells; D. Cav1.2 knockdown and apoptosis rate in HEY cells. E. Statistical analysis of apoptosis rate in STC2-overexpressing cells; F. Statistical analysis of apoptosis rate in Cav1.2-knockdown cells.

[0035] Figure 6 Figure 2 shows the interaction between STC2 and Cav1.2 in A2780 and A2780-resistant cells. A. Effect of cisplatin on the survival of A2780 and A2780cis cells harboring STC2 cDNA and shRNA; B. Effect of cisplatin on the survival of A2780 and A2780cis cells harboring Cav1.2 cDNA and shRNA; C. Effect of cisplatin on the apoptosis of A2780 and A2780cis cells harboring STC2 cDNA and shRNA; D. Effect of cisplatin on the apoptosis of A2780 and A2780cis cells harboring Cav1.2 cDNA and shRNA; E. Statistical analysis of the apoptosis of A2780 and A2780cis cells harboring STC2 cDNA and shRNA; F. Statistical analysis of the apoptosis of A2780 and A2780cis cells harboring STC2 cDNA and shRNA. DETAILED DESCRIPTION

[0036] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1 Experimental methods and materials

[0038] 1.1 RNA extraction and RT-PCR detection

[0039] RNA extraction and purification: The cells were collected when they were in good condition and at an appropriate density. SKOV3 cells with a growth density of about 80% were washed twice with PBS, and 1 mL of Trizol was added to the cells in a 6-well plate. The cells were allowed to stand at room temperature for 5 minutes to fully lyse the cells. The cells were then pipetted into a 1.5 mL EP tube using a 1 mL pipette. 200 μL of chloroform was added and mixed using a vortex shaker for 20 seconds. The cells were allowed to stand at room temperature for 10 minutes and centrifuged at 12,000 rpm for 15 minutes at 4°C. 500 μL of the upper aqueous phase was gently aspirated and transferred to another fresh 1 To a .5mL EP tube, add 500μL of isopropanol, gently invert and mix, let stand at room temperature for 10 minutes, and centrifuge at 12000rpm at 4℃ for 10 minutes; aspirate the supernatant and observe carefully. A white precipitate appears at the bottom of the centrifuge tube, add 1mL of 75% ethanol to wash the precipitate, and centrifuge at 12000rpm at 4℃ for 10 minutes; aspirate the supernatant, open the EP tube cap and dry it at room temperature, add 40μL RNase-free water to dissolve the RNA; use NanoDrop2000 to determine the RNA concentration and purity.

[0040] Reverse transcription: Genomic DNA was removed using the PrimeScript RT Master Mix kit (Takara). The mRNA reverse transcription experiment was performed using the following system: To remove genomic DNA: 1 μg of Total RNA, 2 μL of 5× gDNA Eraser Buffer, 1 μL of gDNA Eraser, and 10 μL of RNase-Free H2O. The above reaction system was added to a PCR tube, pipetted to mix, and placed in a PCR instrument at 42°C for 2 minutes. Reverse transcription system (20 μL): 10 μL of the first step reaction solution, 1 μL of RT Primer Mix, 24 μL of 5× PrimeScript Buffer, 11 μL of PrimeScript Enzyme Mix, and 4 μL of RNase-Free H2O. The above reaction system was added to a PCR tube, pipetted to mix, and placed in a PCR instrument at 37°C for 15 minutes. The tube was then stored in a -20°C refrigerator.

[0041] RT-PCR or PCR: System: dNTPs (2.5 mM each) 0.6 μL, 10× PCR Buffer (containing Mg 2+) 2μL, 0.6μL Forward Primer (10μM), 0.6μL Reverse Primer (10μM), 1μL cDNA, and 0.1μL Taq diluted to 20μL in ddH2O. Add the reaction mixture to a PCR tube, mix thoroughly by pipetting, and place in a PCR instrument. Reaction conditions are as follows: initial denaturation at 94°C for 4 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a complete extension at 72°C for 7 minutes, and storage at 4°C. Gene expression was detected by agarose gel electrophoresis. RT-PCR primer sequences are listed in Appendix 1.

[0042] Table 1. RT-PCR primer sequences

[0043]

[0044] 1.2 Site-directed mutagenesis vector construction experiment

[0045] The site-directed mutagenesis of phosphorylation sites and ubiquitination sites was performed using the MutExpressII site-directed mutagenesis kit (Vazyme). The specific steps are as follows: (1) Design of mutation primers: forward primer The 5' end contains a 21 bp complementary region, the mutation site is located in the middle of the sequence, and there is a 15 bp non-complementary region at the 3' end; the reverse primer The 5' end contains a 21 bp complementary region to the forward primer, including the mutation site, and the 3' end contains a 15 bp non-complementary region. (2) Amplify the target plasmid: dNTPMix (10 mM each) 1 μL, 2×MaxBuffer 2 μL, Forward Primer (10 μM) 2 μL, Reverse Primer (10 μM) 2 μL, template DNA (≤1 ng) optional, DNA polymerase 1 μL, ddH2O to 50 μL. (3) After the system is prepared, perform PCR amplification. The reaction conditions are as follows: pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 15 seconds, annealing at 62°C for 15 seconds, extension at 72°C for 30-60 seconds / kb, 30 cycles, complete extension at 72°C for 7 minutes, and store at 4°C. After the reaction is completed, take 5 μL of the amplified product for agarose gel electrophoresis. If the amplified band is specific and the size is correct, proceed to the next step of the experiment. (3) DpnI digestion: Since the above reaction contains a template plasmid, in order to prevent the generation of false positive transformants, the inventors performed DpnI digestion on the above reaction products. The reaction system is as follows: DpnI 1μL, reaction product 45μL mixed evenly, 37℃, reaction for 1.5h. (4) Recombination reaction: The 5' end of the forward and reverse primers contains a reverse complementary sequence. Under the catalysis of ExnaseII, the 5' and 3' ends of the amplified product can undergo homologous recombination and circularization. In an ice water bath, add the following components of the system to the PCR tube in sequence: 5×CEIIBuffer 4μL, DpnI digestion product 50-400ng ExnaseII 2μL, and ddH2O to 50μL. The amount of DpnI digestion product used = [0.02×the number of base pairs of the target plasmid]ng. Too much or too little will affect the efficiency of circularization. After the system is prepared, blow evenly. React at 37℃ for 30min, and immediately place in an ice water bath for 5min. (5) Transformation of reactants, plating, and identification of bacteria: Take 10 μL of the cooled reactants and add them to 100 μL of DH5α competent cells. Gently mix with a pipette tip, avoiding pipetting. Place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and ice-water bath for 2 minutes. Add 900 μL of LB medium and shake at 37°C for 1 hour. Take 200 μL of the bacterial solution and spread it on a plate containing antibiotics. Place the plate upside down at 37°C incubator and incubate overnight. The next day, pick clones and shake the cells.

[0046] The primer sequences for N73A site-directed mutagenesis are:

[0047] Forward:

[0048] Reverse:

[0049] 1.3 Western Blot Experiment

[0050] Protein extraction: Observe the cell status and take cells in the logarithmic growth phase for lysis. Aspirate the original culture medium and add pre-cooled PBS to wash twice; Aspirate the PBS and place the cells to be collected on ice. Add an appropriate volume of RIPA lysis buffer (with protease inhibitors, phosphatase inhibitors, and PMSF added in advance) according to the cell quantity to lyse the cells; Use a cell scraper to scrape the cells from the cell culture dish and transfer them to a 1.5mL EP tube with a pipette. Use a 1mL syringe to blow back and forth 6-8 times until the cell lysate is no longer viscous. Let it stand on ice for 30 minutes to fully lyse; Place in a 4℃ centrifuge at 12000rpm for 10 minutes; Aspirate the supernatant, which is the lysed protein, and transfer it to a new 1.5mL EP tube.

[0051] Protein concentration was measured by BCA method: Standard curve was drawn: a new 96-well plate was taken and 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard (concentration was 0.5 μg / μL, BSA) were added respectively. Then, 20, 19, 18, 16, 12, 8, 4, and 0 μL of cell lysate were added to each well to make up to 20 μL, so that the concentration of protein standard in each well was 0, 25, 50, 100, 200, 300, 400, and 500 μg / mL respectively. Three replicate wells were set for each concentration. 1 μL of protein sample to be tested and 19 μL of protein lysate were added to the 96-well plate. Each sample was set Set up three replicate wells; prepare BCA working solution: prepare BCA working solution according to the instructions at a ratio of 50:1 of solution A: solution B, and add 200 μL to each well; place the above 96-well plate in a 37°C incubator in the dark for 30 minutes; ensure that there are no bubbles in each well, and read the absorbance value at a wavelength of 562 nm using a microplate reader; draw a standard curve based on the concentration of the measured protein standard, and calculate the concentration of the measured protein sample; add 1× loading buffer to the measured protein sample to adjust to the same concentration, mix well, place in a metal bath, boil the sample at 100°C for 4-8 minutes according to the sample volume, and store in a -80°C refrigerator for long-term storage.

[0052] Electrophoresis: Load the prepared gel into the electrophoresis tank, add electrophoresis buffer, and adjust the sample loading well with a syringe to ensure that there are no impurities and bubbles in the well; prepare protein samples and markers, ensuring that the sample amount in each well is consistent; perform electrophoresis at a constant voltage of 80V in the stacking gel and at a constant voltage of 120V in the separation gel.

[0053] Transfer: Prepare 1x transfer buffer in advance and pre-chill in a 4°C cold room. Immerse the PVDF membrane in methanol for 15 seconds, then in ddH2O for 2 minutes, and finally immerse it in transfer buffer. Prepare a transfer cassette, sponge, and filter paper. Clean them with ddH2O and immerse them in transfer buffer. Remove the glass plate and rinse it with ddH2O. Remove the gel and immerse it in transfer buffer. Place the transfer cassette with the black side facing down and place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge on top. Remove all bubbles. Secure the transfer cassette and place it in a transfer tank. Pour in the pre-chilled transfer buffer. Transfer the membrane at a constant current of 200 mA for approximately 2 hours, depending on the molecular weight of the target protein.

[0054] Blocking: Remove the membrane from the transfer tank, place it in the prepared 5% skim milk powder, place it on a horizontal shaker and shake slowly, and block it at room temperature for 2 hours.

[0055] Antibody incubation: The membrane was removed from the blocking solution and rinsed twice with 1×TBST; the membrane was incubated with the primary antibody (anti-HA antibody <#3724S>, anti-GST antibody <#2624S>, and anti-β-actin antibody <#3700S> purchased from CellSignaling; or anti-Cav1.2 antibody purchased from Sigma). <c1241>and anti-STC2 antibodies <hpa045372>) Incubation: Place the membrane in the prepared primary antibody and immerse it completely. Place it on a horizontal shaker with slow shaking, incubate overnight at 4°C or at room temperature for 2 hours; after the incubation, transfer it to a membrane wash box, rinse twice with 1×TBST, and then wash 3 times, 10 minutes each time. Secondary antibody incubation: Select the corresponding HRP-labeled secondary antibody according to the species of the primary antibody (including HRP-conjugated anti-rabbitIgG<#7074S> or HRP-conjugated anti-mouseIgG<#7076S> purchased from CellSignaling). Prepare it according to the proportions in the instructions. Place the membrane in it and immerse it completely. Place it on a horizontal shaker at room temperature and incubate slowly for 2 hours; after the incubation, transfer it to a membrane wash box, rinse twice with 1×TBST, and wash 3 times, 15 minutes each time.

[0056] HRP luminescence color development: According to the luminescence solution instructions, mix solution A and solution B in a ratio of 1:1 to prepare the luminescence solution, and protect from light; place the membrane on a luminescence plate and add an appropriate amount of luminescence solution; place it in a pre-cooled exposure instrument for exposure, and observe the analysis results.

[0057] 1.4 Coomassie Brilliant Blue Staining

[0058] (1) Take out the SDS-PAGE gel after running, gently put it into the staining box, and pour in R250 staining solution to completely immerse the gel. (2) Staining: Place it on a horizontal shaker and slowly incubate at room temperature for 30 minutes or longer to fully stain. The staining time varies depending on the thickness of the gel and the temperature during staining. (3) When the gel is stained uniformly and in the same color as the staining, recover the staining solution and add double-distilled water to rinse off the attached staining solution. (4) Decolorization: Discard the rinsing solution, add decolorizing solution to fully cover the gel, put it on a horizontal shaker, adjust the speed to a slightly faster speed, and elute at room temperature. When the staining solution turns blue, replace the staining solution, or use absorbent paper to absorb the dye. (5) Place the gel in double-distilled water until the blue background of the gel is basically removed and the protein bands are clearly visible, and take a picture. Alternatively, use a clean blade to cut the target band and send it to the company for mass spectrometry analysis.

[0059] 1.5 Lentiviral packaging and construction of stable cell lines

[0060] Observe the density of 293T cells and perform transfection when it reaches more than 80%; Take EP tube No. 1, add the expression plasmids including full-length Cav1.2, STC2 cDNA and truncated Cav1.2 / STC2 cDNA fragments (all p) and viral packaging plasmids psPAX2 and pMD2.G in a ratio of 4:3:1 to 500 μL serum-free culture medium; Take EP tube No. 2, draw 8 μL of Lipofectamine 2000 and add it to 500 μL serum-free culture medium, mix well, and let it stand at room temperature for 5 minutes; Use a pipette to mix the liquid in the above two tubes thoroughly, let it stand at room temperature for 20 minutes; Take out 293T cells, remove the original culture medium, add 2 mL serum-free culture medium, and add the above mixture to 6c m culture dish; placed in a 37°C cell culture incubator, incubated for 6 hours, the original culture medium was aspirated and 4 mL of serum-containing culture medium was added; after 48 hours and 72 hours, the viral supernatant was collected, centrifuged at 6000 rpm for 10 minutes, and the viral liquid was filtered with a 0.45 μm filter; the target cells to be infected were taken out, the original culture medium in the 6-well plate was aspirated and 1 mL of cell culture medium was aspirated, 1 mL of viral liquid and 2 μL of polybrane (final concentration of 8 μg / mL) were added, and mixed evenly; placed in a 37°C cell culture incubator, infected for 48 hours, digested the cells, transferred to a 6 cm culture dish, and added 3 μg / mL puromycin (different target cells have different drug concentrations) for drug screening to obtain a stably transfected cell line.

[0061] 1.6 Co-immunoprecipitation (Co-IP)

[0062] Prepare cell lysate: aspirate the culture medium in a 10 cm cell culture dish and wash once with pre-chilled 1× PBS; aspirate the PBS, add 500 μL of pre-chilled 1× cell lysis buffer, and place on ice for 5 min; scrape the cells from the cell culture dish with a cell scraper, transfer to a 1.5 mL EP tube, and place on ice; pipette back and forth with a 1 mL syringe until the cell lysate is no longer viscous; centrifuge at 12,000 rpm at 4°C for 10 min, transfer the supernatant to a new 1.5 mL EP tube; determine the protein concentration.

[0063] Cell lysate pre-clearing: 500 μg of cell lysate was added to 20 μL of 50% protein A / G agarose bead slurry; the mixture was incubated on a shaker at 4°C for 30 min; centrifuged at 12,000 rpm at 4°C for 10 min, and the supernatant was transferred to a new 1.5 mL EP tube.

[0064] Immunoprecipitation reaction: add the primary antibody (anti-HA antibody <#3724S> and anti-GST antibody <#2624S> purchased from CellSignaling) to the pretreated cell lysate in proportion and incubate at 4°C shaker overnight; add 20 μL of 50% protein A / G agarose bead slurry and incubate at 4°C shaker for 2 h; centrifuge at 4°C, 12000 rpm, for 30 s; discard the supernatant, add 500 μL of 1× cell lysis buffer, mix and wash; repeat the above steps (3) and (4) three times.

[0065] Sample analysis: Add 20-40 μL of 2×SDS lysis buffer to the above precipitate, vortex and centrifuge for 30 seconds; boil the sample in a 100°C metal bath for 2-5 minutes; centrifuge at 12,000 rpm for 1 minute; aspirate the supernatant for SDS-PAGE analysis.

[0066] 1.7 Eukaryotic GST-pulldown experiments

[0067] Collect cells in a 10 cm dish, lyse them, and determine the protein concentration; take 500 μg of protein and add cell lysis buffer to 1 mL; take 20 μL of Glutathione Sepharose 4B and add it to the above cell lysate; incubate on a shaker at 4°C for 2 hours, then centrifuge at 500g for 5 minutes at 4°C, discard the supernatant, add 1 mL of cell lysis buffer, invert and wash, and centrifuge at 500g for 5 minutes at 4°C, and discard the supernatant; add 20-40 μL of 2×SDS loading buffer to the above precipitate, and cook the sample in a metal bath at 100°C for 2-4 minutes; aspirate the supernatant for SDS-PAGE detection.

[0068] 1.8 Immunofluorescence

[0069] (1) Prepare the well-grown ovarian cancer cell line SKOV3, place the cell slides in a 12-well plate, and irradiate with ultraviolet light in a biosafety cabinet for half an hour. Digest the target cells and count them using a cell counter. Plant 1×10 cells per well. 5 cells (different cells have different growth rates, so the number of cells planted is slightly different). Three replicate wells were set up for each experimental group and cultured in a cell culture incubator at 37°C overnight. (2) Fixation: 1 mL of 1×PBS was added to each well to wash 3 times, PBS was aspirated, and 4% PFA was added to fix at room temperature for 30 minutes. (3) Permeabilization: 1 mL of 1×PBS was added to each well to wash 3 times, PBS was aspirated, 0.5% TritonX-100 was added, and the cells were treated at room temperature for 10 minutes. (4) Blocking: 1 mL of 1×PBS was added to each well to wash 3 times, PBS was aspirated, 5% BSA was added, and the cells were blocked at room temperature for 2 hours. (5) Anti-Cav1.2 antibody purchased from Sigma was used. <c1241>or anti-STC2 antibody <hpa045372>) Incubation as the first antibody: Take a clean glass slide, add 30 μL of the primary antibody diluted in proportion, and place the cell slide on the antibody, and incubate at 4°C overnight. (6) Incubation with the second antibody (FITC or Texas red labeled anti-rabbit or anti-mouse IgG purchased from Jackson ImmunoResearchLaboratory, catalog numbers 315-097-003 and 115-076-075 respectively): Transfer the cell slide coated with the primary antibody to a 6-well plate, add 0.1% PBST and wash 3 times, 6 minutes each time, and discard the PBST. Take a clean glass slide, add 30 μL of the secondary antibody diluted in proportion, and place the cell slide on the antibody (dilution ratio 1:200), and incubate at room temperature for 50 minutes. (7) Sealing: Wash 3 times, 8 minutes each time, and discard the PBST. Add 1 drop of mounting medium containing DAPI to the glass slide, and place the cell slide on the mounting medium. (8) Photography: Observe and take photos using a fluorescence microscope.

[0070] 1.9 Proximity Ligation Analysis Technology

[0071] Cells were seeded in 24-well plates and cultured overnight. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Trition X-100 for 10 minutes, and then Block at 37°C for 30 minutes using the blocking buffer provided in the InSitu Red Starter Kit (Sigma, St. Louis, MO). Dilute the primary antibody (same as in the immunofluorescence assay in step 1.8) to the appropriate concentration in antibody diluent, add the primary antibody, and incubate overnight at 4°C. The next day, dilute the two probes 1:5 using the antibody diluent provided in the kit. For a 40 μL reaction, add 8 μL of PLA probe PLUS and 8 μL of PLA probe MINUS to 24 μL of antibody diluent. Wash twice with wash buffer, add the PLA probe solution, and incubate at 37°C for 60 minutes. Dissolve the ligation stock solution in a 1:5 ratio with high-purity water. For a 40 μL reaction, add 8 μL of the 5* ligation stock solution to 32 μL of high-purity water. Add the PLA probe dropwise, gently wash twice with wash buffer, remove the ligase from -20°C, and add the ligation system at a 1:40 ratio, vortexing. Add the ligation system to each sample and incubate at 37°C for 30 minutes. Dilute the amplification solution at a ratio of 1:5 with high-purity water. Add the ligation reaction solution dropwise, gently wash twice, add the polymerase at a ratio of 1:80, and vortex. Add the amplification enzyme and incubate at 37°C for 100 minutes. Add the amplification enzyme dropwise, wash twice with 1*Wash Buffer B, and wash for 1 minute with 0.01% Wash Buffer B. Dry the slides at room temperature in the dark. Use a minimum volume of Duolink InSituMounting Medium and DAPI to mount the slides, avoiding bubbles, and seal the edges with nail polish. Incubate in the dark for 15 minutes. Then, perform the antibody incubation, ligation, and amplification procedures according to the kit instructions.

[0072] 1.10 IC50 determination

[0073] Ovarian cancer cell lines HEY, SKOV3, A2780, and A2780cis, as well as their derivatives, were inoculated with cells. The next day, the target cells were digested and pipetted to a single-cell suspension. Counted using a cell counter, 5,000 cells / 100 μL were plated in a 96-well plate, with four replicates per well. The cells were cultured overnight at 37°C. 100 μL of the designated drug was added to each well, and the cells were cultured for 48 hours. CCK8 working solution was prepared by adding 10 μL of CCK8 solution to every 100 μL of culture medium and mixing thoroughly with a pipette. After 0, 24, 48, 72, and 96 hours of cell attachment, the 96-well plate was removed, the culture medium carefully aspirated, and 100 μL of the prepared CCK8 working solution was added to each well, avoiding bubbles and gently mixing. The cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. IC50 values ​​were calculated.

[0074] 1.11 Subcutaneous tumor transplantation experiment

[0075] Animal experiment location: Experimental Animal Center, East China Normal University; Breed: BALB / c nude mice, 5 weeks old, female, subcutaneous injection. Six nude mice per group were injected subcutaneously with 100 μL of cell suspension inoculated into the right posterior axilla of the nude mice using a syringe. 5×10 cells of the SKOV3 cell line and its derivatives were inoculated per mouse. 6 HEY cell lines and their derivatives were seeded at 3×10 6 After inoculation, the mice were observed and measured every 5 days. At the end of the experiment, the mice were killed by cervical dislocation, and the tumors were removed and weighed.

[0076] 1.12 Cell apoptosis assay

[0077] Remove the culture medium from the dish and wash with PBS. Then, add 0.25% trypsin to the cell culture dish and incubate in a 37°C incubator for approximately 2 minutes until the cells become round. Add 2 ml of culture medium to neutralize the trypsin and pipette the cells into single cells. Transfer the cells and culture medium to a flow cytometer and centrifuge at 1000 rpm for 5 minutes. Remove the supernatant, add 4 ml of PBS to the centrifuge tube, pipette the cells, and centrifuge at 1000 rpm for 5 minutes. Remove the supernatant and add 100 μl of 1× buffer (10× buffer diluted with ddH2O) to the cell pellet. Mix the cells thoroughly. Then, add 5 μl of Annexin-V FITC and 5 μl of PI to the buffer. Incubate at room temperature for 15 minutes in the dark. Flow cytometric analysis: Use 488 nm excitation wavelength. Detect FITC fluorescence using a 515 nm bandpass filter and detect PI using a filter with a wavelength greater than 560 nm.

[0078] Experimental results of Example 2

[0079] 2.1 Isolation and identification of STC2 receptor

[0080] SKOV3pLVX-STC2-HA cells were established by lentiviral infection (ovarian cancer cell line SKOV3 was infected with pLVX-Tet-OnAdvanced virus and pLVX-Tight-Puro virus, and drug screening yielded SKOV3pLVX-Tight-Puro-STC2, referred to as SKOV3pLVX-STC2). Adding doxycycline to the culture medium of SKOV3pLVX-STC2-HA cells induced the expression and secretion of STC2-HA protein. Trypsinized SKOV3 ovarian cancer cells were then incubated with conditioned medium (CM, RPMI1640 medium that had been cultured for approximately 48 hours, Gibco product number 11875119) containing STC2-HA. Cells incubated with normal serum-free medium (NM, general RPMI1640 medium, Gibco product number 11875119) served as a negative control for immunofluorescence analysis. The results showed that STC2 could bind to the plasma membrane ( Figure 1 A) To further confirm the binding ability of STC2, doxycycline-treated SKOV3pLVX-STC2-HA cells were incubated with NM and CM, respectively. Cells incubated with NM but not treated with doxycycline served as negative controls for immunofluorescence analysis. The results showed that STC2 was significantly expressed in the cytoplasm of doxycycline-treated SKOV3pLVX-STC2-HA cells, and incubation with CM resulted in STC2 accumulation in the plasma membrane ( Figure 1 B) These results indicate the presence of STC2 receptors in the plasma membrane.

[0081] To isolate the STC2 receptor, whole-cell lysates were incubated with CM, and the STC2-bound complex was purified by IP using protein A / G agarose beads. HA antibody (from the same source) was used as bait. The cells were then eluted with loading buffer, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue R250. The results showed significant differences between the CM- and NM-cultured groups ( Figure 1 C) Then different protein bands were collected and their components were identified by mass spectrometry. Figure 1 As shown in D.

[0082] To confirm the interaction between STC2 and STC2-interacting proteins, the inventors collected doxycycline-induced SKOV3pLVX-STC2-HA cells and performed immunoprecipitation using HA antibody as bait. Using cells without doxycycline as a negative control, the results showed that Cav1.2 accumulated significantly in the doxycycline-treated group, indicating that STC2-HA can interact with Cav1.2 ( Figure 1 E). To further verify its binding ability, the inventors established a stable STC2 overexpressing cell line (SKOV3STC2-HA) and performed immunoprecipitation experiments using Cav1.2 and HA as bait (normal IgG was used as a negative control). The results showed that STC2 indeed interacted with Cav1.2 ( Figure 1 F and 1G). IF detection also confirmed the co-localization of Cav1.2 and STC2 ( Figure 1 H).

[0083] 2.2 Peptides Binding to Cav by STC2

[0084] In order to identify which region of Cav1.2 is required for interaction with STC2, the inventors introduced a prokaryotic GST fusion protein expression system. Previous studies have shown that exogenous STC2 can bind to the plasma membrane, suggesting that the binding site of STC2 is located in the extracellular region of Cav1.2. Since Cav1.2 is a multi-transmembrane protein with 12 extracellular segments, the inventors selected five extracellular regions with relatively long amino acid sequences (Ca-1, Ca-2, Ca-3, Ca-4 and Ca-5, respectively) (Table 2), cloned them into a GST expression vector, expressed them in Escherichia coli, and then performed a GST pull-down experiment. The results showed that only the Ca-1 and Ca-2 fragments could significantly interact with STC2 ( Figure 2 A). Next, the interaction was verified in a eukaryotic GST fusion protein expression system, and it was found that the Ca-1 fragment could interact with STC2-HA in the eukaryotic cell line 293T ( Figure 2 B). The data further confirmed that the binding sites of STC2 are located in the extracellular region of Cav1.2, with the corresponding amino acid sequences being aa291-380 and aa674-728, respectively.

[0085] Table 2. Cav1.2 fragments

[0086] name Location length area Ca-1 291-380 90 Extracellular Ca-2 674-728 55 Extracellular Ca-3 1072-1161 90 Extracellular Ca-4 1321-1372 52 Extracellular Ca-5 1431-1499 69 Extracellular

[0087] In addition, to explore the binding sites on STC2, STC2 cDNA was divided into 4 fragments (S1, S2, S3, and S4) (Table 2) and directional ligated into the pEGFP-N1 vector. 293T cells were then transfected with pEBG-Ca-1 and GST pull-down experiments were performed. The results showed that in the pull-down group, only the S2 fragment (containing the N73 glycosylation site) was stably detected, indicating that S2 is involved in the interaction between STC2 and Cav1.2 ( Figure 2 C).

[0088] 2.3 Colocalization of STC2 and Cav1.2

[0089] PLA experiments further confirmed the colocalization of STC2 and Cav1.2 ( Figure 3 A). To further explore the function of STC2, the inventors established an STC2 mutant containing A73 instead of N73 and tested its effect. The results showed that STC2 containing the N73A mutation failed to bind to Cav1.2 in the co-IP experiment ( Figure 3 B), GST pull-down experiments also verified that after N73A mutation, STC2 could not bind to Cav1.2 ( Figure 3 C).

[0090] 2.4 Regulatory effects of STC2 and Cav1.2 on tumorigenesis and progression

[0091] The growth of tumor cells was detected in a xenograft tumor model, and the results showed that knockdown of STC2 in HEY cells reduced the tumor growth rate and tumor weight ( Figure 4 AC), while overexpression of STC2 in SKOV3 increased the tumor growth rate and tumor weight ( Figure 4 DF). In HEY cells ( Figure 4 In GI and SKOV3 cells, knockdown of Cav1.2 led to accelerated tumor growth and increased tumor size and weight, indicating that in vivo, knockdown of Cav1.2 promoted tumorigenesis. Taken together, these data indicate that both STC2 overexpression and Cav1.2 knockdown directly affect ovarian cancer proliferation and tumor progression.

[0092] 2.5STC2 and Cav1.2 regulate tumor cell chemotherapy resistance

[0093] To explore the role of STC2 and Cav1.2 in chemotherapy sensitivity, the IC50 values ​​of established cell lines were calculated, and it was found that STC2 overexpression promoted the chemotherapy resistance of SKOV3 cells to cisplatin ( Figure 5 A), while Cav1.2 knockdown promoted cisplatin-resistant HEY cells ( Figure 5 B). Flow cytometry showed that apoptosis was involved in cisplatin-induced cell death. STC2 overexpression inhibited CDDP-induced apoptosis ( Figure 5 C and 5E), Cav1.2 knockdown inhibited CDDP-induced apoptosis ( Figure 5 D and 5F).

[0094] Cisplatin-sensitive A2780 and cisplatin-resistant A2780 cells (A2780Cis) were used to further investigate the effects of STC2 and Cav1.2 on chemoresistance. A2780 cells were transfected with STC2 cDNA and Cav1.2 shRNA, A2780Cis was transfected with STC2 shRNA, and A2780Cis was transfected with Cav1.2 cDNA. IC50 assay results showed that STC2 promoted chemoresistance ( Figure 6 A), Cav1.2 promotes chemosensitivity ( Figure 6 B). Flow cytometry analysis showed that STC2 inhibited cisplatin-induced cell apoptosis ( Figure 6 C, E) and Cav1.2 promote cisplatin-induced apoptosis ( Figure 6 D, F).

[0095] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from all points of view, the embodiments should be regarded as illustrative and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and range of equivalents of the claims are included in the present invention. Any reference signs in the claims should not be construed as limiting the claim to which they relate.

[0096] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Use of an agent targeting the interaction between STC2 and Cav1.2 in the preparation of a drug for tumor prevention or treatment; wherein: The reagent targeting the interaction between STC2 and Cav1.2 is selected from a Cav1.2 expression promoting reagent or a combination thereof with an STC2 expression inhibiting reagent; the tumor is ovarian cancer resistant to cisplatin; the STC2 expression inhibiting reagent is STC2shRNA; and the Cav1.2 expression promoting reagent is a vector or cell carrying Cav1.2 cDNA.

2. A pharmaceutical composition for preventing or treating tumors, characterized in that: The pharmaceutical composition includes an STC2 expression inhibitory agent and a Cav1.2 expression promoting agent; wherein, the tumor is ovarian cancer resistant to cisplatin, the STC2 expression inhibitory agent is STC2 shRNA; the Cav1.2 expression promoting agent is a vector or cell carrying Cav1.2 cDNA, and the STC2 expression inhibitory agent and Cav1.2 expression promoting agent can be administered sequentially or simultaneously.

Images