Application of GPR3 or GPR3 downstream signal molecule XPC as target spot in preparation of product for treating melanoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a G-protein-coupled receptor 3 (GPR3) or GPR3 downstream signal molecule XPC as a target spot in preparation of a medicine for treating melanoma. The GPR3 is taken as a target spot, and the expression of the GPR3 is knocked out or knocked down, so that the proliferation ability, migration ability and invasion ability of melanoma cells can be remarkably reduced, the Ki67 level can be remarkably reduced, the Cleved-Caspase3 level can be improved, the sensitivity of tumor cells to ultraviolet rays can be improved, the genomes of the tumor cells are damaged, apoptosis occurs, and the melanoma can be treated; the overexpression of the GPR3 promotes the characteristics of tumor stem cells to cause proliferation and metastasis of the tumor cells, and the expression of XPC and a stemness marker can be inhibited and tumor proliferation can be inhibited by knocking out or knocking down the expression of the GPR3. Therefore, GPR3 or a GPR3 downstream signal molecule XPC is an important target spot for treating melanoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of GPR3 ​​or GPR3 ​​downstream signal molecule XPC as a target in the preparation of a product for treating melanoma. Background Art

[0002] Melanoma is a highly malignant tumor derived from melanocytes. In recent years, the incidence and mortality of malignant melanoma have increased year by year. Existing treatment methods have limitations, so there is an urgent need to find new therapeutic targets and develop new anti-tumor drugs.

[0003] G protein-coupled receptor 3 (GPR3) is a class A orphan member of the GPCRs (GPCRs) superfamily, and is widely expressed in neurons in different brain regions such as the cerebral cortex, thalamus, hypothalamus, amygdala, hippocampus, pituitary gland, etc. GPR3 ​​is also expressed in tissues such as skin, eyes, lungs, kidneys, liver, and ovaries. At present, research on GPR3 ​​mainly focuses on nervous system diseases, obesity, follicle development, and oocyte maturation. GPR3 ​​has not yet been found to have a definite endogenous ligand, and there is no record of its involvement in melanoma. Summary of the invention

[0004] The purpose of the present invention is to provide the use of GPR3 ​​or GPR3 ​​downstream signal molecule XPC as a target in the preparation of a product for treating melanoma, thereby reducing the proliferation, migration and invasion abilities of melanoma cells, increasing the sensitivity of tumor cells to ultraviolet rays, and effectively treating melanoma.

[0005] The present invention provides the use of GPR3 ​​or GPR3 ​​downstream signal molecule XPC as a target in the preparation of a product for treating melanoma.

[0006] The present invention also provides the use of shRNA for knocking out or knocking down a target in preparing a product for treating melanoma, wherein the target includes GPR3 ​​or a GPR3 ​​downstream signal molecule XPC.

[0007] Preferably, the product for treating melanoma includes a product that reduces the proliferation ability of melanoma cells.

[0008] Preferably, the product for treating melanoma includes a product that reduces the migration ability of melanoma cells.

[0009] Preferably, the product for treating melanoma includes a product that reduces the invasive ability of melanoma cells.

[0010] Preferably, the product for treating melanoma includes a product that reduces the level of Ki67 and / or increases the level of Cleaved-Caspase3.

[0011] Preferably, the product for treating melanoma includes a product that increases the sensitivity of melanoma cells to ultraviolet rays, thereby causing apoptosis of melanoma cells.

[0012] Preferably, the melanoma comprises a tumor derived from human melanoma cells A875 and / or a tumor derived from mouse melanoma cells B16.

[0013] Preferably, the target sequence of the shRNA for knocking out or knocking down the GPR3 ​​is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5;

[0014] The target sequence of the shRNA for knocking out or knocking down the GPR3 ​​downstream signaling molecule XPC is shown in SEQ ID NO:14 or SEQ ID NO:15.

[0015] The present invention also provides a shRNA for treating melanoma, wherein the shRNA comprises one or more of shGPR3-1, shGPR3-2, shGpr3-1, shGpr3-2, shXPC-1 and shXPC-2;

[0016] The target sequence of shGPR3-1 is shown in SEQ ID NO: 1;

[0017] The target sequence of the shGPR3-2 is shown in SEQ ID NO: 2;

[0018] The target sequence of shGpr3-1 is shown in SEQ ID NO:4;

[0019] The target sequence of shGpr3-2 is shown in SEQ ID NO: 5;

[0020] The target sequence of shXPC-1 is shown in SEQ ID NO: 14;

[0021] The target sequence of shXPC-2 is shown in SEQ ID NO:15.

[0022] Beneficial effects:

[0023] The present invention confirms for the first time the mechanism of action of GPR3. GPR3 ​​can significantly reduce the proliferation, migration and invasion ability of melanoma cells by regulating the DNA damage repair pathway and the expression of XPC protein, significantly reduce the level of Ki67, increase the level of Cleaved-Caspase3, increase the sensitivity of tumor cells to ultraviolet rays, cause damage to the tumor cell genome, and cause apoptosis, thereby treating melanoma. GPR3 ​​or GPR3 ​​downstream signal molecule XPC is an important target for the treatment of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 RT-qPCR detection of the mRNA expression level of GPR3 ​​stably knocked down in A875 cells (A) and B16 cells (B);

[0026] Figure 2 CCK-8 was used to detect the proliferation ability of A875 cells (A) and B16 cells (B) with stable knockdown of GPR3;

[0027] Figure 3 The clone formation ability of A875 cells (A) and B16 cells (B) with stable knockdown of GPR3 ​​was tested for clone formation assay;

[0028] Figure 4 The growth ability of A875 cells (A) and B16 cells (B) with stable knockdown of GPR3 ​​was detected for EdU experiments;

[0029] Figure 5 Transwell migration assay to detect the migration ability of A875 cells (A) and B16 cells (B) with stable knockdown of GPR3;

[0030] Figure 6 The ability of GPR3 ​​in stably knocked-down cells was tested for Transwell invasion in A875 cells (A) and B16 cells (B);

[0031] Figure 7 To verify the effect of GPR3 ​​knockdown on melanoma cells in animal experiments;

[0032] Figure 8 Flow cytometry was used to detect the NER repair efficiency of A875 cells with overexpression and knockdown of GPR3;

[0033] Fig. 9 Western Blot was used to detect the expression of key proteins of the NER pathway, stemness markers, and XPC mRNA in A875 cells with GPR3 ​​knockdown;

[0034] Fig.10 The clone formation ability of A875 cells with stable knockdown of XPC was tested for clone formation experiment;

[0035] Fig.11 The migration ability of A875 cells with stable knockdown of XPC was tested for Transwell migration;

[0036] Fig.12 CCK-8 was used to detect the proliferation ability of A875 cells with stable knockdown of XPC;

[0037] Fig.13 Western Blot was used to detect the protein expression of A875 cells with knockdown of XPC;

[0038] in, Figures 1 to 13 **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05. DETAILED DESCRIPTION

[0039] The present invention provides the use of GPR3 ​​or GPR3 ​​downstream signal molecule XPC as a target in the preparation of a product for treating melanoma. The GPR3 ​​downstream signal molecule XPC described in the present invention is a DNA damage repair factor.

[0040] The present invention uses GPR3 ​​as a target, and by knocking out or knocking down the expression of GPR3, the proliferation ability, migration ability and invasion ability of melanoma cells can be significantly reduced, the Ki67 level can be significantly reduced, the Cleaved-Caspase3 level can be increased, the sensitivity of tumor cells to ultraviolet rays can be increased, and the tumor cell genome damage and apoptosis can be caused, and melanoma can be treated; overexpression of GPR3 ​​promotes tumor stem cell characteristics, leading to tumor cell proliferation and metastasis, and by knocking out or knocking down the expression of GPR3, the expression of XPC and stemness markers can be inhibited, and tumor proliferation can be inhibited. In addition, the present invention uses the GPR3 ​​downstream signal molecule XPC as a target, and by knocking out or knocking down the expression of XPC, it is found that the number of clones and migration ability of melanoma cells are significantly reduced, the value-added ability is significantly reduced, and the expression of stemness markers SOX2 and Nanog protein is significantly reduced. The present invention confirms the mechanism of action of GPR3 ​​for the first time. GPR3 ​​regulates the expression of XPC protein by regulating the DNA damage repair pathway. Therefore, GPR3 ​​or the GPR3 ​​downstream signal molecule XPC is an important target for the treatment of melanoma.

[0041] As one embodiment, the melanoma described in the present invention includes a tumor derived from human melanoma cells A875 and / or a tumor derived from mouse melanoma cells B16; as another embodiment, the melanoma is a tumor derived from human melanoma cells A875.

[0042] As an embodiment, the use of GPR3 ​​or GPR3 ​​downstream signal molecule XPC as a target in the preparation of a product for treating melanoma is the use of shRNA for knocking out or knocking down the target in the preparation of a product for treating melanoma, and the target includes GPR3 ​​or GPR3 ​​downstream signal molecule XPC. As an embodiment, the target sequence of the shRNA for knocking out or knocking down the GPR3 ​​of the present invention is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; as another embodiment, the target sequence of the shRNA for knocking out or knocking down the GPR3 ​​of the present invention is as shown in SEQ ID NO: 1 or SEQ ID NO: 5. In the present invention, the targets described in SEQ ID NO: 1 and SEQ ID NO: 2 correspond to the GPR3 ​​gene in human melanoma cells A875; the targets described in SEQ ID NO: 4 and SEQ ID NO: 5 correspond to the Gpr3 gene in mouse melanoma cells B16 (i.e., mouse GPR3 ​​gene). As an embodiment, the target sequence of the shRNA for knocking out or knocking down the GPR3 ​​downstream signaling molecule XPC of the present invention is as shown in SEQ ID NO:14 or SEQ ID NO:15; as an embodiment, the target sequence of the shRNA for knocking out or knocking down the GPR3 ​​downstream signaling molecule XPC of the present invention is as shown in SEQ ID NO:14.

[0043] As an embodiment, the product of the present invention includes a drug or a reagent.

[0044] The present invention also provides an shRNA for treating melanoma, wherein the shRNA comprises one or more of shGPR3-1, shGPR3-2, shGpr3-1, shGpr3-2, shXPC-1 and shXPC-2; the target sequence of the shGPR3-1 is shown in SEQ ID NO: 1; the target sequence of the shGPR3-2 is shown in SEQ ID NO: 2; the target sequence of the shGpr3-1 is shown in SEQ ID NO: 4; the target sequence of the shGpr3-2 is shown in SEQ ID NO: 5; the target sequence of the shXPC-1 is shown in SEQ ID NO: 14; and the target sequence of the shXPC-2 is shown in SEQ ID NO: 15.

[0045] To further illustrate the present invention, the present invention belongs to the field of biomedicine technology, and specifically relates to the use of GPR3 ​​or GPR3 ​​downstream signal molecule XPC as a target in the preparation of drugs for treating melanoma. However, they should not be construed as limiting the scope of protection of the present invention.

[0046] GraphPadPrism (v.9.2.0) was used for data processing and statistical analysis in the embodiment of the present invention. The calculated data were expressed as mean ± standard deviation (mean ± SD). The F test was used for variance homogeneity test, and the two-tailed Student'T-test was performed for data that met the normal distribution. P < 0.05 was considered to be significantly different. Each group of experiments was repeated at least 3 times.

[0047] Example 1

[0048] 1. Experimental Materials

[0049] (1) Experimental instruments. See Table 1 for details.

[0050] Table 1 Experimental instruments

[0051]

[0052] (2) Antibodies, see Table 2 for details.

[0053] Table 2 Main antibody information

[0054]

[0055]

[0056] (3) Experimental reagents, see Table 3 and Table 4 for details.

[0057] Table 3 Configuration of experimental reagents

[0058]

[0059] Table 4 Other experimental reagents

[0060]

[0061]

[0062] (4) Experimental cells

[0063] Melanoma cell lines A875 and B16: both were obtained from the Translational Research Center of the Affiliated Obstetrics and Gynecology Hospital of Tongji University; all cells were regularly tested for mycoplasma to ensure that the cells were free of mycoplasma contamination;

[0064] 293T cell line: derived from human embryonic kidney cells, purchased from ATCC.

[0065] Example 2

[0066] Cell experiment-construction of stable cell lines

[0067] 1. Construction of shGPR3 plasmid

[0068] (1) shRNA sequences were designed on the official website of Sigma (http: / / www.sigamaldrich.com / ) against the GPR3 ​​gene of the human melanoma cell line A875 (accession number in NCBI is GeneID: 2827) and the Gpr3 gene of the mouse melanoma cell line B16 (accession number in NCBI is GeneID: 14748), and RFP gene (accession number in NCBI is GeneID: 5987) was used as a control, and primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; among them, shRNA against human melanoma cell line A875 is represented by shGPR3, and shRNA against mouse melanoma cell line B1 is represented by shGpr3. The specific synthetic primers and shRNA target sequence information are shown in Table 5.

[0069] Table 5 shRNA sequences and primer sequence information for synthesizing shRNA (5'-3')

[0070]

[0071]

[0072] Mix the primer sequence and 10×NEB buffer 2 in a 1.5mL centrifuge tube, seal the centrifuge tube with a sealing film, insert the sealed centrifuge tube on the floating plate, heat it in a 95℃ water bath for 5 minutes, turn off the water bath, and let it cool naturally to room temperature. If the annealed shRNAoligo is not used immediately, it needs to be stored in a -20℃ refrigerator. After annealing, the above product forms a DNA double strand containing Age I and EcoR I sticky ends, which is connected to the pLKO.1 vector cut by Age I and EcoR I by T4 ligase. The shGPR3 plasmid is obtained by transformation, plating, picking monoclonal bacteria, sending to the company for sequencing identification, and extracting plasmids after amplification.

[0073] (2) Plasmid transformation: First, take 20 μl of NcmDH5α competent cells melted on ice, add the ligation product from the previous step, mix well and place on ice for 30 min. React in a 43°C metal bath for 45 s, quickly transfer to ice, place for 5 min, add 500 μl of LB medium, and place on a 37°C shaker for 45 min. Take 50 μl of the recovery solution from the previous step and apply it on the ampicillin-resistant LB agar medium, and spread it evenly with a sterile glass rod. Finally, invert the plate in a 37°C incubator overnight.

[0074] (3) Plasmid amplification: Take 32 ml of LB medium and add 32 μl of ampicillin (1000×) to prepare bacterial culture medium. Pick a single colony from the culture dish in the previous step, gently mix it with the culture medium, and place it on a shaker at 37°C overnight.

[0075] (4) Plasmid extraction: The experiment was carried out according to the instructions of the endotoxin-free plasmid mini-extraction kit. The details are as follows: collect the bacterial culture medium from the previous step, centrifuge at 4000 rpm for 10 min at room temperature, discard the supernatant, and retain the precipitate; add 500 μL P1 solution (RNase A has been added) to the precipitate, fully shake on a vortex for 30 s, and transfer the solution to a 2 mL EP tube; add 500 μL P2 solution to the EP tube, gently turn it upside down 8 times, at which point the solution becomes clear and viscous; quickly add 500 μL P4 solution to the EP tube, gently turn it upside down 8 times, at which point a white flocculent precipitate appears in the solution. Let stand at room temperature for 10 minutes, then centrifuge at 12000rpm for 10 minutes at room temperature; transfer the supernatant to the filter column in batches, centrifuge at 12000rpm for 1 minute at room temperature, collect the supernatant into a 2mL EP tube, repeat several times to filter and collect all the supernatant; add 0.3 times isopropanol to the solution, mix it upside down, and let stand at room temperature for 5 minutes; column balance: add 500μL BL balance solution to the adsorption column, centrifuge at 12000rpm for 1 minute at room temperature, and discard the solution; add the mixed solution in the first two steps to the adsorption column in batches, centrifuge at 12000rpm for 1 minute at room temperature, and discard the solution; add 500μL deproteinized solution PD to the adsorption column , centrifuge at 12000rpm at room temperature for 1min, and discard the liquid; add 600μL of rinsing solution PW (with anhydrous ethanol added) to the adsorption column, let it stand for 3min, centrifuge at 12000rpm at room temperature for 1min, discard the liquid, and repeat twice; put the adsorption column into a collection tube, centrifuge at 12000rpm at room temperature for 1min, discard the liquid, open the lid, and dry it thoroughly for 10min; put the adsorption column into a new EP tube, add 70μL of preheated ddH2O in the middle of the adsorption membrane, let it stand at room temperature for 10min, centrifuge at 12500rpm at room temperature for 3min, collect the DNA solution into the EP tube, detect the concentration and store it at -20℃ or use it for transfection.

[0076] 2. Construction of Stable Knockdown Cell Lines

[0077] ① 293T cell packaging of lentivirus: Digested 293T cells were plated in a 10cm culture dish. When the cell density reached about 80%, the auxiliary plasmid pMD2.G, psPAX2 and the constructed target plasmid (shGPR3 plasmid) were transfected at a mass ratio of 2:1:3. After 4 hours, the liquid in the 10cm culture dish was replaced with DMEM medium. After 72 hours, the 293T cell supernatant was filtered with a 0.45μm pore filter. The virus solution obtained after filtration was stored in a -80℃ refrigerator.

[0078] ②Infect cells with virus solution: Digest the cells to be infected and spread them in a 6cm culture dish. Wait until the cells adhere to the wall and the cell density is about 50%. Add virus solution to infect the cells in proportion (1.5mL complete medium + 1.5mL virus solution + 9μL polybrene). After 24 hours, change the solution and use appropriate concentration of puromycin to remove cells not infected with the virus. Wait for 2 to 3 days.

[0079] 3. Construction of stable overexpression cell lines

[0080] The cDNA fragment of GPR3 ​​(accession number 2827 in NCBI) was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and primers were designed for PCR on the synthetic gene, upstream primer F: 5'-AAAGAATTCATGGGAGACTACAAGGACGATGAT-3' (SEQ ID NO: 16), downstream primer R: 5'-AAACTCGAGCTAGACATCACTGGGGGAGCGGGA-3' (SEQ ID NO: 17), restriction sites EcoR I and Xho I were added through the primers, and the target gene and PLVX vector of the PCR product were double-digested by these two restriction sites, and the target gene was connected to the vector PLVX plasmid to obtain a GPR3 ​​overexpression plasmid (PLVX-FLAG-GPR3 plasmid);

[0081] According to step 2, the GPR3 ​​overexpression plasmid was packaged into lentivirus and infected in 293T cells to obtain an overexpression stable cell line.

[0082] Test Example 1

[0083] 1. mRNA expression level detection

[0084] (1) Total RNA extraction: Take out the cell culture dish in which the stable knockdown cells constructed in Example 2 were cultured from the cell culture incubator, discard the culture medium, wash with 1×PBS pre-cooled in a 4°C refrigerator, and repeat twice. Add 1 mL of Trizol to lyse the cells and collect them in a 1.5 mL RNase-free EP tube. Add 200 μL of chloroform, mix thoroughly, and let stand on ice for 10 minutes. Centrifuge at 12000 rpm in a 4°C centrifuge for 15 minutes. Take the upper aqueous phase and place it in a new 1.5 mL RNase-free EP tube, add an equal volume of isopropanol pre-cooled in a 4°C refrigerator, vortex and mix, and place in a -80°C refrigerator overnight. Centrifuge at 12000 rpm in a 4°C centrifuge for 10 minutes, discard the supernatant, add 1 mL of 75% ethanol pre-cooled in a 4°C refrigerator to wash, centrifuge at 12000 rpm in a 4°C centrifuge for 5 minutes, discard the supernatant, and repeat twice. Place the EP tube on ice, open the lid and dry it, add 30 μL of RNase-free ddH2O, and store it in a -80℃ refrigerator.

[0085] (2) cDNA synthesis:

[0086] ① Removal of residual genomic DNA

[0087] Prepare the residual genomic DNA removal system (5×gDNAdigesterMix 3μL, Total RNA 10pg-5μg (or mRNA 4 pg-500ng), RNase free ddH2O to 15μL) in an RNase free centrifuge tube, mix gently with a pipette, and incubate at 42℃ for 2min.

[0088] ② Reverse transcription to generate cDNA

[0089] Directly add 5 μL of IIISuperMix plus, mix gently by pipetting; 25℃ for 5 min, 55℃ for 15 min, 85℃ for 5 min.

[0090] (3) RT-qPCR: The obtained cDNA was diluted to 200 μL (1:10 dilution) with RNase Free ddH2O, and then the reaction solution was prepared using the following system. Samples were spotted in an optical 384-well PCR plate, with 3 replicate wells set for each sample, and RT-qPCR reaction was performed.

[0091] Reaction solution: qPCR SYBR Green Master Mix (Low Rox Plus) 5 μL, 10 μM upstream detection primer 0.4 μL, 10 μM downstream detection primer 0.4 μL, cDNA template 1 μL, sterile ultrapure water to 10 μL; wherein the detection primer sequences are shown in Table 6;

[0092] Table 6 RT-qPCR primer sequences

[0093]

[0094] RT-qPCR reaction program: 95°C, 30 s, 95°C, 5 s, 60°C, 30 s, 72°C, 10 s, 95°C, 60 s, 60°C, 30 s, 95°C, 15 s.

[0095] (4) Statistical analysis: The CT value of the target gene relative to the internal reference gene GAPDH was calculated by the method. The expression level of the gene after shRFP knockout relative to the internal reference gene GAPDH was recorded as 1, and normalization was performed for statistical analysis. The results are as follows Figure 1 shown.

[0096] according to Figure 1 It can be seen that the GPR3 ​​knockdown cell lines shGPR3-1 and shGPR3-2 were constructed in the human melanoma cell line A875, and compared with the control group shRFP, they had a good knockdown effect on A875 cells ( Figure 1 Middle A); Gpr3 knockdown cell lines shGpr3-1 and shGpr3-2 were constructed in the mouse melanoma cell line B16. Compared with the control group shRFP, they had a good knockdown effect on B16 cell Gpr3 ( Figure 1 (middle B).

[0097] 2.CCK-8 cell proliferation assay

[0098] (1) Cell preparation: Take out the cell culture dish from the incubator, digest the stable knockdown cells constructed in Example 2, centrifuge, add culture medium and resuspend, and count with a counting plate to make the cell concentration about 1×10 4 Pieces / mL.

[0099] (2) Cell plating: Prepare 5 96-well plates, with 3 to 6 replicate wells for each sample and 1,000 cells / well, and plate 5 replicate 96-well plates for 5 days of data measurement. Add 100 μL of cell suspension to each well and place in a 37°C constant temperature incubator with 5% CO2 for 24 h.

[0100] (3) Add reaction solution: Prepare CCK-8 reaction solution by adding 10 μL CCK-8 dye solution + 90 μL blank culture medium to each well and mix well in a centrifuge tube. Take out the 96-well plate, remove the culture medium, add 100 μL CCK-8 reaction solution per well, and incubate in a 37°C constant temperature incubator for 2 h.

[0101] (4) Detection: Use an enzyme-labeled instrument to detect the absorbance of each well at a wavelength of 492 nm, derive the value and calculate and analyze the results. Figure 2shown.

[0102] according to Figure 2 It can be seen that the absorbance value of the GPR3 ​​knockdown group in A875 cells was reduced and cell proliferation was inhibited ( Figure 2 Middle A), the absorbance value of Gpr3 knockdown group in B16 cells was reduced, and cell proliferation was inhibited ( Figure 2 Middle B). Knockdown of GPR3 ​​and Gpr3 significantly reduced the proliferation capacity of A875 and B16 cells.

[0103] 3. Colony formation

[0104] (1) Cell preparation: Take out the cell culture dish from the incubator, digest and count the stable knockdown cells constructed in Example 2, take an appropriate amount of cells into a new 15 mL centrifuge tube, centrifuge, remove the upper layer of liquid, and resuspend the cells in full culture medium to a cell concentration of 1×10 4 Pieces / mL.

[0105] (2) Cell plating: Prepare a 6-well plate, set up 3 replicate wells for each sample, and plate 1,000 cells in each well. Gently shake the 6-well plate to evenly distribute the cells. Place it in a 37°C constant temperature incubator with 5% CO2 and culture for about 14 days.

[0106] (3) Cell fixation and staining: Take out the 6-well plate, remove the culture medium, add 1 mL of 4% paraformaldehyde to each well, and fix at room temperature for 30 min. Remove the paraformaldehyde, add 1 mL of 0.1% crystal violet to each well, and leave at room temperature for 20 min. Recover the crystal violet, rinse the 6-well plate with running water for 1 min, rinse with clean water until the liquid is completely transparent, and then dry the 6-well plate.

[0107] (4) Photographing: Place the 6-well plate on a white plate, take a photo, and use Image J to count the number of clones. The results are as follows: Figure 3 shown.

[0108] according to Figure 3 It can be seen that the number of A875 cell clones decreased significantly after knocking down GPR3 ​​( Figure 3 Middle A), the number of B16 cell clones was significantly reduced after knocking down Gpr3 ( Figure 3 (middle B).

[0109] 4. Edu cell proliferation assay

[0110] (1) Reagent preparation: a. Fixative: 4% paraformaldehyde; b. Washing solution: 3% BSAc permeabilization solution, dilute TritonX-100 with PBS to a final concentration of 0.3%.

[0111] (2) EdU labeling

[0112] a. Cell preparation: Stable knockdown cells constructed in Example 2 were cultured in well plates or culture dishes, and transfection or drug addition was performed, followed by digestion and resuspending of cells (the drug-added group was resuspended in a drug-containing medium), cell counting was performed, and 10 5 Cells / ml cell suspension;

[0113] b. Cell plating: Use a spray gun to load the sample into a 96-well plate at 100 μl / well, with 3 replicates per group, and place in a 37°C incubator for 24 hours;

[0114] c. Prepare EdU reaction solution: dilute EdU (10mM) with culture medium at 1:500 to obtain 2× EdU working solution (20μM). Preheat at 37℃ for 5 minutes, then use a sample gun to add 100μl EdU reaction solution to each well of the cell culture dish. Place in a 37℃ incubator and incubate for 2 hours;

[0115] d. Fixation: Take out the plate, discard the liquid, add 100 μl 4% paraformaldehyde to each well, fix for 20 minutes at room temperature, then wash 3 times with washing solution, 5 minutes each time;

[0116] e. Permeabilization: Add 100 μl of permeabilization solution to each well and incubate at room temperature for 15 min. Discard the solution and wash three times with washing solution, 5 min each time.

[0117] (3) EdU detection

[0118] a. Prepare the detection solution: Calculate and prepare the total reaction solution according to 50 μl Click reaction solution (43 μl Click Reaction Buffer + 2 μl CuSO4 + 0.1 μl Azide 555 + 5 μl Click Additive Solution) per well and store in the dark;

[0119] b. Staining: Discard the solution, add 50 μl Click reaction solution to each well, and gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample. Incubate at room temperature in the dark for 30 minutes. Then wash with washing solution 3 times, 5 minutes each time;

[0120] c. Nuclear staining: add 50 μl of 1× Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 min. Discard the solution and wash three times with washing solution, 5 min each time;

[0121] d. Chromogenic film: Use 10x fluorescence microscope, use 555nm and 346nm to detect the film, count the cells and perform statistical analysis. The results are as follows Figure 4 shown.

[0122] according to Figure 4It can be seen that the proliferation ability of A875 cells was significantly reduced after knocking down GPR3 ​​( Figure 4 Middle A), the proliferation ability of B16 cells was significantly reduced after knocking down Gpr3 ( Figure 4 (middle B).

[0123] 5. Transwell migration assay

[0124] (1) Cell preparation: Take out the cell culture dish from the incubator, digest and count the stable knockdown cells constructed in Example 2 respectively, take equal amount of cells from different samples, centrifuge and take appropriate amount of empty culture to resuspend the cells so that each 200 μL empty culture contains 5-10w cells.

[0125] (2) Cell plating: Add 200 μL of the above cell suspension to the upper chamber of the Transwell and 800 μL of complete culture medium to the lower chamber. Set up 3 replicate wells for each sample, gently shake the well plate to evenly distribute the cells in the upper chamber, and place it in a 37°C constant temperature incubator containing 5% CO2 for 16 to 24 hours.

[0126] (3) Color development and photography: Prepare fluorescent dye (10 mL sterile PBS + 12.5 μL Calcein-AM). Take out the Transwell plate from the incubator, remove the culture medium in the lower chamber, add fluorescent dye to the lower chamber at 800 μL / well, and put the Transwell chamber back. Incubate them in a 37°C constant temperature incubator for 20 min, take pictures under a fluorescence microscope, use Image J to count the cells, and statistically analyze the results. Figure 5 shown.

[0127] according to Figure 5 It can be seen that the migration ability of A875 cells was significantly reduced after knocking down GPR3 ​​( Figure 5 Middle A), the migration ability of B16 cells was significantly reduced after knocking down Gpr3 ( Figure 5 (middle B).

[0128] 6. Transwell invasion assay

[0129] (1) Chamber preparation: Place Matrigel in a 4°C refrigerator to melt, and pre-cool a 200 μL pipette tip and a 1.5 mL EP tube at -20°C. Take out the Matrigel and place it on an ice block. Dilute the Matrigel at a volume ratio of 1:10 between Matrigel and basic culture medium. Add 100 μL of the diluted Matrigel to each chamber and place it in a 37°C constant temperature incubator for 2 h.

[0130] (2) Cell preparation: Take out the cell culture dish from the incubator, digest and count the stable knockdown cells constructed in Example 2 respectively, take equal amount of cells from different samples, centrifuge and take appropriate amount of empty culture to resuspend the cells so that each 200 μL empty culture contains 5-10w cells.

[0131] (3) Cell plating: Gently remove excess liquid from the upper chamber, add 200 μL of cell suspension to the upper chamber of each well, add 800 μL of complete culture medium to the lower chamber, and set up 3 replicate wells for each sample. Gently shake the plate to evenly distribute the cells in the upper chamber, and then place it in a 37°C constant temperature incubator with 5% CO2 and culture for 16 to 24 hours.

[0132] (4) Cell fixation and staining: Remove the Transwell plate from the incubator, discard the culture medium, add 1 mL of 4% paraformaldehyde to the lower chamber of each well, and fix at room temperature for 30 min. After discarding the paraformaldehyde, add 800 μL of 0.1% crystal violet to each well and leave at room temperature for 20 min. Recover the crystal violet, rinse the chamber with clean water, and gently wipe the inside of the chamber with a cotton swab.

[0133] (5) Filming: Place the chamber on a 6 cm dish, add 100 μL of pure water to the center of the chamber, and film it under a 10x microscope. J counted the cells and the results were statistically analyzed. Figure 6 shown.

[0134] according to Figure 6 It can be seen that the invasive ability of A875 cells was significantly reduced after knocking down GPR3 ​​( Figure 6 Middle A), the invasive ability of B16 cells was significantly reduced after knocking down Gpr3 ( Figure 6 (middle B).

[0135] Example 3

[0136] Animal experiments

[0137] Take out the cell culture dish from the incubator, digest and count the B16 cell Gpr3 stable knockdown cells constructed in Example 2, and resuspend the cells in pre-cooled 1× PBS. The amount of cells injected for tumor formation in each nude mouse was 3×10 5 The cell suspension was drawn up with a 1 mL syringe and the cells were injected subcutaneously into the abdominal area on both sides of the nude mice.

[0138] Every 3 days, use a vernier caliper to measure the long and wide diameters of the tumor and record the data. After 2 to 3 weeks, dissect the mice, remove the tumor tissue, and weigh the tumor. According to the formula V = l × s 2 / 2 to calculate the tumor volume, where l is the long diameter and s is the short diameter. The removed tumor tissue was fixed with 4% paraformaldehyde and entrusted to Wuhan Saiweier Biotechnology Co., Ltd. for paraffin embedding and sectioning, and immunohistochemical detection. The results are as follows Figure 7As shown; A is the tumor condition of nude mice bearing subcutaneous tumors for 15 days, n=5; B is the tumor weight; C is the tumor growth curve in the nude mouse tumor model; D is the immunohistochemical detection of Ki67 and Cleaved Caspase3 levels in nude mouse tumor tissues.

[0139] according to Figure 7 It can be seen that knocking down Gpr3 can significantly reduce the tumor volume and weight of nude mice; the level of Ki67, a proliferation marker, in the Gpr3 knockdown group was lower than that in the control group, indicating that GPR3 ​​promotes the proliferation of melanoma cells; the level of Cleaved-Caspase3, apoptosis marker, was higher than that in the control group, indicating that GPR3 ​​inhibits the apoptosis of melanoma cells.

[0140] Example 4

[0141] Transfect cells by electroporation

[0142] (1) NER reporter vector treatment: dilute the pmax-GFP (positive control plasmid in the Loza electroporation kit) plasmid to 0.01 μg / μl with ddH2O. Cut a piece of sealing film and drop 50 μl of the diluted plasmid on the sealing film. Turn on the power button of the UV crosslinker, press the "Energy" button, enter the irradiation dose, and then press the "Enter" button (the irradiation dose of the plasmid is 1200 J / m 2 ). Open the instrument door, place the plasmid in the UV crosslinker, and press the "start" button to start irradiation. After the end, open the door, remove the sealing film, suck the plasmid into the EP tube, and turn off the power of the UV crosslinker.

[0143] (2) Electroporation: prepare electroporation solution and plasmids, digest the A875 cells GPR3 ​​stable knockdown cells and GPR3 ​​overexpression stable cells constructed in Example 2 with trypsin, suspend them with a certain volume of culture medium, mix the cells and calculate the cell density. The amount of cells transfected in each electroporation cup is about 1 million to 2 million. According to the number of transfected cells required for each electroporation cup, a certain volume of culture medium is taken to suspend and mix the cells in a 1.5mL centrifuge tube and centrifuge at low speed (1000rmp, 3min), discard the culture medium to collect the cells, add 200μL of electroporation solution to resuspend and mix the cells. Take the condition of 5μg target plasmid / 1 million cells (can be adjusted appropriately) and add 15ng DsRed plasmid to the mixture of cells and electroporation solution, and add the above system to the electroporation cup. Insert the electroporation cup into the small slot of the gene introduction instrument, and set the appropriate capacitor voltage value to electroshock the cells according to the type of cells. The electroporated cells were transferred out of the cup, the cells in the electroporation cup were fully mixed, and approximately one electroporation cup of cells were evenly seeded in three wells of a six-well plate.

[0144] Test Example 2

[0145] DNA repair efficiency analysis

[0146] 1. After the cells electroporated in Example 4 were cultured in a 6-well plate for 48 to 72 hours, the cells were resuspended in 200 μL 1×PBS and flow cytometry was performed on a BD flow cytometer to analyze GFP. + and DsRed + Cell ratio. At least 2 × 10 4 All results were analyzed using FlowJo software. Figure 8 As shown. Figure 8 It can be seen that the NER repair efficiency was significantly increased in the GPR3 ​​overexpression group (pGPR3) compared with the control group (Ctrl). Similarly, when two independent shRNAs were used to knock down the expression of GPR3, a decrease in NER repair efficiency was observed. GPR3 ​​is involved in DNA damage repair.

[0147] 2. In order to further clarify the specific molecular mechanism of GPR3 ​​involved in DNA damage repair, Western Blot experiments were performed to examine the expression of stemness factors and NER pathway proteins regulated by GPR3. The specific steps are as follows:

[0148] (1) Total cell protein extraction: Remove the cell culture dish from the cell culture incubator, digest with trypsin, take 20 μL into a cell counting plate, count and calculate, take an equal amount of cells into a 1.5 mL EP tube, resuspend and wash with 1× PBS pre-cooled in a 4°C refrigerator, centrifuge to obtain a cell pellet, repeat twice, add an equal amount of 1× SDS solution, vortex and mix, heat in a 100°C metal bath for 10 min, store in a -80°C refrigerator or use directly in Western Blot experiments.

[0149] (2) Preparation of PAGE gel: Wash the glass plate required for gel preparation with pure water, dry it naturally, fix the glass plate on the gel preparation rack, and check the sealing of the glass plate with pure water. Take out the PAGE gel rapid preparation kit stored in a 4°C refrigerator, take the lower gel solution and lower gel buffer in equal proportions, mix them thoroughly, add the improved coagulant (Yazyme PG112 PAGE gel rapid preparation kit) at a volume ratio of 1:100, mix them well and inject them into the gel plate, and cover the lower gel with 2mL of anhydrous ethanol. Wait for 15 minutes, pour out the anhydrous ethanol, take the upper gel solution and upper gel buffer in equal proportions, mix them thoroughly, add the improved coagulant at a volume ratio of 1:100, mix them well and inject them into the gel plate, and immediately insert the 15-hole comb into the upper gel solution. Wait for 15 minutes and install the prepared gel plate into the electrophoresis tank.

[0150] (3) Sample loading and electrophoresis: Add a certain volume of 1× Running buffer to the electrophoresis tank, gently pull out the 15-hole comb, gently blow each loading well with the tip of the gun, and add the same volume of protein sample and appropriate amount of protein marker to the loading well. Use 80V constant voltage electrophoresis for 120 minutes, and remove the PAGE gel from the glass plate after the electrophoresis is completed.

[0151] (4) Electrotransfer: Cut the PVDF membrane into 9 cm × 6 cm size and activate it by soaking it in methanol for 5 min. Place the transfer clip in the transfer tray, pour the pre-cooled 1× Transfer buffer into the transfer tray, install the transfer clip in the order of positive electrode-sponge pad-filter paper-PVDF membrane-PAGE gel-filter paper-sponge pad-negative electrode, then place it in the transfer tank and fill it with transfer solution. Place it in a 4°C refrigerator and use 250 mA constant current electrotransfer for 90 min.

[0152] (5) Blocking: Take 40 mL of 1×TBST solution, add 2 g of skim milk powder to it to prepare a 5% blocking solution, mix thoroughly and pour into a blocking box, take out the electro-transferred PVDF membrane and put it into the blocking box, block at room temperature for 1 hour. Then wash with 1×TBST for 5 minutes, repeat 3 times.

[0153] (6) Incubation with primary antibody: Use antibody diluent to dilute the primary antibody according to the ratio recommended in the antibody manual and pour it into the antibody incubation box. Then cut the PVDF membrane according to the position of the protein band corresponding to the antibody and place it in the diluted primary antibody. Place it in a shaker at 4°C and incubate for 6 to 8 hours.

[0154] (7) Membrane washing: Recover the primary antibody in the incubation box and rinse the PVDF membrane with 1× TBST three times, 5 min each time.

[0155] (8) Incubation with secondary antibody: Use antibody diluent to dilute the secondary antibody according to the ratio recommended in the antibody manual. Incubate the secondary antibody at room temperature for 1 h. Recover the secondary antibody and rinse the PVDF membrane with 1× TBST 3 times, 5 min each time.

[0156] (9) Exposure: Prepare ECL exposure solution at a volume ratio of 1:1, immerse the PVDF membrane in the exposure solution, incubate in the dark for 10 seconds, and then take a photo. Record the grayscale value of the target protein band and perform statistical plotting. The results are shown in Figure 2. Fig. 9 shown.

[0157] according to Fig. 9 It can be seen that in melanoma cells with GPR3 ​​knockdown, the expression of stemness markers Nanog and SOX2 decreased, and both XPC transcription and protein expression decreased.

[0158] Example 5

[0159] 1. Construction of shXPC plasmid

[0160] (1) The shRNA sequence was designed for the XPC gene of the human melanoma cell line A875 (the accession number in NCBI is GeneID: 7508) on the official website of Sigma (http: / / www.sigamaldrich.com / ), and the RFP gene (the accession number in NCBI is GeneID: accession number 5987) was used as a control. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific information of the synthetic primers and shRNA target sequences is shown in Table 7.

[0161] Table 7 shRNA sequences and primer sequence information for synthesizing shRNA (5'-3')

[0162]

[0163]

[0164] 2. Referring to step 2 of Example 2, 293T cells were packaged with lentivirus and infected to obtain an XPC stable knockdown cell line.

[0165] Test Example 3

[0166] According to Test Example 1, the XPC stable knockdown cell line obtained in Example 5 was subjected to a plate clone formation experiment and a Transwell migration experiment to detect changes in the proliferation and migration ability of melanoma cells after knockdown of XPC. The results show that the number of clones of knockdown XPC cells ( Fig.10 ) and cell migration ability ( Fig.11 ) was significantly reduced. According to Test Example 1, the XPC stable knockdown cell line obtained in Example 5 was tested with CCK-8 to explore the effect of XPC on the malignant proliferation of melanoma cells. It was found that knocking down XPC reduced the proliferation ability of cells ( Fig.12 ).

[0167] Referring to Test Example 2, the XPC stable knockdown cell line obtained in Example 5 was subjected to Western Blot detection, and it was found that XPC knockdown reduced the expression of stemness markers SOX2 and Nanog proteins in the cell line ( Fig.13 ).

[0168] Based on the above content, it can be seen that GPR3 ​​or the GPR3 ​​downstream signaling molecule XPC is an important target for the treatment of melanoma. Melanoma can be effectively treated by knocking out or knocking down GPR3 ​​or XPC.

[0169] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of GPR3 ​​or GPR3 ​​downstream signaling molecule XPC as a target in the preparation of products for the treatment of melanoma.

2. Use of shRNA for knocking out or knocking down a target in the preparation of a product for treating melanoma, characterized in that: The targets include GPR3 ​​or GPR3 ​​downstream signaling molecule XPC.

3. The use according to claim 1 or 2, characterized in that: The product for treating melanoma includes a product for reducing the proliferation ability of melanoma cells.

4. The use according to claim 1 or 2, characterized in that: The product for treating melanoma includes a product for reducing the migration ability of melanoma cells.

5. The use according to claim 1 or 2, characterized in that: The product for treating melanoma includes a product for reducing the invasive ability of melanoma cells.

6. The use according to claim 1 or 2, characterized in that: The products for treating melanoma include products that reduce Ki67 levels and / or increase Cleaved-Caspase3 levels.

7. The use according to claim 1 or 2, characterized in that: The product for treating melanoma includes a product that increases the sensitivity of melanoma cells to ultraviolet rays and causes apoptosis of melanoma cells.

8. The use according to claim 1 or 2, characterized in that: The melanoma includes a tumor derived from human melanoma cells A875 and / or a tumor derived from mouse melanoma cells B16.

9. The use according to claim 2, characterized in that: The target sequence of the shRNA for knocking out or knocking down the GPR3 ​​is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; The target sequence of the shRNA for knocking out or knocking down the GPR3 ​​downstream signaling molecule XPC is shown in SEQ ID NO:14 or SEQ ID NO:

15.

10. A shRNA for treating melanoma, characterized in that: The shRNA includes one or more of shGPR3-1, shGPR3-2, shGpr3-1, shGpr3-2, shXPC-1 and shXPC-2; The target sequence of shGPR3-1 is shown in SEQ ID NO: 1; The target sequence of the shGPR3-2 is shown in SEQ ID NO: 2; The target sequence of shGpr3-1 is shown in SEQ ID NO:4; The target sequence of shGpr3-2 is shown in SEQ ID NO: 5; The target sequence of shXPC-1 is shown in SEQ ID NO: 14; The target sequence of shXPC-2 is shown in SEQ ID NO:15.

Images