Use of CBX3 in the preparation of a drug for diagnosing cutaneous melanoma, predicting the prognosis of cutaneous melanoma, and treating cutaneous melanoma

Abstract

The present invention discloses the application of CBX3 in the preparation of drugs for diagnosing cutaneous melanoma, predicting the prognosis of cutaneous melanoma, and treating cutaneous melanoma. Knocking down the expression of CBX3 has the effect of inhibiting the proliferation and migration of melanoma cells. The advantages of the present invention include: providing a new target for the treatment of SKCM, and it is expected to screen and prepare new drugs for the effective treatment of SKCM based on this target, effectively reducing the proliferation and migratory motility of SKCM cells, and providing a new direction for the development of drugs for treating SKCM.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor therapeutic drugs, and particularly to drugs for treating melanoma. Background Art

[0002] Melanoma originates from melanocytes in the epidermis, mucosa, and other tissues. Melanoma grows faster than any other solid tumor and has a relatively high mortality rate, with approximately 230,000 new cases and 20,000 deaths each year. Cutaneous melanoma (SKCM) is the most lethal malignant tumor among skin cancers. Although modern diagnostic techniques such as dermoscopy have greatly improved the diagnostic accuracy of cutaneous melanoma, the current diagnosis of cutaneous melanoma still faces many challenges. The diagnostic results often highly rely on the clinical experience and expertise of doctors, with strong subjectivity, resulting in misdiagnosis or missed diagnosis of some melanoma cases. In addition, the complex and time-consuming diagnostic process, the difficulty of early diagnosis, and the high diagnostic cost are also important factors restricting some patients from receiving timely examinations and treatments. In clinical practice, surgery is the treatment option for most patients. However, for advanced patients, due to the characteristics of high proliferation and metastasis rate of melanoma, it is easy to lead to poor treatment prognosis. Gene therapy has been gradually emphasized in recent years and has been reported to effectively improve the prognosis of patients in various diseases. However, for melanoma, there is currently a lack of effective molecular targets, which limits the research progress of melanoma gene therapy technology. There is an urgent need for a new therapeutic target to develop treatment regimens that can improve the prognosis of patients.

[0003] Chromobox protein homolog 3 (CBX3), also known as heterochromatin protein 1 gamma (HP1γ), is a member of the heterochromatin protein 1 family and can silence transcription. After CBX3 is trimethylated at H3 Lys-9, the chromatin where it is located is transformed into a heterochromatin-like (inhibitory state) and is recruited to sites of ultraviolet-induced DNA damage and double-strand breaks, thereby affecting cell growth and development. Recent studies have shown that CBX3 is involved in multiple biological processes such as the cell cycle, apoptosis, and immune infiltration, thus affecting the progression of various malignant tumors, such as liver cancer, pancreatic cancer, clear cell renal cell carcinoma, breast cancer, etc.

[0004] So far, the role of CBX3 in cutaneous melanoma (SKCM) is still unclear, and there have been no relevant reports on treating cutaneous melanoma with CBX3 as a therapeutic target. Therefore, analyzing the correlation between CBX3 and SKCM patients, constructing lentiviral vectors with CBX3 knockdown and overexpression, and further exploring the role of CBX3 in SKCM cell proliferation, migration, etc. have potential clinical application value. Summary of the Invention

[0005] The object of the present invention is to provide the application of CBX3 in the preparation of drugs for diagnosing cutaneous melanoma, predicting the prognosis of cutaneous melanoma and treating cutaneous melanoma, so as to solve the problems of complex diagnosis technology, difficult prognosis evaluation and single treatment method in the prior art for cutaneous melanoma.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The application of CBX3 in the preparation of products for diagnosing cutaneous melanoma or predicting the prognosis of cutaneous melanoma.

[0008] Furthermore, the products include products for diagnosing cutaneous melanoma or predicting the prognosis of cutaneous melanoma by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization or gene chip technology.

[0009] The present invention also provides the application of CBX3 in the preparation of drugs for treating cutaneous melanoma.

[0010] The present invention also provides a method for constructing a CBX3 knockdown lentivirus. The construction process is as follows: Synthesize a cDNA fragment of the CBX3 interference target sequence, digest the BamHI / EcoRI sites of the lentiviral vector, and then insert the cDNA fragment of the CBX3 interference target sequence into the digested linearized lentiviral vector to obtain a CBX3 knockdown lentiviral vector; Mix the CBX3 knockdown lentiviral vector, the lentiviral packaging auxiliary vector psPAX 2 and the lentiviral packaging auxiliary vector pMD2.G evenly with Opti-MEM and incubate to obtain the diluted three vectors; Mix the Lipofectamine 2000 reagent evenly with Opti-MEM and incubate to obtain the diluted Lipofectamine 2000; Quickly mix the diluted three vectors with the diluted Lipofectamine 2000 to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution. Incubate the transfection complex with cells, and then culture the cells transfected with the CBX3 knockdown lentivirus, and purify and concentrate the cell supernatant to obtain a CBX3 knockdown lentivirus concentrate.

[0011] Furthermore, the forward sequence of the CBX3 interference target sequence is as shown in SEQ ID NO.1, and its reverse sequence is as shown in SEQ ID NO.2.

[0012] Furthermore, the A260 / A280 of the plasmid DNA extracted from the CBX3 knockdown lentiviral vector, the lentiviral packaging auxiliary vector psPAX 2 and the lentiviral packaging auxiliary vector pMD2.G are all between 1.8 and 2.0;

[0013] The cells are human embryonic kidney cells 293T cells;

[0014] Further, during the construction of the CBX3 knockdown lentivirus, 24 h before transfection, the cells were digested, the cell density was adjusted, and the cells were cultured at 37 °C and 5% CO2 for 24 h. 2 h before transfection, the cell medium was replaced with serum-free medium. The FV055-CBX3 knockdown lentiviral vector, the lentiviral packaging helper vector pSPAX2, the lentiviral packaging helper vector pMD2.G, and Opti-MEM were mixed evenly and incubated at room temperature for 5 minutes. The Lipofectamine 2000 reagent was gently shaken, and an aliquot of it was mixed with 2.4 mL of Opti-MEM and incubated at room temperature for 5 minutes. The diluted three vectors were mixed with the diluted Lipofectamine 2000 within 5 minutes and then incubated at room temperature for 2 minutes to form a transfection complex of the three vectors and the Lipofectamine 2000 diluent. The transfection complex of the three vectors and the Lipofectamine 2000 diluent was transferred to the cell-containing culture medium, mixed evenly, washed after culturing at 37 °C and 5% CO2 for 8 h, the medium was added, and the cells were further cultured in an incubator at 37 °C and 5% CO2 for 48 h and 72 h. The cell supernatants were collected at 48 h and 72 h respectively, purified and concentrated to obtain a virus concentrate, which was stored at -80 °C for a long time.

[0015] Further, 24 h before transfection, human embryonic kidney 293T cells in the logarithmic growth phase were digested, and the cell density was adjusted to 1.2×10 7Cells / 20 mL were inoculated into cell culture dishes and cultured at 37 °C and 5% CO2. After 24 h, when the cell density reached 70% - 80%, they could be used for transfection. Two hours before transfection, the cell culture medium was replaced with serum-free medium. 10 μg of the FV055-CBX3 knockdown lentiviral vector, 10 μg of the lentiviral packaging helper vector pSPAX2, and 10 μg of the lentiviral packaging helper vector pMD2.G were mixed evenly with the corresponding volume of Opti-MEM, and the total volume was adjusted to 2.5 mL and incubated at room temperature for 5 minutes. 100 μL of Lipofectamine 2000 reagent was mixed with 2.4 mL of Opti-MEM and incubated at room temperature for 5 minutes. The diluted three vectors and the diluted Lipofectamine 2000 were gently inverted and mixed within 5 minutes without shaking. After mixing, it was incubated at room temperature for 2 minutes to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution. The transfection complex of the three vectors and the Lipofectamine 2000 dilution was transferred to the culture medium containing 293T cells and mixed evenly. After culturing at 37 °C and 5% CO2 for 8 h, the culture medium containing the transfection complex was poured off and washed. 25 mL of cell culture medium containing 10% serum was added to each bottle of cells and continued to be cultured in an incubator at 37 °C and 5% CO2 for 48 h. The supernatant of 293T cells was collected at 4 °C and centrifuged at 4000 g for 10 min to remove cell debris. The supernatant was filtered through a 0.45 μm filter into a 40 mL ultracentrifuge tube and centrifuged at 4000 × g until the required virus concentration volume was reached. The CBX3 knockdown lentivirus in the filter cup was transferred to the sample collection cup to obtain the virus concentrate of the CBX3 knockdown lentivirus, and the virus concentrate was stored at -80 °C for a long time.

[0016] The present invention also provides a CBX3 knockdown lentivirus obtained by the construction method described above.

[0017] The present invention also provides an application of the CBX3 knockdown lentivirus described above in the preparation of a drug for treating cutaneous melanoma.

[0018] The advantages of the present invention include: providing a new target for treating SKCM, and it is expected to screen and prepare new drugs effectively targeting this target, effectively reducing the proliferation and migration ability of SKCM cells, and providing a new direction for the development of drugs for treating SKCM. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and do not constitute an improper limitation to the present invention. In the drawings:

[0020] Figure 1 It is a statistical chart of detecting CBX3 expression by immunohistochemical staining of 192 samples;

[0021] Figure 2 It is a statistical chart showing that the expression level of CBX3 is higher in tumor tissues as indicated by the sequencing data results;

[0022] Figure 3 It is a statistical chart of the overall survival time of patients in the high CBX3 expression group;

[0023] Figure 4 It is a Western blot result chart of the expression level of CBX3 in different melanoma cells;

[0024] Figure 5 It is the DNA map of the FV055-CBX3 knockdown lentiviral vector;

[0025] Figure 6 It is the DNA map of the PDS407_pL-CMV-RFP-puro-CBX3 overexpression lentiviral vector;

[0026] Figure 7 It is the DNA map of the helper vector psPAX 2 plasmid;

[0027] Figure 8 It is the DNA map of the helper vector pMD2.G plasmid;

[0028] Figure 9 It is a test chart of the CBX3 protein expression level in cells of the CBX3-sh, CBX3-nc, and CBX3-oe groups;

[0029] Figure 10 It is a statistical chart showing that CBX3 has the ability to regulate the proliferation of SKCM cells as demonstrated by the CCK-8 experiment;

[0030] Figure 11 It is an experimental chart showing that CBX3 has the ability to regulate the proliferation of SKCM cells as demonstrated by the colony formation experiment;

[0031] Figure 12 It is an experimental chart showing that CBX3 has the ability to regulate the migration of SKCM cells as demonstrated by the scratch experiment;

[0032] Figure 13 It is an experimental chart showing that CBX3 has the ability to regulate the migration of SKCM cells as demonstrated by the Transwell experiment;

[0033] Figure 14 It is an experimental chart showing that knockdown of CBX3 inhibits the in vivo proliferation ability of SKCM cells as demonstrated by the xenograft tumor experiment;

[0034] Figure 15 It is an experimental chart showing that overexpression of CBX3 promotes the in vivo proliferation ability of SKCM cells as demonstrated by the xenograft tumor experiment. Detailed implementation manners

[0035] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. Here, the illustrative embodiments and descriptions of the present invention are used to explain the present invention, but do not limit the present invention.

[0036] 1. Expression of CBX3 in cutaneous melanoma and its relationship with prognosis

[0037] The tissue microarray purchased from Alenabio contained 204 skin tissue samples. Each skin tissue sample was processed using an anti-CBX3 antibody (1:400; catalog number: 11650-2-AP, Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Twelve samples were shed and excluded, and the remaining 192 samples were subjected to immunohistochemical staining to detect CBX3 expression. Transcriptome data related to SKCM included in TCGA and GTEx were downloaded from the UCSC Xena website, and two melanoma-related datasets (GSE133713, GSE19234) were downloaded from the GEO database for exploration, visualization, and analysis of cancer genomics data. Three SKCM cell lines, A2058, A375, and Mewo (Cell Bank of the Chinese Academy of Sciences), were cultured in a humidified environment at 37 °C, 5% CO2, and 95% air, and Western blot experiments were used to determine the expression of CBX3 in the cell lines.

[0038] As Figure 1 shown, the expression level of CBX3 was elevated in most cutaneous melanoma tissues (166 / 176), and not elevated in most normal skin tissues (14 / 16). The difference was statistically significant. As Figure 2 shown, Figure 1 the results shown were also verified in the sequencing data collected from the TCGA database. The tumor tissues included in TCGA, GSE133713, and GSE19234 were grouped according to the high or low expression level of CBX3. As Figure 3 shown, the results of the prognostic analysis indicated that the overall survival time of patients in the high CBX3 expression group was shorter. The Western blot results were as Figure 4 shown. CBX3 was most highly expressed in the A375 cell line and least expressed in A2058. Based on the experimental results, in subsequent experiments, the CBX3 gene was knocked down or overexpressed in the three cell lines respectively.

[0039] 2. Construction of a lentiviral vector for knocking down CBX3 and a lentiviral vector for overexpressing CBX3

[0040] Obtain the CBX3 gene sequence from the National Center for Biotechnology Information (NCBI) website, as shown in Table 1, and design relevant overexpression and knockdown sequences based on this sequence.

[0041] Table 1 CBX3 cDNA gene sequence

[0042]

[0043] Entrust Hanheng Biotechnology (Shanghai) Co., Ltd. to construct a CBX3 knockdown lentiviral vector. The general construction process is as follows: According to the CBX3 sequence shown in Table 1, artificially synthesize the CBX3 interference target sequence cDNA fragment in vitro. The interference target sequence is shown in Table 2. Digest the BamHI / EcoRI sites of the lentiviral vector FV055, and then insert the CBX3 interference target sequence cDNA fragment into the digested linearized lentiviral vector FV055 to obtain the CBX3 knockdown lentiviral vector, namely the FV055-CBX3 knockdown lentiviral vector. Its map is shown in Figure 5 . Perform sequencing alignment on the MCS region of the FV055-CBX3 knockdown lentiviral vector. The correctly aligned clone is the successfully constructed lentiviral expression vector containing the CBX3 interference target sequence.

[0044] Table 2 CBX3 interference target sequence

[0045]

[0046] Entrust Tsingke Biotechnology Co., Ltd. to construct a CBX3 overexpression lentiviral vector. The general construction process is as follows: Digest the NheI / AscI sites of the lentiviral vector PDS407_pL-CMV-RFP-puro, and insert the full-length CBX3 cDNA product into the linearized lentiviral vector PDS407_pL-CMV-RFP-puro to obtain the PDS407_pL-CMV-RFP-puro-CBX3 overexpression lentiviral vector. Perform a PCR identification experiment on the CBX3 sequence in the PDS407_pL-CMV-RFP-puro-CBX3 overexpression lentiviral vector. The primer sequences are shown in Table 3, and the results are shown in Table 4. The DNA map of the PDS407_pL-CMV-RFP-puro-CBX3 overexpression lentiviral vector is shown in Figure 6 , perform sequencing and alignment on the clones positive for PCR identification. The correctly aligned clone is the successfully constructed overexpression lentiviral vector containing the CBX3 gene sequence.

[0047] Table 3 CBX3 cDNA PCR amplification primers

[0048]

[0049] PCR identification results of CBX3 gene sequence in PDS407_pL-CMV-RFP-ccdB-puro lentiviral overexpression vector (Table 4)

[0050]

[0051]

[0052] 3. Construction of CBX3 knockdown lentivirus, CBX3 overexpression lentivirus and negative control lentivirus

[0053] Prepare the lentiviral vector, lentiviral packaging helper vector psPAX2 and lentiviral packaging helper vector pMD2.G in advance. The DNA map of the lentiviral packaging helper vector psPAX2 is as Figure 7 shown, and the DNA map of the lentiviral packaging helper vector pMD2.G is as Figure 8 shown. Extract the plasmid DNA of the above vectors and dissolve it in sterile TE. Determine its concentration and purity by ultraviolet light absorption method, and ensure that the A260 / A280 of the plasmid DNA extracted is between 1.8 and 2.0. Meeting this standard is qualified and can be used for subsequent cell transfection.

[0054] Twenty-four hours before transfection, digest human embryonic kidney cells 293T cells in logarithmic growth phase, and adjust the cell density to 1.2×10 7Cells / 20 mL were inoculated into cell culture dishes and cultured at 37 °C with 5% CO2. After 24 h, when the cell density reached 70% - 80%, they could be used for transfection. The cell medium was replaced with serum-free medium 2 h before transfection. 10 μg of the prepared FV055-CBX3 knockdown lentiviral vector DNA or PDS407_pL-CMV-RFP-puro-CBX3 overexpression lentiviral vector was added to a sterilized centrifuge tube, along with 10 μg of the lentiviral packaging auxiliary vector pSPAX2 and 10 μg of the lentiviral packaging auxiliary vector pMD2.G. They were mixed evenly with the corresponding volume of Opti-MEM, and the total volume was adjusted to 2.5 mL and incubated at room temperature for 5 minutes. The Lipofectamine 2000 reagent was gently shaken, and 100 μL was taken and mixed with 2.4 mL of Opti-MEM in another tube and incubated at room temperature for 5 minutes. The above three diluted vectors were mixed with the diluted Lipofectamine 2000, gently inverted and mixed evenly, without shaking, and must be mixed within 5 minutes. After mixing, it was incubated at room temperature for 2 minutes to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution. The transfection complex of the three vectors and the Lipofectamine 2000 dilution was transferred to the culture medium containing 293T cells and mixed evenly, and cultured at 37 °C with 5% CO2. After continuing to culture for 8 h, the culture medium containing the transfection complex was poured out, and the residual transfection complex was washed with PBS solution and then poured out. 25 mL of cell medium containing 10% serum was added to each bottle of cells, and they were continued to be cultured in an incubator at 37 °C with 5% CO2 for 48 h, and the 293T cell supernatant was collected. Fresh complete medium was replaced and continued to be cultured until 72 h, and the 293T cell supernatant was collected again. The two collected 293T cell supernatants were centrifuged at 4 °C and 4000 g for 10 min to remove cell debris; the supernatant was filtered through a 0.45 μm filter into a 40 mL ultracentrifuge tube and centrifuged at 4000×g to the required virus concentration volume to obtain the virus concentrate of CBX3 knockdown lentivirus or CBX3 overexpression lentivirus. Its viral biological titer was measured, and the virus concentrate was stored at -80 °C for a long time. In the same way, the lentivirus constructed by replacing the FV055-CBX3 knockdown lentiviral vector and the PDS407_pL-CMV-RFP-puro-CBX3 overexpression lentiviral vector with the FV055 empty vector and the PDS407_pL-CMV-RFP-puro empty vector was used as the negative control virus.

[0055] 4. Infection of skin melanoma cell lines with CBX3 knockdown lentivirus and CBX3 overexpression lentivirus

[0056] Trypsinize the SKCM cells in the logarithmic growth phase to make the cell density about 5×10 4The suspension of cells / mL was inoculated into 6-well plates and cultured until the cell confluence reached about 50%.

[0057] Knockdown experiment: According to the multiplicity of infection (MOI) value, the MOI of the CBX3 knockdown lentivirus was 6.0, and the MOI of the negative control lentivirus was 2.5. Appropriate amounts of the CBX3 knockdown lentivirus were added to A375 and Mewo respectively. After culturing for 12 h, the medium was changed. After 48 h of infection, the expression of the reporter gene GFP (green fluorescent protein) on the lentivirus was observed, and the infection fluorescence rate was greater than 80%. In the control group, the CBX3 knockdown lentivirus was replaced with the negative control, and the lentivirus was experimented in the same way. The SKCM cells infected with the CBX3 knockdown lentivirus and the negative control virus were respectively named CBX3-sh and CBX3-nc (A375-sh / A375-nc, Mewo-sh / Mewo-nc).

[0058] Overexpression experiment: According to the multiplicity of infection (MOI) value, the MOI of the CBX3 overexpression lentivirus was 8.0, and the MOI of the negative control lentivirus was 3.0. An appropriate amount of the CBX3 overexpression lentivirus was added to A2058. After culturing for 12 h, the medium was changed. After 48 h of infection, the expression of the reporter gene RFP (red fluorescent protein) on the lentivirus was observed, and the infection fluorescence rate was greater than 80%. In the control group, the CBX3 overexpression lentivirus was replaced with the negative control lentivirus and experimented in the same way. The SKCM cells infected with the CBX3 overexpression lentivirus and the negative control lentivirus were respectively named CBX3-oe and CBX3-nc (A2058-oe / A2058-nc).

[0059] 5. Detection of CBX3 protein knockdown / overexpression by Western blot

[0060] Extract the CBX3-sh, CBX3-nc and CBX3-oe cell proteins; quantify the extracted proteins according to the BCA kit instructions; use Image J for quantitative analysis to record the protein expression differences in each group. The expression levels of CBX3 protein in the CBX3-sh, CBX3-nc and CBX3-oe groups of cells were as Figure 9 shown. It can be seen that the relative expression level of CBX3 protein in the CBX3-sh group was significantly down-regulated compared with the corresponding CBX3-nc group, and the relative expression level of CBX3 protein in the CBX3-oe group was significantly up-regulated compared with the corresponding CBX3-nc group, proving successful knockdown / overexpression of CBX3 protein.

[0061] 6. Effects of CBX3 knockdown / overexpression on the proliferation ability of cutaneous melanoma cells

[0062] CCK-8 assay: Prepare a 96-well plate. After digesting, centrifuging, and resuspending the SKCM cells in the logarithmic growth phase of CBX3-sh, CBX3-nc, CBX3-oe, and CBX3-nc, take 10 μL of the cell suspension and mix it with 10 μL of trypan blue stain. Take 10 μL of the mixed solution for counting and record the cell concentration. According to the cell growth status, 2 - 5×10 3 cells can be added to each well to keep the cell quantity in each group consistent, and the culture medium is supplemented to 100 μL. Set 27 replicate wells for each group of cells and culture them in an incubator at 37 °C and 5% CO2. At 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h of culture respectively, add 10 μL of CCK8 solution to three replicate wells. After incubating in the incubator for 2 h, use a Bio-Tek microplate reader to detect the absorbance at OD450, and calculate the proliferation effect according to the formula.

[0063] Colony formation assay: Gently pipette and mix the counted CBX3-sh, CBX3-nc, CBX3-oe, and CBX3-nc cells. Add 600 cells to each well in a 6-well plate, supplement the culture medium to 1 mL, and culture them in an incubator for 14 days, changing the medium every 2 - 3 days. Fix the cells with 4% paraformaldehyde and 0.5% crystal violet, take pictures with a camera, and count and analyze with Image J software.

[0064] According to the results of the CCK-8 assay and the colony formation assay, as Figures 10 - 11 shown, the proliferation rate of the cells in the CBX3-sh group is significantly lower than that in the CBX3-nc group, and the proliferation rate of the cells in the CBX3-oe group is significantly higher than that in the CBX3-nc group, proving that CBX3 has the ability to regulate the proliferation of SKCM cells, and knocking down CBX3 inhibits the proliferation of SKCM cells.

[0065] 7. Effect of CBX3 knockdown / overexpression on the migration ability of SKCM cells

[0066] Wound healing assay: Take the SKCM cells in the logarithmic growth phase of CBX3-sh, CBX3-nc, CBX3-oe, and CBX3-nc. After digestion and centrifugation, resuspend the cells with serum-free culture medium. Take 10 μL for trypan blue staining and then count. According to the cell growth status, add 2 - 5×10 3 cells to each well to keep the cell quantity in each group consistent, and supplement the culture medium to 1 mL. Culture in a 6-well plate in serum-free medium for 12 hours. Use a sterile pipette tip to create a scratch. Wash the cells with PBS and culture them in a medium supplemented with 2% fetal bovine serum. Obtain microscopic images of the designated area at 0 and 24 h of culture and perform statistical analysis.

[0067] Transwell assay: Adjust the cell quantity in each chamber to 3 - 8×10 5Cells were cultured in a 37 °C, 5% CO2 incubator for 24 - 72 h. Cultivation was terminated before the end of one cell growth cycle, and the culture medium was removed. The upper and lower layers of the chamber were washed with PBS, fixed with a fixative (4% paraformaldehyde or a methanol: glacial acetic acid mixture 1:3), and then washed with PBS. Crystal violet staining solution was added to both the upper and lower layers of the chamber to immerse the cells for staining. The cells that did not pass through the chamber pores on the upper layer of the chamber were wiped off, and the excess staining solution in the chamber was removed. After the chamber was air-dried or immersed in water, each group was photographed and recorded using a microscope, and subsequent statistical analysis was performed.

[0068] The results were as Figures 12 - 13 shown. The migration speed of cells in the CBX3 knockdown group decreased significantly, while the migration speed of cells in the CBX3 overexpression group increased. It can be seen that knockdown of CBX3 inhibits the migration of SKCM cells.

[0069] 8. Verification of the effect of CBX3 knockdown / overexpression on the proliferation of SKCM cells by subcutaneous xenograft tumor experiments in nude mice

[0070] Cells A375-sh, A375-nc, A2058-oe, and A2058-nc were subcutaneously injected into BALB / c nude mice (Hunan SJA Laboratory Animal Co., Ltd., Changsha, China) at a dose of 2×10 6 cells / mouse to establish a xenograft model. Monitoring was performed every 4 days, and the tumor volume was measured using a sliding caliper according to the formula (width 2 × length) / 2. Four weeks after implantation, the mice were euthanized, and the subcutaneous tumor tissues were surgically removed. The tumor diameters and weights in different groups were measured. The results were as Figure 14 shown. In animals implanted with A375 cells with CBX3-sh, the growth rate and tumor weight of melanoma were significantly lower than those of the control group. On the contrary, as Figure 15 shown, upregulating the expression of CBX3 in A2058 cells, both the tumor volume and weight were larger than those of the control group. Knockdown of CBX3 inhibits the growth of melanoma cells.

[0071] In the examples of the present invention, a lentiviral vector for CBX3 gene knockdown was constructed to efficiently infect SKCM cells and significantly downregulate the expression of the CBX3 gene in the latter, verifying at the cellular level that knockdown of CBX3 has the effect of inhibiting the proliferation and migration of melanoma cells. The present invention verified in an animal model of in vivo tumors that knockdown of CBX3 inhibits the proliferation of melanoma cells in vivo.

[0072] The above has introduced in detail the technical solutions provided by the embodiments of the present invention. Specific examples are used herein to elaborate on the principles and implementation manners of the embodiments of the present invention. The description of the above embodiments is only applicable to helping understand the principles of the embodiments of the present invention. At the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. Use of a CBX3 gene knockdown lentivirus targeting cutaneous melanoma in the preparation of a drug for treating cutaneous melanoma, characterized in that, The construction process of the CBX3 gene knockdown lentivirus is as follows: Synthesize the CBX3 interference target sequence, digest the BamHI / EcoRI sites of the lentiviral vector, and then insert the CBX3 interference target sequence into the digested linearized lentiviral vector to obtain the CBX3 knockdown lentiviral vector; Mix the CBX3 knockdown lentiviral vector, the lentiviral packaging auxiliary vector psPAX 2, and the lentiviral packaging auxiliary vector pMD2.G evenly with Opti-MEM and incubate to obtain the three diluted vectors; Mix the Lipofectamine 2000 reagent with Opti-MEM and incubate to obtain the diluted Lipofectamine 2000; Quickly mix the three diluted vectors with the diluted Lipofectamine 2000 to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution. Incubate the transfection complex with cells, and then culture the cells transfected with the CBX3 knockdown lentivirus, purify and concentrate the cell supernatant to obtain the CBX3 knockdown lentivirus concentrate; The forward sequence of the CBX3 interference target sequence is shown as SEQ ID NO.1, and the reverse sequence is shown as SEQ ID NO.

2.

2. The application of a CBX3 gene knockdown lentivirus targeting cutaneous melanoma according to claim 1 in the preparation of a drug for treating cutaneous melanoma, characterized in that: The A260 / A280 of the plasmid DNA extracted from the CBX3 knockdown lentiviral vector, the lentiviral packaging auxiliary vector psPAX 2, and the lentiviral packaging auxiliary vector pMD2.G are all between 1.8 and 2.0; The cells are human embryonic kidney cells 293T cells.

3. The application of a CBX3 gene knockdown lentivirus targeting cutaneous melanoma according to claim 1 in the preparation of a drug for treating cutaneous melanoma, characterized in that: During the construction of the CBX3 knockdown lentivirus, 24 h before transfection, the cells were digested, the cell density was adjusted, and the cells were cultured at 37 °C and 5% CO2 for 24 h. 2 h before transfection, the cell culture medium was replaced with serum-free medium. The CBX3 knockdown lentiviral vector, the lentiviral packaging helper vector pSPAX2, the lentiviral packaging helper vector pMD2.G, and Opti-MEM were mixed evenly and incubated at room temperature for 5 minutes; Lipofectamine 2000 reagent was gently shaken, taken and mixed with 2.4 mL of Opti-MEM, and incubated at room temperature for 5 minutes; the diluted three vectors were mixed with the diluted Lipofectamine 2000 within 5 minutes, and then incubated at room temperature for 2 minutes to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution; the transfection complex of the three vectors and the Lipofectamine 2000 dilution was transferred to the cell-containing culture medium and mixed evenly, washed after culturing at 37 °C and 5% CO2 for 8 h, the medium was added, and the cells were continued to be cultured in a 37 °C and 5% CO2 incubator. The cell supernatants were collected at 48 h and 72 h respectively, purified and concentrated to obtain a virus concentrate, which was stored at -80 °C for a long time.

4. The application of a CBX3 gene knockdown lentivirus targeting cutaneous melanoma according to claim 3 in the preparation of a drug for treating cutaneous melanoma, characterized in that: Twenty-four hours before transfection, human embryonic kidney cell line 293T cells in logarithmic growth phase were digested and the cell density was adjusted to 1.2×10 7 cells / 20 mL with a medium containing 10% serum, and then inoculated into cell culture dishes and cultured at 37 °C and 5% CO2. After 24 h, when the cell density reached 70% - 80%, the cells were ready for transfection. Two hours before transfection, the cell medium was replaced with serum-free medium. 10 μg of CBX3 knockdown lentiviral vector, 10 μg of lentiviral packaging helper vector pSPAX2, and 10 μg of lentiviral packaging helper vector pMD2.G were mixed evenly with the corresponding volume of Opti-MEM, and the total volume was adjusted to 2.5 mL and incubated at room temperature for 5 minutes. 100 μL of Lipofectamine2000 reagent was mixed with 2.4 mL of Opti-MEM and incubated at room temperature for 5 minutes. The diluted three vectors and the diluted Lipofectamine 2000 were gently inverted and mixed within 5 minutes without shaking. After mixing, it was incubated at room temperature for 2 minutes to form a transfection complex of the three vectors and Lipofectamine 2000 dilution. The transfection complex of the three vectors and Lipofectamine 2000 dilution was transferred to the culture medium containing 293T cells and mixed evenly. After culturing at 37 °C and 5% CO2 for 8 h, the culture medium containing the transfection complex was removed and the cells were washed. 25 mL of cell medium containing 10% serum was added to each bottle of cells and continued to be cultured in an incubator at 37 °C and 5% CO2 for 48 h. The supernatant of 293T cells was collected at 4 °C and centrifuged at 4000 g for 10 min to remove cell debris; The supernatant was filtered through a 0.45 μm filter into a 40 mL ultracentrifuge tube and centrifuged at 4000×g until the required virus concentration volume was reached; the CBX3 knockdown lentivirus in the filter cup was transferred to the sample collection cup to obtain a virus concentrate of the CBX3 knockdown lentivirus, and the virus concentrate was stored at -80 °C for a long time.

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