CN114569725A

CN114569725A - Application of YKL-40 in treatment of melanoma

Info

Publication number: CN114569725A
Application number: CN202210319185.7A
Authority: CN
Inventors: 王玉冰; 王艺; 石梦琪; 高志芹
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-06-03
Anticipated expiration: 2042-03-29
Also published as: CN114569725B

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to an application of YKL-40 in melanoma treatment. According to the invention, the treatment effect of the CHI3L1/YKL-40 gene and the expressed YKL-40 protein on a human melanoma model is evaluated through in vivo and in vitro experiments, and researches show that the CHI3L1/YKL-40 gene and the expressed YKL-40 protein have a good anti-melanoma effect. The technical scheme lays an experimental foundation for developing high-efficiency drugs related to treating melanoma and provides a new visual field, so that the technical scheme has good practical application value.

Description

Application of YKL-40 in treatment of melanoma

Technical Field

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of YKL-40 in melanoma treatment.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Melanoma is a malignant tumor with strong proliferation capacity and high invasion and metastasis rate, and clinically, lymph node, brain, liver and lung metastasis frequently occurs. Melanoma causes 65% -75% of skin cancer deaths, and metastatic melanoma patients have 5% -10% of 5-year survival rates.

Currently, the treatment for melanoma is mainly surgical resection, and radiotherapy and chemotherapy are used for prolonging the survival time of patients after surgery, in recent years, immunotherapy and targeted therapy have a certain effect, but for melanoma patients, the risk of cancer recurrence after treatment is high, and the combination of an immune checkpoint inhibitor and a signal transduction inhibitor can reduce the recurrence risk to a certain extent, so that better therapeutic targets and targeted drugs are still needed for clinical treatment of melanoma.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of YKL-40 in the treatment of melanoma. According to the invention, the study finds that the CHI3L1/YKL-40 gene can be used as a target for resisting melanoma, and further, the YKL-40 protein expressed by the gene has the effect of inhibiting the proliferation, invasion and metastasis of melanoma.

Specifically, the invention relates to the following technical scheme:

according to the invention, researches show that YKL-40 can inhibit the proliferation and invasion of melanoma cells and increase the infiltration of immune cells through MAPK and NF-kB signal pathways, thereby inhibiting the proliferation in melanoma cells and increasing the sensitivity of the melanoma cells to drugs. Therefore, in a first aspect of the present invention, there is provided a use of a substance for promoting improvement of at least one of CHI3L1/YKL-40 gene and its expression product and/or activity in at least one of the following a1) -a 5):

a1) inhibiting the proliferation of tumor cells or preparing products for inhibiting the proliferation of tumor cells;

a2) inhibiting the migration and invasion of tumor cells or preparing products for inhibiting the migration and invasion of tumor cells;

a3) inhibiting tumor growth or preparing a product for inhibiting tumor growth;

a4) promoting the sensitivity of the tumor to the therapeutic drugs or preparing products for promoting the sensitivity of the tumor to the therapeutic drugs;

a5) treating tumor or preparing tumor treating product.

In a second aspect of the present invention, there is provided a use of a substance for inhibiting a decrease in at least one of CHI3L1/YKL-40 gene and an expression product and/or activity thereof in at least one of b1) -b5) below:

b1) promoting proliferation of tumor cells or preparing products for promoting proliferation of tumor cells;

b2) promoting the migration and invasion of tumor cells or preparing products for promoting the migration and invasion of tumor cells;

b3) promoting tumor growth or preparing products for promoting tumor growth;

b4) inhibiting the sensitivity of the tumor to the therapeutic drugs or preparing products for inhibiting the sensitivity of the tumor to the therapeutic drugs;

b5) constructing tumor cells or animal models.

In a third aspect of the present invention, a composition is provided, wherein the active ingredients of the composition at least comprise a substance promoting the improvement of the CHI3L1/YKL-40 gene and its expression product and/or activity, and at least one other chemotherapeutic drug.

In a fourth aspect of the invention, there is provided the use of a composition as described above in any one or more of:

a3) inhibiting tumor growth or preparing a product for inhibiting tumor growth;

a5) treating tumor or preparing tumor treating product.

The tumor may be melanoma.

The product may be a pharmaceutical or experimental reagent that is effective for basic research.

In a fifth aspect of the present invention, there is provided a method for treating melanoma, which comprises administering to a subject a therapeutically effective amount of the above-mentioned substance promoting improvement of CHI3L1/YKL-40 gene and its expression product and/or activity or the above-mentioned composition.

The beneficial technical effects of one or more technical schemes are as follows:

according to the technical scheme, the treatment effect of the CHI3L1/YKL-40 gene and the expressed YKL-40 protein on a human melanoma cell model and a melanoma animal model is evaluated through in vivo and in vitro experiments, and researches show that the CHI3L1/YKL-40 gene and the expressed YKL-40 protein have a good anti-melanoma effect; the technical scheme lays an experimental foundation for developing high-efficiency drugs related to treating melanoma and provides a new visual field, so that the technical scheme has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows the results of the protein expression verification in human melanoma cells A375 and murine melanoma cells B16-F10 overexpressing YKL-40 used in the present invention; wherein A is human melanoma cells A375, and B is murine melanoma cells B16-F10;

FIG. 2 shows the cell proliferation assay (MTS) and cell clonogenic assay of the present invention to detect the proliferation inhibitory effect of YKL-40 on melanoma cells A375 and B16-F10; wherein A is the statistical result of A375 cell proliferation experiments, B is the experimental result of A375 and B16-F10 cell clonogenic experiments, C is the statistical result of A375 cell clonogenic experiments, and D is the statistical result of B16-F10 cell clonogenic experiments; **: p < 0.01; ***: p < 0.001.

FIG. 3 is a graph showing that YKL-40 inhibits the migration of melanoma cells A375 and B16-F10 according to the present invention using a cell scratch assay; wherein A is the A375 cell scratch experiment result, B is the A375 cell scratch experiment statistical result, C is the B16-F10 cell scratch experiment result, D is the B16-F10 cell scratch experiment statistical result, x: p < 0.01; ***: p < 0.001.

FIG. 4 shows that YKL-40 inhibits the invasion of melanoma cells A375 and B16-F10 according to the invention by using a Transwell invasion assay; wherein, A is the result of the A375 cell Transwell invasion experiment, B is the statistical result of the A375 cell Transwell invasion experiment, and the ratio of A: p < 0.01.

FIG. 5 is a flow cytometry experiment of the present invention to detect that YKL-40 increases the sensitivity of melanoma cells A375 and B16-F10 to dacarbazine; wherein, A is the apoptosis experiment result of flow cytometry detection A375 and B16-F10 when dacarbazine stimulates, B is the statistical result of flow cytometry detection A375, and C is the statistical result of flow cytometry detection B16-F10, x: p < 0.01.

FIG. 6 is a graph showing that YKL-40 increases the sensitivity of melanoma cells A375 and B16-F10 to metrinib by the cell resistance assay (MTS) according to the present invention; wherein, A is the statistical result of the A375 cell MTS experiment, B is the statistical result of the B16-F10 cell MTS experiment, and the following components are adopted: p < 0.05.

FIG. 7 shows an in vivo subcutaneous tumor-bearing model of the present invention, which verifies the ability of YKL-40 to inhibit the tumorigenesis of melanoma cells B16-F10 in C57BL/6 mice; wherein, A is a comparative graph of the results of subcutaneous tumors formed in mice by B16-F10 melanoma cells over-expressing YKL-40 and control cells, B is the change of the subcutaneous tumor volume with time, C is the weighing result of tumor tissues, and the weight ratio of the tumor tissues is as follows: p < 0.05.

FIG. 8 shows an in vivo lung metastasis model of the present invention, which verifies that YKL-40 inhibits lung metastasis ability of melanoma cells B16-F10 in a C57BL/6 mouse; wherein, A is a graph comparing the lung metastasis results of melanoma of B16-F10 cells over-expressing YKL-40 with cells of a control group, and B is the counting statistical result of the number of nodes of lung metastasis, which is: p < 0.05.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. Unless the experimental methods of specific conditions are specified in the following detailed description, the conventional methods and conditions of molecular biology within the skill of the art are generally followed, and such techniques and conditions are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; reagents, consumables, cells, laboratory animals, and the like used in the examples are commercially available unless otherwise specified.

As mentioned above, there is still a need for better therapeutic targets and targeted drugs for the clinical treatment of melanoma.

In view of the above, the research of the invention finds that YKL-40 can inhibit the proliferation and invasion of melanoma cells and increase the infiltration of immune cells through MAPK and NF-kB signal pathways, thereby inhibiting the proliferation of melanoma cells in vivo and increasing the sensitivity of the melanoma cells to drugs.

In a typical embodiment of the present invention, there is provided a use of a substance for promoting improvement of at least one of CHI3L1/YKL-40 gene and its expression product and/or activity in at least one of the following a1) -a 5):

a3) inhibiting tumor growth or preparing a product for inhibiting tumor growth;

a5) treating tumor or preparing tumor treating product.

In another embodiment of the invention, the substances for promoting the improvement of the expression product and/or activity of the CHI3L1/YKL-40 gene include, but are not limited to, substances for up-regulating the expression and/or activity of CHI3L1/YKL-40 based on gene-specific Mimics technology; such as short hairpin RNA (shRNA) artificially synthesized from CHI3L1/YKL-40, or promoter or lentivirus for up-regulating the expression of CHI3L 1/YKL-40; compound accelerators are also included; the compound promoter can be used for activating the expression of YKL-40 protein.

In another embodiment of the invention, the sequence of the CHI3L1/YKL-40 gene is shown in SEQ ID NO. 1; the expression product of the CHI3L1/YKL-40 gene can be YKL-40 protein, and the amino acid sequence of the expression product is shown as SEQ ID NO. 2.

In yet another embodiment of the present invention, the tumor may be melanoma.

In still another embodiment of the present invention, there is provided a use of a substance for inhibiting a decrease in at least one of the CHI3L1/YKL-40 gene and an expression product and/or activity thereof in at least one of b1) -b5) below:

b3) promoting tumor growth or preparing products for promoting tumor growth;

b5) and (3) constructing tumor cells or animal models.

In still another embodiment of the present invention, the substance for inhibiting the CHI3L1/YKL-40 gene and the expression product and/or activity reduction thereof includes, but is not limited to, RNA interference molecules or antisense oligonucleotides against CHI3L1/YKL-40, small molecule inhibitors, shRNA (small hairpin RNA), small interfering RNA, substances for performing lentiviral infection or gene knock-out, and specific antibodies against YKL-40 itself or molecules upstream and downstream thereof, such as anti-YKL-40 antibody.

In yet another embodiment of the present invention, the tumor may be melanoma.

In still another embodiment of the present invention, there is provided a composition comprising as active ingredients at least a substance promoting the improvement of CHI3L1/YKL-40 gene and its expression product and/or activity, and at least one other chemotherapeutic agent.

In another embodiment of the present invention, the substance promoting the improvement of the expression and/or activity of the CHI3L1/YKL-40 gene comprises but is not limited to a substance that uses gene-specific Mimics technology to up-regulate the expression and/or activity of CHI3L 1/YKL-40; such as short hairpin RNA (shRNA) artificially synthesized from CHI3L1/YKL-40, or promoter or lentivirus for up-regulating the expression of CHI3L 1/YKL-40; compound accelerators are also included; the compound promoter can be used for activating the expression of YKL-40 protein.

In another embodiment of the present invention, the chemotherapeutic agent is not specifically limited, and in one embodiment of the present invention, the chemotherapeutic agent may be one or more BRAF inhibitors such as bimatonil and one or more common chemotherapeutic agents such as dacarbazine.

In a further embodiment of the invention, there is provided the use of the above composition in any one or more of:

a3) inhibiting tumor growth or preparing a product for inhibiting tumor growth;

a5) treating tumor or preparing tumor treating product.

In yet another embodiment of the present invention, the tumor may be melanoma.

In yet another embodiment of the invention, the product may be a pharmaceutical or a test agent that is effective for basic research.

According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.

The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In still another embodiment of the present invention, the subject to which the medicament is administered may be a human or non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, orangutan, or the like.

In still another embodiment of the present invention, there is provided a method for treating melanoma, which comprises administering to a subject a therapeutically effective amount of the above-mentioned substance promoting the improvement of CHI3L1/YKL-40 gene and its expression product and/or activity or the above-mentioned composition.

In yet another embodiment of the present invention, the melanoma treatment may be any one or more of immunotherapy, gene therapy, and inhibitory antibody therapy.

In yet another embodiment of the present invention, the subject is an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment. By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It will be appreciated that the optimum dosage and interval for administration of the active ingredients of the invention will be determined by the nature and external conditions, such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLE 1 establishment of cell line stably overexpressing YKL-40 protein

Culturing A375 cells and B16-F10 cells in DMEM complete culture solution containing 10% fetal calf serum and 1% penicillin-streptomycin, culturing in a constant-temperature incubator at 37 ℃ and 5% CO2 and saturated humidity, after the monolayer cells are full, digesting with 1mL of 0.25% trypsin (containing EDTA) until the cell connection is broken to be circular, adding the complete culture solution to stop digestion, resuspending the cells, and continuing to culture by passage at the ratio of 1: 3.

Constructing a DNA fragment of a YKL-40 gene sequence (the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2), carrying out BamHI and EcoRI double enzyme digestion on a vector pBABE and a target fragment, connecting the enzyme digestion product to a p-BABE recombinant plasmid through T4 ligase, transforming DH-5 alpha competent cells through the connection product, coating the cells on an ampicillin screening plate, incubating the cells at 37 ℃ for 14 hours in a incubator, picking out positive clones, and extracting the plasmids according to the method of the specification of a plasmid extraction kit. After the plasmid is successfully extracted, transfecting 293T cells by using a Lipofectamine2000 liposome transfection method, collecting virus liquid after 72 hours, centrifuging, taking supernatant and DMEM complete culture medium according to the proportion of 3:4, adding the supernatant and the DMEM complete culture medium into the adherent cells, changing the culture medium into a culture medium containing 0.5ug/ml of Puromycin for screening after 24 hours, and finally continuously subculturing the obtained cells. The stable overexpression of YKL-40 is verified by Western blot, and the result is shown in figure 1.

Example 2 inhibition of proliferation of melanoma cell models by YKL-40

(1) Cell proliferation assay

The A375 cells over expressing YKL-40 and the control cells in the logarithmic growth phase were treated at 1X 10⁴Inoculating each well into a 96-well plate, culturing in a constant-temperature incubator at 37 ℃ for 24h, adding drugs, continuously culturing for 72h, removing the culture medium in the well, mixing PMS, MTS and DMEM colorless culture medium according to the ratio of 1:20:100 to prepare MTS working solution, adding 100 mu of LMTS working solution into each well, incubating at 37 ℃ for 10min, uniformly mixing, and measuring OD (optical density) by using a microplate reader_490nmAbsorbance (A) value.

As a result, as shown in fig. 2(a), the proliferation capacity of a375 cells over-expressing YKL-40 was significantly lower than that of the control cells.

(2) Cell clone formation assay

Respectively inoculating 200 cells/dish of A375 cells and B16-F10 cells which over-express YKL-40 and cells of a control group, changing the solution every three days, culturing for two weeks, sucking off the culture medium, washing with PBS twice, fixing PFA for 15min, washing with PBS twice, staining with 0.1% crystal violet for 15min, washing with PBS until the background is colorless, and photographing to calculate the average area of formed clones.

The results are shown in fig. 2(b.c), and the average clone area formed by a375 cells and B16-F10 cells over-expressing YKL-40 is significantly reduced compared to the control group (P < 0.01). YKL-40 inhibited clonogenic formation of A375 and B16-F10 cells.

Example 3 inhibition of migration and invasion of melanoma cell models by YKL-40

(1) Cell scratch test

Seeding cells in six well cell culture plates 3X 10⁶And (2) sucking out the culture medium in a DMEM culture solution until the cell fusion rate exceeds 80%, adding PBS, drawing two parallel straight lines in each hole by using a 200ul pipette tip, washing cell fragments by using the PBS, adding the culture medium containing 1% serum, taking pictures at fixed time points, and recording the healing condition of scratches.

As shown in FIG. 3, the scratch healing rate was significantly slowed in the A375 cells (P <0.001) and B16-F10 cells (P <0.01) that over-expressed YKL-40. YKL-40 inhibited the migration of A375 and B16-F10 cells.

(2) Transwell cell migration experiment

Taking B16-F10 cells in logarithmic growth phase at 1X 10⁶Density of cells/well was added to atrigel Matrigel coated Transwell chamber (upper chamber), then each set of upper chambers was put into a well of cell culture plate containing DMEM culture solution (lower chamber), taken out after culturing for 36h, cells on Matrigel and membrane surface were gently wiped off with a wet cotton swab, fixed with paraformaldehyde, disrupted with 0.2% Triton, DAPI stained, washed with PBS, and counted in 5 fields under a fluorescence microscope.

The results are shown in fig. 4, the number of B16-F10 cells overexpressing YKL-40 invading metastatic cells was significantly reduced (P < 0.01). YKL-40 inhibited the invasion of B16 cells.

Example 4YKL-40 increases the sensitivity of melanoma cells to drugs

(1) Apoptosis assay

Taking cells in logarithmic growth phase, and culturing at 3 × 10⁵And inoculating the cells/well density into a six-well cell culture plate, treating the cells for 24h by 150uM Dacarbazine, collecting the cells, washing the cells twice by PBS, resuspending the cells, adding 5 mu L of Annexin V-FITC and 10 mu L of PI, incubating the cells for 15min in a dark place, and detecting the apoptosis rate of each group of cells by using a flow cytometer.

The results are shown in FIG. 5, where the apoptosis of cells overexpressing YKL-40 was more pronounced upon dacarbazine stimulation. YKL-40 increases the sensitivity of melanoma cells to dacarbazine.

(2) Cell resistance assay (MTS)

Taking cells in logarithmic growth phase at 1 × 10⁴The density of each well was seeded in 96-well cell culture plates at a Binimetinib concentration of 0.3125 μm, control groups were supplemented with equal amounts of DMSO, blank groups were wells without cells and drug, and 3 duplicate wells were set for each group. After 72h of culture, the medium was discarded, 100. mu.L of MTS working solution was added to each well, incubation was carried out at 37 ℃ for 10min, and the optical density (D) value at 490nm was measured with a microplate reader.

Cell activity (%). gtoreq (experimental D-blank D)/(control D-blank D) × 100%

The results are shown in fig. 6, where cells overexpressing YKL-40 show a significant decrease in cell viability upon stimulation with bimatinib (P < 0.05). YKL-40 increased the sensitivity of melanoma cells to bimatinib.

Example 5YKL-40 inhibits melanoma cells from tumorigenesis and metastasis in animal models

(1) Establishment of melanoma subcutaneous tumor-bearing mouse model

B16-F10 cells over-expressing YKL-40 in logarithmic growth phase and control B16-F10 cells were taken and prepared in cold physiological saline at a concentration of 5X 10⁵Cell suspension per mL. Injections were made outside the hind limb of six week old wild type C57BL/6 male mice, with each mouse injected with 0.2mL of cell suspension. Tumor volumes were measured every other day from day 13 and tumors were weighed out by sacrifice on day 19.

The results are shown in FIG. 7, the tumor formation rate and volume of B16-F10 cells over-expressing YKL-40 in mice are obviously different from those of a control group, and YKL-40 can inhibit the tumor formation capability of melanoma cells in mice.

(2) Melanoma lung metastasis mouse model establishment

B16-F10 cells over-expressing YKL-40 in logarithmic growth phase and control group B16-F10 cells were prepared in cold physiological saline at a concentration of 4X 10⁵Cell suspension per mL. Six weeks old wild type C57BL/6 female mice were injected rat vein with 0.5mL cell suspension per mouse. Lungs were dissected out by sacrifice on day 15 and lung melanoma nodules were counted.

The results are shown in FIG. 8, the number of tumors formed by the B16-F10 cells which over-express YKL-40 and metastasize to the lung in mice is obviously reduced compared with the cells in the control group, and YKL-40 can inhibit the metastasizing capability of melanoma cells in mice.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Weifang medical college

Application of <120> YKL-40 in melanoma treatment

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Val Gly Tyr Met Leu Arg Leu Gly Ala Pro Ala Ser Lys Leu Val Met

245 250 255

Gly Ile Pro Thr Phe Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr

260 265 270

Gly Val Gly Ala Pro Ile Ser Gly Pro Gly Ile Pro Gly Arg Phe Thr

275 280 285

Lys Glu Ala Gly Thr Leu Ala Tyr Tyr Glu Ile Cys Asp Phe Leu Arg

290 295 300

Gly Ala Thr Val His Arg Ile Leu Gly Gln Gln Val Pro Tyr Ala Thr

305 310 315 320

Lys Gly Asn Gln Trp Val Gly Tyr Asp Asp Gln Glu Ser Val Lys Ser

325 330 335

Lys Val Gln Tyr Leu Lys Asp Arg Gln Leu Ala Gly Ala Met Val Trp

340 345 350

Ala Leu Asp Leu Asp Asp Phe Gln Gly Ser Phe Cys Gly Gln Asp Leu

355 360 365

Arg Phe Pro Leu Thr Asn Ala Ile Lys Asp Ala Leu Ala Ala Thr

370 375 380

Claims

1. Application of substances for promoting improvement of CHI3L1/YKL-40 gene and expression products and/or activity thereof in at least one of the following a1) -a 5):

a3) inhibiting tumor growth or preparing a product for inhibiting tumor growth;

a5) treating tumor or preparing tumor treating product.

2. The use according to claim 1, wherein the substance promoting the improvement of the expression and/or activity of the CHI3L1/YKL-40 gene comprises a substance up-regulating the expression and/or activity of CHI3L1/YKL-40 based on gene-specific mics technology; further comprises short hairpin RNA artificially synthesized from CHI3L1/YKL-40 or a promoter or a lentivirus for up-regulating the expression of CHI3L 1/YKL-40; compound accelerators are also included; the compound promoter is used for activating the expression of YKL-40 protein.

3. The use according to claim 1, wherein the CHI3L1/YKL-40 gene sequence is as shown in SEQ ID No. 1; the expression product of the CHI3L1/YKL-40 gene is YKL-40 protein, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.

4. The use of claim 1, wherein the tumor is melanoma;

the product is a drug or an experimental reagent for basic research.

5. The application of substances for inhibiting CHI3L1/YKL-40 gene and expression products and/or activity reduction thereof in at least one of the following b1) -b 5):

b1) promoting tumor cell proliferation or preparing products for promoting tumor cell proliferation;

b3) promoting tumor growth or preparing products for promoting tumor growth;

b5) constructing tumor cells or animal models.

6. The use according to claim 5, wherein the substance inhibiting the CHI3L1/YKL-40 gene and its expression products and/or reduction in activity comprises RNA interfering molecules or antisense oligonucleotides against CHI3L1/YKL-40, small molecule inhibitors, shRNA, small interfering RNA, substances performing lentiviral infection or gene knock-out, and specific antibodies against YKL-40 itself or molecules upstream and downstream thereof, including anti-YKL-40 antibodies;

the CHI3L1/YKL-40 gene sequence is shown in SEQ ID NO. 1; the expression product of the CHI3L1/YKL-40 gene is YKL-40 protein, and the amino acid sequence of the protein is shown as SEQ ID NO. 2;

the tumor is melanoma.

7. A composition, characterized in that the active ingredients of the composition at least comprise a substance promoting the improvement of CHI3L1/YKL-40 gene and its expression product and/or activity, and at least one other chemotherapeutic drug.

8. The composition according to claim 7, wherein the substance promoting the improvement of the expression and/or activity of the CHI3L1/YKL-40 gene comprises a substance up-regulating the expression and/or activity of CHI3L1/YKL-40 based on gene-specific mics technology; such as artificially synthesized short hairpin RNA of CHI3L1/YKL-40 or a promoter or lentivirus for up-regulating the expression of CHI3L 1/YKL-40; compound accelerators are also included; the compound accelerator is used for activating the expression of YKL-40 protein;

preferably, the chemotherapeutic agent is a BRAF inhibitor, including bimatonib; or dacarbazine.

9. Use of a composition according to claim 7 or 8 in any one or more of:

a3) inhibiting tumor growth or preparing a product for inhibiting tumor growth;

a5) treating tumors or preparing products for treating tumors;

wherein the tumor is melanoma;

preferably, the product is a medicament or an experimental reagent, and the experimental reagent is used for basic research;

it is further preferred that when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.

10. A method for treating melanoma, which comprises administering to a subject a therapeutically effective amount of a substance promoting an increase in CHI3L1/YKL-40 gene and expression products and/or activity thereof or a composition according to claim 7 or 8.