CN113577283A

CN113577283A - Application of Siglec-15 inhibitor in preparation of medicines for preventing and treating giant cell tumor of bone

Info

Publication number: CN113577283A
Application number: CN202110815412.0A
Authority: CN
Inventors: 张奇; 王玲; 康富标; 陈伟; 齐莉莉
Original assignee: Third Hospital of Hebei Medical University
Current assignee: Third Hospital of Hebei Medical University
Priority date: 2021-07-19
Filing date: 2021-07-19
Publication date: 2021-11-02

Abstract

The invention provides an application of a Siglec-15 inhibitor in preparation of a medicine for preventing and treating giant cell tumor of bones. The inhibitor is a substance for inhibiting the expression and/or function of Siglec-15. The inhibitor is an antibody against Siglec-15 or an interfering RNA against the Siglec-15 gene. The invention also provides a pharmaceutical composition for preventing and treating giant cell tumor of bone. In-vitro cell experiments prove that the ability of Hs737.T cells to proliferate, clone, invade and migrate can be obviously reduced by silencing the Siglec-15 gene. The invention provides a new idea and scheme for the clinical treatment of GCTB, and the Siglec-15 inhibitor provided by the invention can effectively down-regulate the expression of Siglec-15, thereby playing a role in negatively regulating the functions of proliferation, migration, invasion and the like of tumor cells.

Description

Application of Siglec-15 inhibitor in preparation of medicines for preventing and treating giant cell tumor of bone

Technical Field

The invention relates to the technical field of tumor treatment, in particular to application of a Siglec-15 inhibitor in preparation of a medicine for preventing and treating giant cell tumor of a bone.

Background

Giant Cell Tumor of Bone (GCTB) is a potential malignancy that occurs well in the knee joint, often manifested as locally aggressive osteolysis. In asia, it accounts for approximately 5% of primary bone tumors, and around 20% of benign tumors. Like most bone tumors, GCTB occurs well in young and strong years, and most patients are between 20 and 40 years of age. For GCTB, surgical resection is the main treatment mode, but the postoperative recurrence rate is as high as 25-50%. At present, phenol, methyl methacrylate and the like are often used as auxiliary agents in surgical treatment, and have a certain inhibiting effect on tumor cells, but the influence on the postoperative recurrence rate is not large. Dinomizumab is a targeted inhibitor of a receptor activator of nuclear factor kappa B (RANK) pathway, and effectively slows down the osteolytic reaction of GCTB by preventing the formation and activation of osteoclasts. However, some patients often have serious adverse reactions, such as hypophosphatemia and jaw necrosis. Therefore, it is important to actively explore new therapeutic strategies to improve the prognosis of GCTB patients.

Sialic Acid binding Immunoglobulin-like Lectins (Siglecs) are a family of specific membrane receptors that modulate innate and adaptive immune functions by recognizing glycan ligands, and this family plays a regulatory role in a variety of disease states in the body, such as infection, neuropathy, autoimmune disease, tumors, and the like. Siglec-15, also known as CD33L3/HsT1361, maps to chromosome 18q 12.3. The Siglec-15 gene is over-expressed in various cancer tissues such as bladder cancer, kidney cancer, lung cancer, liver cancer and the like. Siglec-15 was positive for 90% of tumor cell expression in tissue samples from human non-small cell lung cancer patients. Whereas Siglec-15 expression was higher in epidermal growth factor receptor mutant lung cancer. It has been reported that Siglec-15 expressed on myeloid cells/macrophages can inhibit the activity of T cells. Furthermore, in a mouse tumor model, knockout of Siglec-15 expression resulted in retarded tumor growth and prolonged mouse survival. Therefore, Siglec-15 expression plays a very important role in cancer progression and can be considered as a novel potential anti-tumor target.

No report is available on whether Siglec-15 is expressed in GCTB tissues and whether the Siglec-15 is involved in the biological behavior of tumor cells. Therefore, our research objective was to verify that Siglec-15 plays a key role in the progression of GCTB, and its expression in relation to clinical information data. Since GCTB mononuclear stromal cells are tumor components, the influence of Siglec-15 on the biological properties of GCTB mononuclear stromal cells is further discussed, so that a new scheme is provided for the treatment of GCTB.

Disclosure of Invention

The invention aims to provide application of a Siglec-15 inhibitor in preparation of a medicine for preventing and treating giant cell tumor of bone, so as to solve the problem that the medicine for treating giant cell tumor of bone in the prior art is easy to cause serious adverse reaction of a patient, and provide a new scheme for treating GCTB.

The purpose of the invention is realized as follows:

application of the Siglec-15 inhibitor in preparing medicines for preventing and treating giant cell tumor of bone.

The inhibitor is a substance for inhibiting the expression and/or function of Siglec-15.

The inhibitor is an antibody against Siglec-15 or an interfering RNA against the Siglec-15 gene.

The inhibitor is a monoclonal antibody against Siglec-15.

The inhibitor is siRNA selected from the following sequences: SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

A pharmaceutical composition for preventing and treating giant cell tumor of bone comprises a Siglec-15 inhibitor.

In the pharmaceutical composition, the inhibitor is an antibody against Siglec-15 or an interfering RNA against the Siglec-15 gene.

In the pharmaceutical composition, the inhibitor is siRNA selected from the following sequences: SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

The pharmaceutical composition further comprises:

a pharmaceutically acceptable carrier and/or adjuvant;

other active ingredients for preventing or treating giant cell tumor of bone.

The other active ingredients include: chemotherapeutic agents, radiotherapeutic agents or other drugs having the function of down-regulating the expression of Siglec-15.

Histologically, GCTB is composed of osteoclast-like multinucleated giant cells, mononuclear stromal cells, which are tumor cells of GCTB, and monocytes. The malignancy of the mononuclear stromal cells is closely related to the invasion, recurrence and metastasis of GCTB. Siglec-15 is expressed in human GCTB tissue mononuclear stromal cells and Hs737.T cells, which are cells closely related to tumor progression, and the invention has proved that the expression of Siglec-15 in human GCTB tissue is related to tumor progression and recurrence.

In-vitro cell experiments prove that the ability of Hs737.T cells to proliferate, clone, invade and migrate can be obviously reduced by silencing the Siglec-15 gene. The invention provides a new idea and scheme for the clinical treatment of GCTB, and the Siglec-15 inhibitor provided by the invention can effectively down-regulate the expression of Siglec-15, thereby playing a role in negatively regulating the functions of proliferation, migration, invasion and the like of tumor cells.

Drawings

FIG. 1 is the expression of Siglec-15 in GCTB tissues and mononuclear stromal cells. Wherein A is Siglec-15 protein immunohistochemical staining in GCTB tissue (I: control; II: +; III: + +; IV: + + +) (400X); b is Hs737. immunofluorescent staining for Siglec-15 protein in T cells (I: Siglec-15 protein expression; II: DAPI; III: Merge) (400X).

FIG. 2 shows the transfection state by fluorescence microscopy.

FIG. 3 is RT-PCR assay of silencing efficiency of Siglec-15-siRNA sequence (P < 0.001).

FIG. 4 is a graph showing the result of Western blot assay. Wherein, A is Western blot to detect protein expression of Siglec-15 in siRNA transfected Hs737.T cells; b is the gray level of protein measured by Image J software.

FIG. 5 is the effect of silencing Siglec-15 on the proliferative capacity of GCTB mononuclear stromal cells (P < 0.05).

FIG. 6 is a graph of the effect of silencing Siglec-15 on the clonality of GCTB mononuclear stromal cells. A is a picture of Hs737.T cells shown in a cloning experiment, the left is an NC group, and the right is a Siglec-15-siRNA group; b is a statistical result chart, which shows that the number of T cell clones in Siglec-15-siRNA group Hs737. is obviously less than that in NC group (P < 0.001).

Fig. 7 is a graph showing the results of the scratch test.

FIG. 8 is a statistical plot of the area remaining for scratches within the images obtained by Image J software (P <0.05and P < 0.001).

FIG. 9 is a graph of the effect of silencing Siglec-15 on the invasive potential of GCTB mononuclear stromal cells. Wherein, A is a migration ability determination graph, and the migration ability of the Hs737.T cells transfected with Siglec-15siRNA is reduced compared with negative control cells (100 x) at 48 h; b is a migration capacity statistical chart, and compared with negative control cells, the number of migrated cells is reduced after Siglec-15-siRNA transfection (P < 0.001); c is an invasion capacity determination chart, and Hs737.T cells transfected with Siglec-15siRNA have reduced invasion capacity compared with negative control cells (100 x) at 24h and 48 h; d is a statistical map of invasion capacity, and compared with negative control cells, the invasion cells are reduced when Siglec-15-siRNA is transfected for 48h (P <0.05and P < 0.01).

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific examples. The test conditions and procedures not mentioned in the examples of the present invention were carried out according to the conventional methods in the art or the conditions suggested by the manufacturer.

In the implementation of the invention, the immunohistochemical secondary antibody (goat anti-rabbit IgG) is purchased from China fir Jinqiao biology in Beijing; western Blot secondary antibody (goat anti-rabbit IgG) and fluorescent secondary antibody (goat anti-rabbit IgG) were purchased from Boaosen organisms (Beijing, China); the GAPDH primary antibody, Siglec-15 antibody, was purchased from Abcam, UK. The other reagents are all common commercial reagents.

The study of the present invention has been approved by the ethical committee of my hospital. Clinical data and tumor specimen pathological sections of giant cell tumor patients treated by pathologists department of third hospital of Hebei medical university from 2012 1 month 1 to 2017 12 months 31 are collected. Patients who were in pregnancy, those who were in lactation, those who had other benign and malignant tumors, and those who had severe cardiovascular and renal diseases were excluded and finally included in 56 patients. All section tissue specimens have complete clinical information data, are verified to be giant cell tumor tissues of bones through strict clinical, imaging and pathological staining, and are subjected to anacci's staging. Clinical data were collected from patients including age, sex, tumor localization, tumor size, and tumor recurrence, etc. (see table 1 for details).

Primary human bone cytomegalovirus mononuclear stromal cells (Hs737.T) were purchased from American Type Culture Collection, USA. High-glucose DMEM medium containing 15% FBS and 1% penicillin/streptomycin in 5% CO₂And culturing at 37 ℃.

Example 1: immunohistochemical staining

1) Before operation, the slices are placed into a preheated slice baking machine, and the slices are baked for 30min at 65 ℃ to ensure that the paraffin is in tear-drop flow, so as to prevent slice detachment.

2) Soaking the slices in transparent liquid I (containing alkane compound as main component) for 20 min; soaking transparent liquid II (containing isoparaffin compound as main ingredient) for 10 min; soaking in 100% ethanol for 2 times for 10min respectively; soaking in 95%, 90%, 80%, 70% ethanol and distilled water for 10min respectively; this was completely dewaxed and then gently rinsed 3 times for 3 min/time with PBS.

3) After sucking residual PBS, the slices are soaked in 0.1mol/L citrate buffer solution prepared in advance, placed in a water bath pot and heated to 98 ℃, kept for about 15min, cooled for 1h at room temperature, and rinsed by PBS for 3 times and 3 min/time gently.

4) After blotting residual PBS, the sliced tissue was incubated with 3% hydrogen peroxide in deionized water at room temperature for about 12min, and then gently rinsed 3 times with PBS for 3 min/time.

5) After the residual PBS was aspirated, goat serum was slowly added dropwise to block non-specific proteins and kept in a 37 ℃ water bath for 15 min.

6) The blocking solution was decanted off, and then the Siglec-15 antibody (primary antibody) was added at a ratio of 1:200 and refrigerated overnight in a refrigerator at 4 ℃.

7) The next day, gently wash 3 times with PBS for 3 min/time. After the residual PBS was aspirated, the tissue surface was confluent with goat anti-rabbit IgG (secondary antibody) containing horseradish peroxidase (HRP), maintained at 37 ℃ for 10min, and gently rinsed 3 times with PBS 3 min/time.

8) After the residual PBS was aspirated, the tissue sections containing HRP-labeled streptavidin working solution were incubated in a 37 ℃ water bath for about 15min, and gently rinsed with PBS 3 times for 3 min/time.

9) After the residual PBS was aspirated, DAB color was developed, observed under an optical microscope for about 5-8 min, and stopped with tap water.

10) After counter-staining with hematoxylin for 20s, the cells were transiently differentiated with 1% HCl and immersed in water.

11) The sections were sequentially washed with gradient ethanol: soaking in 70%, 80%, 90%, and 95% ethanol for 5min respectively; soaking in 100% ethanol for 2 times for 10min respectively; soaking in transparent liquid II for 10 min; soaking in transparent liquid I for 20min to completely dehydrate, and sealing.

12) Primary antibody replaced with PBS and positive results from the preliminary experiment served as negative and positive controls, respectively.

Siglec-15 is mainly expressed on the cytoplasm and cell membrane of GCTB tissue cells. A double blind method is utilized. Randomly selecting 10 high-power lens fields for each section, counting 100 cells in each high-power field, and judging the positive cell rate and the staining intensity score: number of positive cells (I): marking the positive cells with the value of less than or equal to 5 percent as 0 point; marking the score of 1 for 6-25 percent; 26-50% of the total weight is marked as 2 points; recording the score of 51-75% as 3; score 4 for > 75%. Staining intensity (II): no staining was scored as 0 min; weak positive (light yellow) was scored as 1 point, medium positive (tan) was scored as 2 points, and strong positive (tan) was scored as 3 points. And (3) obtaining a final score by the method, wherein 0-1 is negative (-), 2-3 is weak positive (+), 4-5 is positive (+ +), and more than or equal to 6 is strong positive (+ +++). The results are shown in FIG. 1 and Table 1.

Table 1: relationship of Siglec-15 expression to clinical characteristics of GCTB patients

The results showed that 51 of the tumor tissue sections of 56 GCTB patients with Siglec-15 positive expressors had a positive rate of 91.07% (51/56), with a staining scale of mild (+) at 20 (35.71%), moderate (++) at 19 (33.93%) and severe (++) at 12 (21.43%). Siglec-15 positive staining was mostly in mononuclear stromal cells, the cytoplasm and cell membrane of multinucleated giant cells (see FIG. 1). Analysis in combination with the clinical characteristics of GCTB patients revealed that Siglec-15 expression in GCTB tissues was significant (P <0.05) compared to the differences between the stages of anacci's and tumor recurrence, but not compared to the differences between patient gender, age, tumor localization and tumor size (P >0.05), as detailed in table 1. Further, the expression of Siglec-15 protein in GCTB mononuclear stromal cells was examined by in vitro experiments. The Siglec-15 protein was confirmed to be cytoplasmic and cell membrane of Hs737.T cells by immunofluorescence staining techniques. The gene expression level of Siglec-15 in Hs737.T cells is detected by RT-PCR technology, and the result shows that the expression level of Siglec-15mRNA in Hs737.T cells (Siglec-15/. beta-actin) is 1.636 +/-0.012, which belongs to high-level expression.

Example 2: siRNA synthesis and cell transfection

1. SiRNA synthesis

The expression of Siglec-15 in GCTB mononuclear stromal cells was attenuated by transfection with siRNA. Designing and synthesizing siRNA sequence and NC siRNA sequence aiming at Siglec-15, wherein the sequence of Siglec-15siRNA is detailed in Table 2.

Table 2: 3 siRNA sequence names and sequences to Siglec-15

2. Cell transfection

1) Taking GCTB mononuclear stromal cells in logarithmic phase, digesting, centrifuging, and performing 2.5 × 10 on each well⁵cells/mL density in 6-well plates, kept as single as possible, placed in incubators (5% CO)₂37 ℃ C.).

2) When the cells reach 65% -80%, the cell culture medium in the 6-well plate is sucked out, the cells are washed for 2 times by PSB, and 2mL of serum-free high-glucose DMEM medium is put into each well for later use.

3) 4 EP tubes of 1.5mL, 2 EP tubes of 1.5mL as a negative control group (NC) and 2 EP tubes of 1.5mL as a transfection group (Siglec-15-siRNA) were taken. One tube contained serum-free high-glucose DMEM medium + siRNA, and one tube contained serum-free high-glucose DMEM medium + transfection reagent at room temperature for about 3 min.

4) And then, the Siglec-15siRNA and the transfection reagent are mixed uniformly and are kept at room temperature for about 10-15 min.

5) Slowly injecting the mixture into cells along the pore wall, gently shaking the pore plate, and placing in an incubator (5% CO)₂37 ℃ C.).

6) After 6h, the medium was replaced with high-glucose DMEM medium containing 15% FBS. After 24h, the film was observed and photographed. After 48h of transfection, cells were used for subsequent plating.

EXAMPLE 3 Siglec-15 expression assay of transfected cells

The expression of Siglec-15 of the transfected cells is measured by immunofluorescence staining, real-time fluorescence quantitative PCR and Western Blot, and the specific steps are as follows:

(I) immunofluorescence staining

1) Placing the slide on the bottom of 12-well plate, taking GCTB mononuclear stromal cells in logarithmic phase, after trypsinization, according to the ratio of 3.5 × 10 per well⁵cells/mL were plated in wells with slides and placed in an incubator (5% CO)₂And 37 ℃ C. were cultured.

2) After the cells were attached to the slide and stretched, the medium was removed and washed 2 times with PBS for 5 min/time.

3) GCTB mononuclear stromal cells are treated by 500 mu L of 4% paraformaldehyde at room temperature for about 20-25 min, and are lightly washed by PBS for 3 times and 5 min/time.

4) After the residual PBS was aspirated, the cells were permeabilized with 0.1% Triton X-100 for about 5-10min at room temperature, and gently washed 2 times with PBS for 5 min/time.

5) After aspiration of residual PBS, the tissue sections were covered with blocking solution containing 10% goat serum and left at room temperature for about 1 h.

6) The blocking solution was decanted off, and the Siglec-15 antibody (primary antibody) was slowly added dropwise at a ratio of 1:100 and refrigerated overnight in a refrigerator at 4 ℃.

7) On the next day, the cells were gently washed with PBS 3 times for 5 min/time; slowly adding fluorescent secondary antibody (goat anti-rabbit IgG) dropwise at room temperature, keeping away from light for about 1h, and gently washing with PBS for 3 times and 5 min/time.

8) Washing with PBS gently for 3 times, 5 min/time; after blotting residual PBS, mounting (DAPI-containing anti-fluorescence quenching mounting solution). And observing under a mirror and taking a picture.

(II) real-time fluorescent quantitative PCR

Extraction of RNA

1) First, an RNase-free EP tube and a tip were prepared, and a spray for promoting RNase degradation was sprayed around the tube. Pancreatin digestion of tumor cells of NC group and Siglec-15-siRNA group, PSB washing for 2 times, centrifugation at 1500rpm for 5min to obtain cell precipitate, gently shaking the centrifuge tube to loosen it, and adding appropriate amount of cell lysate (containing 1% beta-mercaptoethanol).

2) The tube was shaken vigorously to lyse the cells, and the resulting lysate was transferred to a filtration column and centrifuged at 12000rpm for 2min at 4 ℃. The filtrate obtained was mixed with 70% ethanol of the same volume, packed in an adsorption column, and centrifuged at the above speed for about 35 seconds.

3) Absorbing and discarding the waste liquid, adding a certain amount of protein removal working solution into an adsorption column, and centrifuging at the speed for about 35 s; and (3) absorbing the waste liquid, adding a proper amount of DNase I working solution into an adsorption column, reacting at room temperature for about 10-15 min, adding the same volume of protein removal working solution into the adsorption column, and centrifuging at the speed for about 35 s.

4) Absorbing waste liquid, adding appropriate amount of RNA rinsing liquid into adsorption column, standing at room temperature for about 2min, and centrifuging at the above speed for about 35s

5) Repeat step 4 once.

6) And absorbing and discarding the waste liquid, centrifuging for 2min at the speed, absorbing and discarding the waste liquid in the tube, and standing for about 2min at room temperature.

7) Putting the adsorption column into an EP tube without RNase, and adding 30-50 μ L of RNase Free dH₂And O, standing at room temperature for about 2min, and centrifuging at the speed for 2min to obtain an RNA product.

8) And measuring the purity and concentration of the obtained RNA solution by using a check quantitative detector.

② reverse transcription

1) The following reagents were placed in a 0.2mL RNase-free EP tube on ice.

2) Incubate at 65 ℃ for 5min and cool rapidly on ice.

3) The following reagents were mixed on ice into a 0.2mL RNase-free EP tube.

4) Lightly blowing and uniformly mixing, and keeping the mixture at the temperature of 42 ℃ for 30-60 min; then kept at 95 ℃ for 5min, and put on ice for cooling.

5) The concentration and purity of the cDNA solution were measured with a check meter and stored in a freezer at-20 ℃ for further experiments.

③PCR

1) The following reagents were added to 8-up tubes, 20. mu.L per well in total, and the gene primers are shown in Table 3.

TABLE 3 RT-PCR primers used in the experiments

2) The RT-PCR instrument is turned on, and the reaction system is set as follows:

3) by 2^-△△CtThe calculation yields a relative quantitative value for RT-PCR, with a greater cycle threshold (Ct) representing a lesser amount of mRNA (relative quantitation).

(III) Western Blot

Extraction of protein

1) The NC group and the Siglec-15-siRNA group GCTB mononuclear stromal cells were scraped by cell scraping, washed 2 times with PSB, and centrifuged at 1500rpm for 4min to obtain cell pellets.

2) The RIPA high-efficiency cell lysis solution and a protease inhibitor PMSF are mixed according to the proportion of 1:100, injecting the lysate mixture into the cell sediment, repeatedly blowing and beating, cracking on ice for about 30min, and frequently taking out the lysate mixture during the cracking period to shake so as to fully crack the cells.

3) The lysate was pipetted into a pre-cooled EP tube and centrifuged at 12000g for 10min (4 ℃). The supernatant was pipetted into a fresh precooled EP tube.

4) And diluting the BCA protein quantitative standard protein with pure water according to a certain gradient. And (3) adding 50: 1, preparing two liquids A and B to obtain working liquid.

5) Adding 25 mu L of standard substance (1mg/mL) and the protein sample to be detected after being diluted by 5 times into a 96-well plate, repeating 3 wells, wherein the adding amount is as follows:

6) 200 μ L of the above working solution was added to all the wells, and the wells were shaken for 45s and then shielded from light at 37 ℃ for about 30 min.

7) And detecting the absorbance of the protein at 562nm, and drawing a standard curve to obtain the concentration of the GCTB mononuclear stromal cell protein sample.

8) Adding 1/4 sample of 5 Xloading buffer solution into GCTB mononuclear matrix cell protein sample, mixing, placing in 98 deg.C water bath (about 15min), cooling at room temperature, and storing in-20 deg.C refrigerator.

② glue running, film transferring and developing

1) According to the molecular weight of the target protein, the separation gel is prepared according to the following formula, the separation gel is injected to the position 3/4 of the glass plate, and the distilled water is immediately added for sealing. Then concentrated glue is prepared, and a comb is inserted after the concentrated glue is injected into the glass plate.

2) After 35min, injecting the pre-diluted electrophoresis solution into an electrophoresis tank, and taking out the comb; then, a Marker and a GCTB mononuclear matrix cell protein sample are spotted in the loading hole.

3) Starting a power supply, and operating at a constant voltage of 90V for 25min when the protein is in the concentrated gel; when protein enters the separation gel, the separation gel is operated at constant pressure of 120V for 50min, and after the marked strips are dispersed in motion, the gel is cut.

4) Activating the sheared PVDF membrane in pure methanol for about 15s, washing in distilled water for about 2min, and soaking and buffering in membrane transferring liquid for about 5 min. And buffering the glue with a membrane transferring solution for about 15min, and buffering the filter paper membrane transferring solution for about 10-15 min.

5) The "sandwich" was directly stacked on the plane of the membrane transfer apparatus (semi-dry) in the order: the bottom plate of the film transferring instrument, the filter paper with 3 layers, the PVDF film, the glue, the filter paper with 3 layers and the top plate of the film transferring instrument are covered with the top plate and run for about 8min at a constant pressure of 18V.

6) 3 to 5 percent of skim milk is used for blocking the nonspecific protein interference of the PVDF membrane, and the incubation is carried out for about 60min at room temperature with gentle shaking. The PVDF membrane was not washed.

7) The primary antibody is diluted by TBST solution according to a proportion, and the transferred PVDF membrane is completely immersed in the working solution of the antibody and refrigerated at 4 ℃.

8) The following day, the membrane was washed 3 times 5 min/time with TBST solution, and the PVDF membrane was placed in a secondary antibody diluted with TBST solution (about 60min by a shaker), and washed 3 times 5 min/time with TBST solution.

9) Mixing the raw materials in a ratio of 1:1 mixing the luminous liquid (A liquid and B liquid) to obtain a working liquid, dripping the working liquid on a PVDF membrane, incubating the PVDF membrane at 37 ℃ in the dark for about 3min, draining the residual luminous liquid on the membrane, and developing the membrane by a developing instrument.

10) The grayscale values of the proteins were measured using Image J software.

The results of the above experiments are shown in FIGS. 2-4, and Siglec-15 gene silencing was performed by transferring Siglec-15siRNA into Hs737.T cells. After 48h of the siRNA transferred into Siglec-15, red fluorescence in cytoplasm was observed by a fluorescence microscope, and the transfection efficiency of the Siglec-15-siRNA was more than 75% (as shown in FIG. 2). Expression levels of Siglec-15mRNA and protein from Hs737.T cells after transfection were further examined by RT-PCR and Western Blot (see FIGS. 3 and 4). The results showed that the expression levels of both mRNA and protein of Siglec-15 were reduced (P < 0.05).

EXAMPLE 4 determination of proliferation, migration and invasion function of transfected cells

(I) cell proliferation assay

Taking GCTB mononuclear stromal cells in logarithmic phase, digesting, centrifuging, and taking 3 × 10 cells per well³The individual cells were plated evenly in 96-well plates, 100. mu.L per well of 15% FBS high-glucose DMEM, and placed in an incubator (5% CO)₂37 ℃ C.). At 24h, 48h, 72h and 96h of cell culture, 20. mu.L of MTS proliferating agent was added to each well. The temperature is kept at 37 ℃ for about 60 min. The absorbance of the cells was measured at a wavelength of 462nm and analyzed statistically.

(II) cell cloning experiments

Taking GCTB mononuclear stromal cells in logarithmic phase, digesting, centrifuging, and collecting at 6 × 10 per well²The individual cells were plated evenly in 6-well plates, 2mL per well of 15% FBS high-glucose DMEM, and placed in incubators (5% CO)₂37 ℃ C.). After 14 days, the plates were gently washed 2 times with PBS, then 4% paraformaldehyde was added for about 25min, and the Piemax working solution was stained for about 15 min. After the supernatant was washed off gently with physiological saline, colonies (more than 50 cells) were counted and analyzed statistically.

(III) cell migration experiment

Taking GCTB mononuclear stromal cells in logarithmic phase, digesting, centrifuging, and placing at 4.5 × 10 per well⁵cells/mL were uniformly plated in 6-well plates, 2mL of 15% FBS high-sugar DMEM per well, and placed in an incubator (5% CO)₂37 ℃ C.). After 24h, when the cells were spread flat, a straight line was drawn on the diameter of a 6-well plate using a 10 μ L white pipette tip, and after the scraped cell pieces were washed out with PBS, 2mL of DMEM cell culture medium containing 5% FBS was added to each well, and photographed as 0h by observing under a mirror. At

time points

24h, 48h and 72h, the photographs were observed. And obtaining the residual area of the scratch in the picture by using Image J software, and carrying out statistical analysis.

(IV) Transwell experiment

1. Transwell migration experiment

Taking GCTB mononuclear stromal cells in logarithmic phase, digesting, centrifuging, and placing 3.5 × 10 cells per well⁴The cells were plated evenly in a Transwell chamber, 200. mu.L of high-glucose DMEM (containing 1% FBS) was added to the chamber, and 700. mu.L of high-glucose DMEM was added to the lower chamberGlucose DMEM (containing 15% FBS) placed in incubator (5% CO)₂And 37 ℃ C. were cultured. The chamber was gently washed 3 times with physiological saline at 24h and 48h, respectively, and then kept with 4% paraformaldehyde for about 25 min. After gently washing with physiological saline for 5 times, the cells were stained with Jimusa for about 18 min. After the Jimusa is gently washed away by normal saline, cells which do not pass through the cell are gently wiped off by a cotton swab, and the cells are photographed and counted in 5 random visual fields under a 200-time microscope for statistical analysis.

2. Transwell invasion test

1: matrigel was diluted at a ratio of 8 and gently spread onto the bottom membrane of a Transwell cell (air bubbles were removed) and kept at 37 ℃ for 12 h. Taking GCTB mononuclear stromal cells in logarithmic phase, digesting, centrifuging, and placing 3.5 × 10 cells per well⁴The cells were plated in Transwell chambers, and 200. mu.L of high-glucose DMEM (containing 1% FBS) was added to the chamber, and 700. mu.L of high-glucose DMEM (containing 15% FBS) was added to the lower chamber, and placed in an incubator (5% CO)₂And 37 ℃ C. were cultured. The chamber was gently washed 3 times with physiological saline at 24h and 48h, respectively, and then kept with 4% paraformaldehyde for about 25 min. Gently washed with physiological saline 5 times, and added with Pimex to stain cells for about 18 min. After the Jimusa is gently washed away by normal saline, cells which do not pass through the cell are gently wiped off by a cotton swab, and the cells are photographed and counted in 5 random visual fields under a 200-time microscope for statistical analysis.

The results of the above experiments are shown in FIGS. 5-9, and the proliferation capacity of the silent Siglec-15 group and the control group Hs737.T cells is tested by the MTS method at four time points of 24h, 48h, 72h and 96h in culture. The results show that at three time points of 48h, 72h and 96h, the absorbance of the Siglec-15-siRNA group Hs737.T cells is 0.387 +/-0.006, 0.399 +/-0.013 and 0.455 +/-0.008 respectively, and the absorbance of the NC group is 0.439 +/-0.023, 0.440 +/-0.030 and 0.488 +/-0.012 respectively. The proliferation capacity of Hs737.T cells in the Siglec-15-siRNA group is obviously smaller than that of the NC group, and the two groups have statistical difference (P <0.05), which shows that the proliferation capacity of Hs737.T cells silencing Siglec-15 is obviously reduced (figure 5). The colony formation of two groups of Hs737.T cells was observed by an optical microscope at 14 days of culture, and the cloning ability of the Hs737.T cells was examined. The results show that the number of cell clones of the silent Siglec-15 group is 61.333 +/-2.512, and the number of cell clones of the NC group is 32.333 +/-2.517. There was a statistical difference between the two groups (P <0.001), indicating that the clonality of Hs737.T cells silencing Siglec-15 was significantly reduced (FIG. 6). In conclusion, silencing Siglec-15 decreased both the proliferation and clonogenic capacity of Hs737.T cells, indicating that Siglec-15 expression may play a role in the proliferative growth of GCTB mononuclear stromal cells.

The crawling ability of Hs737.T cells of a silent Siglec-15 group and a control group at three time points of 24h, 48h and 72h is detected through a scratch test, the scratch area of the Siglec-15-siRNA group is 0.499 +/-0.090 and 0.083 +/-0.032 respectively when the silent Siglec-15-siRNA group is cultured for 48h and 72h, the reduction of the NC group is 0.682 +/-0.024 and 0.555 +/-0.053 respectively, and the statistical difference (P <0.05) exists between the two groups, which indicates that the crawling speed of the Hs737.T cells of the silent Siglec-15 is obviously reduced (FIG. 7 and FIG. 8).

The crawling migration capacity of the silent Siglec-15 group and the control group Hs737.T cells was tested by the Transwell method. At 24h of culture, the numbers of transmembrane cells of the silent Siglec-15 group and the control group are 112.000 + -3.600 and 123.00 + -6.000, respectively, and no statistical difference exists between the two groups (P > 0.05). The number of transmembrane cells of the silencing Siglec-15 group and the control group are 126.000 +/-4.000 and 181.000 +/-6.557 respectively when the two groups are cultured for 48 hours, and the statistical difference (P <0.001) between the two groups indicates that the migration capacity of Hs 737-T cells of the silencing Siglec-15 is obviously reduced (FIGS. 9A and 9B). The invasion capacity of the silent Siglec-15 group and the control group Hs737.T cells is detected by a Transwell method. The number of cells passing through Matrigel was 98.000 + -7.550 and 130.667 + -1.528 in the silencing Siglec-15 group and 120.333 + -4.619 and 148.667 + -3.215 in the control group, respectively, when cultured for 24h and 48h, and the statistical difference (P <0.05) was observed between the two groups, indicating that Hs737.T cells silencing Siglec-15 had a significantly reduced invasive capacity (FIGS. 9C and 9D). In conclusion, silencing Siglec-15 decreased both the migration and invasion functions of Hs737.T cells, indicating that Siglec-15 expression may play a role in the local invasion and metastasis processes of GCTB cells.

Sequence listing

Application of <120> Siglec-15 inhibitor in preparation of drugs for preventing and treating giant cell tumor of bone

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gctcatttgt gagaactaa 19

<210> 2

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctacggagaa cttgctcaa 19

<210> 3

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggcccaggag tccaattat 19

Claims

Application of a Siglec-15 inhibitor in preparation of medicines for preventing and treating giant cell tumor of bones.
2. The use of claim 1, wherein the inhibitor is a substance that inhibits the expression and/or function of Siglec-15.
3. The use of claim 1, wherein the inhibitor is an antibody against Siglec-15 or an interfering RNA against the Siglec-15 gene.
4. The use of claim 1, wherein the inhibitor is a monoclonal antibody against Siglec-15.
5. The use according to claim 1, wherein the inhibitor is an siRNA selected from the group consisting of: SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.
6. The pharmaceutical composition for preventing and treating giant cell tumor of bone is characterized by comprising a Siglec-15 inhibitor.
7. The pharmaceutical composition of claim 6, wherein the inhibitor is an antibody against Siglec-15 or an interfering RNA against the Siglec-15 gene.
8. The pharmaceutical composition of claim 6, wherein the inhibitor is an siRNA selected from the group consisting of: SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.
9. The pharmaceutical composition of claim 6, further comprising:

a pharmaceutically acceptable carrier and/or adjuvant;

other active ingredients for preventing or treating giant cell tumor of bone.
10. The pharmaceutical composition of claim 9, wherein the other active ingredients comprise: chemotherapeutic agents, radiotherapeutic agents or other drugs having the function of down-regulating the expression of Siglec-15.