Use of adrm1 gene / protein in preparation of drug for treating osteosarcoma

Abstract

The application relates to the field of biological medicine, and provides application of ADRM1 gene / protein in preparation of a drug for treating osteosarcoma. The ADRM1 gene / protein is applied to preparation of the drug for treating osteosarcoma, can significantly inhibit proliferation, migration and invasion ability of tumor cells, and thus delays occurrence and development of the osteosarcoma. By shRNA intervention technology, the expression of ADRM1 is knocked down, the mRNA and protein levels of ADRM1 in 143b and U2OS osteosarcoma cell lines are significantly reduced, short-term proliferation activity and long-term clone formation ability are inhibited, and migration and invasion characteristics of the cells are effectively weakened. After 143b osteosarcoma cells with the knocked-down ADRM1 are inoculated into nude mice, the growth of the tumor formed by the cells is obviously slowed down, and the expression of a cell proliferation marker KI-67 in the tumor is down-regulated, indicating that the ADRM1 gene / protein also plays a key regulation role in the in-vivo growth process of the osteosarcoma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of ADRM1 gene / protein in preparation of a drug for treating osteosarcoma. BACKGROUND

[0002] Osteosarcoma (OS) is a highly malignant primary bone tumor originating from mesenchymal tissue, and its main pathological feature is that tumor cells directly produce immature bone or bone-like matrix. The disease is mainly seen in adolescents and children, and is the most common primary malignant bone tumor in this population. The osteosarcoma is mainly located at the metaphysis of long bone, and is most commonly seen at the distal femur, proximal tibia and proximal humerus. Some cases can involve the pelvic and other axial bones. The disease has the characteristics of rapid growth, strong local invasiveness, high early metastasis rate, fast clinical progression, poor prognosis, and seriously threatens the life and quality of life of adolescents.

[0003] Although the current standard treatment mode of osteosarcoma includes neoadjuvant chemotherapy, limb-salvage surgery and postoperative adjuvant chemotherapy, the 5-year overall survival rate of patients with localized osteosarcoma has been improved, but for patients with lung metastasis or drug resistance, long-term survival still faces great challenges, and the 5-year survival rate is still low. At the same time, traditional chemotherapy drugs have the problems of high toxicity, large individual differences in efficacy, and some patients have obvious drug resistance to multi-drug combination schemes, and the treatment effect is limited. For patients with postoperative recurrence, long-term metastasis or chemotherapy resistance, there is an urgent need for more effective and safe treatment methods in clinical practice. In addition, signal pathway targeted drugs have achieved certain results in other tumors (such as lung cancer and breast cancer), but their application in osteosarcoma is still in the early stages of research, and there has been no breakthrough progress.

[0004] Therefore, in the background of limited efficacy of existing treatment methods and lack of targeted treatment schemes, it is of great significance to develop new anti-osteosarcoma drugs with clear mechanism of action, accurate efficacy and high safety, identify new molecular targets and convert them into clinical applications for improving the treatment effect of osteosarcoma patients and improving long-term survival, in line with the current development trend of tumor treatment from “general chemotherapy” to “precise targeting” and “individualized treatment”, and has significant clinical value and social demand. SUMMARY

[0005] In view of the deficiencies of the prior art, the application provides the application of ADRM1 gene / protein in preparation of a drug for treating osteosarcoma, which aims to solve the problems mentioned in the background art.

[0006] ADRM1 (Adhesion Regulating Molecule 1) is an important subunit of the 26S proteasome regulatory particle, widely distributed in eukaryotic cells, involved in protein degradation, deubiquitination regulation, cell adhesion and proliferation and other biological processes. ADRM1 recognizes ubiquitin chains through its domain and recruits deubiquitinase UCHL5, regulates protein homeostasis, and is a key molecule for maintaining cell function balance. Studies have shown that ADRM1 has certain biological functions in bone metabolism related diseases. For example, studies have shown that ADRM1 can reduce cartilage degeneration by enhancing UCH37-mediated ALK5 deubiquitination, and thus be used for the treatment of osteoarthritis; studies have also found that inhibition of ADRM1 expression can induce osteoblast differentiation and inhibit osteoclast function, thus having certain effects on regulating bone metabolism and treating osteoporosis.

[0007] However, osteosarcoma is a malignant tumor with high mortality, and its mechanism is significantly different from that of bone metabolism diseases such as osteoarthritis and osteoporosis. Osteoarthritis and osteoporosis mainly involve functional disorders of chondrocytes, osteoblasts and osteoclasts, and are chronic, non-tumorous diseases; while osteosarcoma is highly invasive and metastatic, mainly caused by malignant transformation of bone marrow mesenchymal stem cells, and its core mechanism involves typical malignant tumor molecular characteristics such as activation of proto-oncogenes, inactivation of tumor suppressor genes, and chromosomal instability.

[0008] The present application provides the use of ADRM1 gene / protein in the preparation of a drug for treating osteosarcoma, wherein the drug inhibits the expression of ADRM1 gene or protein to treat osteosarcoma.

[0009] Further, the drug inhibits the expression of ADRM1 gene or protein to inhibit the proliferation, migration and invasion ability of osteosarcoma cells, and delays or blocks the progression of osteosarcoma.

[0010] Further, the drug comprises an RNA interference molecule that inhibits the expression of ADRM1 gene or protein.

[0011] Further, the RNA interference molecule comprises siRNA, miRNA, shRNA or dsRNA.

[0012] Further, the sequence of the shRNA is shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0013] Further, the shRNA is packaged by a lentiviral vector and stably transfected into osteosarcoma cells.

[0014] Further, the osteosarcoma cells are 143b cells or U2OS cells.

[0015] Further, the drug significantly reduces the short-term proliferation ability and long-term proliferation ability of osteosarcoma cells.

[0016] Further, the drug further comprises a small molecule compound that inhibits ADRM1 gene transcription, post-protein translation modification, or directly binds to and inhibits ADRM1 protein activity.

[0017] The present application has the following technical effects: the ADRM1 gene / protein is applied to the preparation of a drug for treating osteosarcoma, which can significantly inhibit the proliferation, migration and invasion ability of tumor cells, thereby delaying the occurrence and development of osteosarcoma. By using shRNA intervention technology to knock down the expression of ADRM1, the mRNA and protein levels of ADRM1 in 143b and U2OS osteosarcoma cell lines can be significantly reduced, thereby inhibiting the short-term proliferation activity and long-term clonogenicity, and effectively weakening the migration and invasion characteristics of the cells. In vivo experiments further show that the growth of tumor bodies formed by 143b osteosarcoma cells with knocked down ADRM1 after being inoculated into nude mice is significantly slowed down, and the expression of cell proliferation marker KI-67 in the tumor bodies is down-regulated, indicating that the ADRM1 gene / protein also plays a key regulatory role in the in vivo growth of osteosarcoma. Therefore, the ADRM1 gene / protein as a potential target for osteosarcoma treatment can be used for drug screening and development, and has good application prospect and transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0018] The exemplary embodiments of the present application can be more completely understood by reference to the following drawings:

[0019] Figure 1 It is an immunohistochemical staining picture of the expression level of ADRM1 protein in osteosarcoma tumor tissues and paraneoplastic normal tissues in Example 1 of the present application; magnification × 200, scale: 50 µm.

[0020] Figure 2 It is a quantitative analysis result of the expression level of ADRM1 protein in osteosarcoma tumor tissues and paraneoplastic normal tissues analyzed by immunohistochemical staining in Example 1 of the present application, wherein *** indicates p<0.001.

[0021] Figure 3 It is a picture of the mRNA expression amount of ADRM1 in normal osteoblast cell line hFOB1.19 and different osteosarcoma cell lines (143b, MG63, U2OS) detected by qRT-PCR in Example 2 of the present application; compared with the osteoblast cell line hFOB1.19, *** indicates p<0.001.

[0022] Figure 4Picture for detecting ADRM1 protein expression in normal osteoblast cell line hFOB1.19 and different osteosarcoma cell lines (143b, MG63, U2OS) by Western blotting in Example 3 of the present application.

[0023] Figure 5 Picture for quantitative analysis of ADRM1 protein expression in normal osteoblast cell line hFOB1.19 and different osteosarcoma cell lines (143b, MG63, U2OS) by Western blotting in Example 3 of the present application; * represents p<0.05, ** represents p<0.01, and *** represents p<0.001 compared with osteoblast cell line hFOB1.19.

[0024] Figure 6 Picture for detecting mRNA expression of ADRM1 in 143b osteosarcoma cells after stable transfection of shRNA by qRT-PCR in Example 4 of the present application; *** represents p<0.001 compared with the negative control group.

[0025] Figure 7 Picture for detecting mRNA expression of ADRM1 in U2OS osteosarcoma cells after stable transfection of shRNA by qRT-PCR in Example 4 of the present application; *** represents p<0.001 compared with the negative control group.

[0026] Figure 8 Picture for detecting ADRM1 protein expression in 143b osteosarcoma cells after stable transfection of shRNA by Western blotting in Example 4 of the present application.

[0027] Figure 9 Quantitative analysis results of ADRM1 protein expression in 143b osteosarcoma cells after stable transfection of shRNA by Western blotting in Example 4 of the present application; *** represents p<0.001 compared with the negative control group.

[0028] Figure 10 Picture for detecting ADRM1 protein expression in U2OS osteosarcoma cells after stable transfection of shRNA by Western blotting in Example 4 of the present application.

[0029] Figure 11 Quantitative analysis results of ADRM1 protein expression in U2OS osteosarcoma cells after stable transfection of shRNA by Western blotting in Example 4 of the present application; *** represents p<0.001 compared with the negative control group.

[0030] Figure 12The CCK-8 cell proliferation experiment was used to detect the influence of the knockdown of ADRM1 on the short-term proliferation ability of 143b osteosarcoma cells in Example 5 of the present application; compared with the negative control group, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0031] Figure 13 The CCK-8 cell proliferation experiment was used to detect the influence of the knockdown of ADRM1 on the short-term proliferation ability of U2OS osteosarcoma cells in Example 5 of the present application; compared with the negative control group, * represents p<0.05, and *** represents p<0.001.

[0032] Figure 14 The plate cell clone formation experiment was used to detect the influence of the knockdown of ADRM1 on the long-term proliferation ability of 143b osteosarcoma cells in Example 6 of the present application.

[0033] Figure 15 The plate cell clone formation experiment was used to detect the influence of the knockdown of ADRM1 on the long-term proliferation ability of 143b osteosarcoma cells in Example 6 of the present application; compared with the negative control group, *** represents p<0.001.

[0034] Figure 16 The plate cell clone formation experiment was used to detect the influence of the knockdown of ADRM1 on the long-term proliferation ability of U2OS osteosarcoma cells in Example 6 of the present application.

[0035] Figure 17 The plate cell clone formation experiment was used to detect the influence of the knockdown of ADRM1 on the long-term proliferation ability of U2OS osteosarcoma cells in Example 6 of the present application; compared with the negative control group, *** represents p<0.001.

[0036] Figure 18 The scratch experiment was used to detect the influence of the knockdown of ADRM1 on the migration ability of 143b osteosarcoma cells in Example 7 of the present application.

[0037] Figure 19 The scratch experiment was used to detect the influence of the knockdown of ADRM1 on the migration ability of 143b osteosarcoma cells in Example 7 of the present application; compared with the negative control group, *** represents p<0.001.

[0038] Figure 20 The scratch experiment was used to detect the influence of the knockdown of ADRM1 on the migration ability of U2OS osteosarcoma cells in Example 7 of the present application.

[0039] Figure 21Quantitative analysis results of the effect of ADRM1 knockdown on the migration ability of U2OS osteosarcoma cells detected by scratch test in Example 7 of the present application; compared with the negative control group, *** represents p<0.001.

[0040] Figure 22 Result graph of the effect of ADRM1 knockdown on the invasion ability of 143b osteosarcoma cells detected by chamber migration test in Example 8 of the present application.

[0041] Figure 23 Quantitative analysis results of the effect of ADRM1 knockdown on the invasion ability of 143b osteosarcoma cells detected by chamber migration test in Example 8 of the present application; compared with the negative control group, *** represents p<0.001.

[0042] Figure 24 Result graph of the effect of ADRM1 knockdown on the invasion ability of U2OS osteosarcoma cells detected by chamber migration test in Example 8 of the present application.

[0043] Figure 25 Quantitative analysis results of the effect of ADRM1 knockdown on the invasion ability of U2OS osteosarcoma cells detected by chamber migration test in Example 8 of the present application; compared with the negative control group, *** represents p<0.001.

[0044] Figure 26 Result graph of the effect of ADRM1 knockdown on the growth of osteosarcoma in vivo evaluated by a nude mouse osteosarcoma subcutaneous tumor model in Example 9 of the present application.

[0045] Figure 27 Quantitative analysis results of the effect of ADRM1 knockdown on the growth of osteosarcoma in vivo evaluated by a nude mouse osteosarcoma subcutaneous tumor model in Example 9 of the present application; compared with the negative control group, ** represents p<0.01, and *** represents p<0.001.

[0046] Figure 28 Result graph of the expression amount of ADRM1 and KI-67 proteins detected by immunohistochemical staining of a tumor body of a nude mouse in Example 10 of the present application; magnification × 200, scale: 50 µm. DETAILED DESCRIPTION

[0047] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0048] Unless otherwise defined, 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; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0049] The present application provides the use of ADRM1 gene / protein in the preparation of a drug for treating osteosarcoma, wherein the drug treats osteosarcoma by inhibiting the expression of ADRM1 gene or protein.

[0050] In some embodiments, the drug delays or blocks the progression of osteosarcoma by inhibiting the proliferation, migration and invasion ability of osteosarcoma cells through inhibiting the expression of ADRM1 gene or protein.

[0051] In some embodiments, the drug comprises an RNA interference molecule that inhibits the expression of ADRM1 gene or protein.

[0052] In some embodiments, the RNA interference molecule comprises siRNA, miRNA, shRNA or dsRNA.

[0053] In some embodiments, the sequence of the shRNA is shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0054] In some embodiments, the shRNA is packaged by a lentiviral vector and stably transfected into osteosarcoma cells.

[0055] In some embodiments, the osteosarcoma cells are 143b cells or U2OS cells.

[0056] In some embodiments, the drug significantly reduces the short-term and long-term proliferation ability of osteosarcoma cells.

[0057] In some embodiments, the drug further comprises a small molecule compound that inhibits the transcription of ADRM1 gene, post-translational modification of protein, or directly binds to and inhibits the activity of ADRM1 protein.

[0058] Embodiment 1:

[0059] It is proved that ADRM1 gene / protein affects the occurrence and development of osteosarcoma through clinical samples.

[0060] Tumor samples and clinical data of 10 patients with osteosarcoma who underwent first surgery and did not undergo radiotherapy and chemotherapy were collected, all patients signed the informed consent form, and the ethical review approval number was: (2025) CDYFYYLK(01-058). Immunohistochemical analysis was performed on tumor tissues and normal tissues adjacent to the tumor of osteosarcoma patients.

[0061] Immunohistochemical analysis is as follows Figure 1 and Figure 2As shown in Table 2, the results show that the expression level of ADRM1 protein in tumor tissues is significantly higher than that in normal tissues adjacent to tumors, which indicates that ADRM1 protein plays a key role in the occurrence and development of osteosarcoma.

[0062] Example 2

[0063] The expression level of ADRM1 gene in normal osteoblast cell lines and different osteosarcoma cell lines was detected by qRT-PCR (quantitative reverse transcription polymerase chain reaction).

[0064] The 143b, MG63, U2OS osteosarcoma cell lines and the normal osteoblast cell line hFOB1.19 were selected, and the above cells were inoculated into a 6-well plate at a density of 2×10 5 cells / well, and after the cells grew to the logarithmic phase, total RNA of the cells was extracted using TRIzol reagent, and 1 μg of RNA was reverse transcribed into complementary DNA using reverse transcriptase. The A260 / A280 ratio was calculated to verify the purity and quality of the cDNA. The mRNA transcription level of the target gene was analyzed using a TB Green Premix Ex Taq II kit, the primer sequences of ADRM1 and β-actin genes before and after were shown in Table 1, β-actin was used as an internal reference gene, and the mRNA relative expression level of ADRM1 gene was calculated by 2−ΔΔCt method.

[0065] Table 1 Primer sequences of ADRM1 and β-actin genes before and after

[0066]

[0067] The calculation results of the mRNA relative expression level of ADRM1 gene are shown in Table 2, and the results show that the expression level of ADRM1 gene in 143b, MG63, U2OS osteosarcoma cell lines is significantly higher than that in the osteoblast cell line hFOB1.19. Figure 3

[0068] Example 3

[0069] The expression level of ADRM1 protein in normal osteoblast cell lines and different osteosarcoma cell lines was detected by Western blotting.

[0070] The 143b, MG63, U2OS osteosarcoma cell lines and the normal osteoblast cell line hFOB1.19 were selected, and the above cells were inoculated into a 6-well plate at a density of 2×10 ​Cells were seeded at a density of 1 cell / well in 6-well plates and grown to the logarithmic phase before extracting proteins. RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was used to extract proteins; the RIPA lysis buffer was incubated on ice for 30 minutes, shaken every 5 minutes, and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected, and the protein concentration was determined using a BCA protein detection kit according to the manufacturer's instructions. Protein samples (30 μg / well) were separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk powder at 4°C overnight, followed by incubation with the primary antibody for at least 8 hours. Then, the membrane was washed with TBST three times, and the secondary antibody (1:5000) was incubated at room temperature for 2 hours. The bands on the membrane were detected using a gel imaging and Western blot imaging system. The band intensity was quantitatively analyzed by ImageJ software, with β-actin as the internal reference.

[0071] The results of the Western blotting detection are shown in Figure 4 and Figure 5 The results show that the expression level of ADRM1 protein in the 143b, MG63, and U2OS osteosarcoma cell lines is significantly higher than that in the osteoblast cell line hFOB1.19.

[0072] In combination with the experimental results of qRT-PCR in Example 2, it is further illustrated that the ADRM1 gene / protein may play a key role in the occurrence and development of osteosarcoma. Therefore, the 143b and U2OS osteosarcoma cell lines with high expression levels of ADRM1 protein were used for cell function research in subsequent examples.

[0073] Example 4:

[0074] The 143b and U2OS osteosarcoma cell lines with knocked-down ADRM1 were constructed by shRNA (short hairpin RNA) stable transfection.

[0075] The 143b and U2OS osteosarcoma cell lines with stably knocked-down ADRM1 expression were constructed by shRNA plasmid lentivirus packaging and stable transfection. In this example, the osteosarcoma cells in the blank control group were not treated, the osteosarcoma cells in the negative control group were transfected with a non-targeting shRNA sequence, and the osteosarcoma cells in experimental groups 1 and 2 were transfected with two different targeting shRNA sequences to knock down the expression of the ADRM1 gene. The shRNA1 sequence was used in experimental group 1, and the shRNA2 sequence was used in experimental group 2. Both sequences were high-efficiency knockdown sequences selected in the pre-experiment. After transfection, the knockdown efficiency was verified by qRT-PCR and Western blotting.

[0076] Table 2 shRNA1 and shRNA2 sequences

[0077]

[0078] The results of qRT-PCR detection are shown in Figures 6-7 , which shows that the expression level of ADRM1 gene in 143b and U2OS osteosarcoma cell lines is successfully reduced by shRNA stable transfection.

[0079] The results of Western blot detection are shown in Figures 8-11 , which shows that the expression level of ADRM1 protein in 143b and U2OS osteosarcoma cell lines is successfully reduced by shRNA stable transfection.

[0080] Example 5:

[0081] The effect of knocking down ADRM1 on the short-term proliferation ability of osteosarcoma cells was detected by CCK-8 cell proliferation experiment. The 143b and U2OS osteosarcoma cells in the negative control group were transfected with non-targeting shRNA sequence, and the 143b and U2OS osteosarcoma cells in the experimental group were transfected with shRNA1 sequence to knock down ADRM1.

[0082] The 143b and U2OS osteosarcoma cells in the logarithmic growth phase were selected, resuspended with complete culture medium and adjusted to a density of cells / mL. 100 μL of the suspension was inoculated into each well of a 96-well plate, and the outer wells were filled with phosphate buffer to reduce edge effects. The culture plate was placed in a constant humidity incubator for continuous culture for 18-24 hours, and then CCK-8 working solution was added after the cells completed adhesion and entered the stable growth phase. The plate was incubated in the dark for 2.5 hours. The absorbance value (OD value) of each well was measured at 450 nm on a microplate reader, and the average value was obtained by repeating the reading 3 times per well.

[0083] The results of CCK-8 cell proliferation experiment are shown in Figure 12 and Figure 13 , which shows that the short-term proliferation ability of 143b and U2OS osteosarcoma cells is significantly inhibited after knocking down ADRM1 compared with the negative control group.

[0084] Example 6:

[0085] The effect of knocking down ADRM1 on the long-term proliferation ability of osteosarcoma cells was detected by plate cell colony formation experiment. The 143b and U2OS osteosarcoma cells in the negative control group were transfected with non-targeting shRNA sequence, and the 143b and U2OS osteosarcoma cells in the experimental group were transfected with shRNA1 sequence to knock down ADRM1.

[0086] Log-phase 143b and U2OS osteosarcoma cells were resuspended in complete culture medium and seeded into 6-well plates at a density of 500 cells / mL. After static incubation for 10-14 days until colony clusters were visible, the cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, rinsed with running water, and dried at 37°C in a laminar flow hood. High-resolution images were captured using a digital camera, and the colony count (≥50 cells per cell) was calculated using ImageJ software to determine the colony formation rate.

[0087] Results of plate cell colony formation experiment as follows Figures 14-17 As shown, the results indicate that, compared with the negative control group, knockdown of ADRM1 significantly inhibited the long-term proliferation of both 143b and U2OS osteosarcoma cells.

[0088] Based on the experimental results of Examples 5 and 6, it is shown that inhibiting the expression of the ADRM1 gene or protein can reduce the activity of osteosarcoma cells in the short term and significantly inhibit their clonogenic ability in the long term, thereby inhibiting the overall proliferation of osteosarcoma cells.

[0089] Example 7:

[0090] The effect of ADRM1 knockdown on the migration ability of osteosarcoma cells was examined using a scratch assay. 143b and U2OS osteosarcoma cells in the negative control group were transfected with a non-targeting shRNA sequence, while 143b and U2OS osteosarcoma cells in the experimental group were transfected with a targeting shRNA1 to knock down ADRM1.

[0091] 143b and U2OS osteosarcoma cells in logarithmic growth phase were resuspended in complete culture medium and their density adjusted to [value missing]. Cells / mL. Inoculate into 6-well plates, with 2 mL of phosphate-buffered saline (PFS) in the edge wells to reduce evaporation interference. Incubate at 37°C and 5% CO2 saturated humidity. Once the monolayer cell confluence reaches 90-95%, use a 200 μL sterile pipette tip perpendicular to the bottom of the plate to draw three parallel linear lesions spaced 8 mm apart at a constant rate. Immediately wash three times with pre-cooled PFS. Replace with serum-free medium, and mark the fixed field of view at the start, middle, and end of the scratches. Take a baseline image and then take another image 24 hours later. The scratch healing rate is calculated based on ImageJ software to measure changes in the scratched area. Specifically, after 24 hours of scratch experiment, divide the area occupied by cell migration within the scratched region by the total area of ​​the scratched region at the start of the experiment; the resulting ratio is the scratch healing rate.

[0092] The results of the scratch test are as follows: Figures 18-21 As shown, the results indicate that, compared with the negative control group, knocking down ADRM1 reduced the scratch healing rate and cell migration ability of 143b and U2OS osteosarcoma cells.

[0093] Example 8:

[0094] The effect of ADRM1 knockdown on the invasive ability of osteosarcoma cells was examined using a chamber migration assay. 143b and U2OS osteosarcoma cells in the negative control group were transfected with a non-targeting shRNA sequence, while 143b and U2OS osteosarcoma cells in the experimental group were transfected with a targeting shRNA1 to knock down ADRM1.

[0095] 143b and U2OS osteosarcoma cells in logarithmic growth phase were resuspended in complete culture medium and their density adjusted to [value missing]. Cells per mL were stored on ice for later use. 600 μL of chemotactic medium containing 10% fetal bovine serum was injected into the lower chamber of the Transwell, and 200 μL of precisely loaded cell suspension was added to the upper chamber. Cells were allowed to migrate continuously at 37°C, 5% CO2, and saturated humidity for 24 h. The upper chamber fluid was then removed, and the cells were gently rinsed three times with phosphate-buffered saline (PFS). The membrane surface was gently wiped with a cotton swab dipped in PFS. The cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 20 minutes. The chambers were inverted on a slide, and the transmembrane cells in the Transwell chambers were counted using ImageJ software. Each experiment was independently repeated three times. Five microscopic fields were randomly selected for counting in each experiment, and the average value was calculated for statistical analysis.

[0096] The results of the cell migration experiment are as follows: Figures 22-25 As shown, the results indicate that, compared with the negative control group, knocking down ADRM1 reduced the number of invasive cells and decreased the invasive ability of 143b and U2OS osteosarcoma cells.

[0097] Example 9:

[0098] To establish a nude mouse subcutaneous osteosarcoma tumorigenesis model and evaluate the effect of ADRM1 knockdown on in vivo osteosarcoma growth.

[0099] Four-week-old SPF-grade BALB / c-nu athymic nude mice were selected and sex-balanced. They were acclimatized for 7 days in an AAALAC-certified barrier environment, receiving sterilized irradiated feed and acidified drinking water. After acclimatization for one week, the mice were randomly divided into four groups of five mice each: Experimental Group 1 (inoculated with 143b osteosarcoma cells with ADRM1 knocked down using the shRNA1 sequence), Experimental Group 2 (inoculated with 143b osteosarcoma cells with ADRM1 knocked down using the shRNA2 sequence), a blank control group (inoculated with untreated 143b osteosarcoma cells), and a negative control group (inoculated with 143b osteosarcoma cells transfected with a non-targeted shRNA sequence). Tumor volume was measured every 3 days after cell inoculation. The three-dimensional diameter of the tumor was measured daily using digital calipers, and data were analyzed according to... The tumor volume was calculated by formula. When the tumor volume reached or exceeded 1500 mm3, the body weight of the nude mice decreased by more than 20%, or the nude mice showed obvious activity disorders, the nude mice were euthanized by cervical dislocation and tumor tissue collection. The experimental plan was approved by the animal ethics committee (approval number: CDYFY-IACUC-202404QR025).

[0100] The volume and weight results of the tumors are shown in Figure 26 and Figure 27 The results show that the growth of osteosarcoma in nude mice is significantly inhibited after knocking down ADRM1 compared with the negative control group.

[0101] Example 10:

[0102] Immunohistochemical analysis was performed on the tumor tissues of the nude mice to detect the important marker of osteosarcoma proliferation.

[0103] KI-67 protein is an important marker of cell proliferation, and its expression level can accurately reflect cell cycle activity. In osteosarcoma, high expression of KI-67 protein often indicates strong proliferative capacity and poor clinical prognosis. Therefore, KI-67 protein is an important reference index for the biological behavior of osteosarcoma. In this embodiment, the expression of KI-67 protein in the tumor tissues of the nude mice in Example 9 was detected by immunohistochemical analysis.

[0104] The results of the immunohistochemical analysis are shown in Figure 28 The results show that the expression of KI-67 protein in the tumor tissues of osteosarcoma is significantly reduced after knocking down ADRM1 compared with the negative control group, and the two are positively correlated, further indicating that ADRM1 plays an important role in the proliferation process of osteosarcoma cells.

[0105] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of ADRM1-targeting shRNA in the preparation of drugs for treating osteosarcoma, characterized by: The sequence of the shRNA is shown in SEQ ID NO.5 or SEQ ID NO.

6.

2. The application as described in claim 1, characterized in that: The shRNA was packaged using a lentiviral vector and stably transfected into osteosarcoma cells.

3. The application as described in claim 2, characterized in that: The osteosarcoma cells are either 143b cells or U2OS cells.

4. The application as described in claim 3, characterized in that: The drug significantly reduces the short-term and long-term proliferative capacity of osteosarcoma cells.

5. The application as described in claim 4, characterized in that: The drug treats osteosarcoma by inhibiting the expression of the ADRM1 gene and reducing the synthesis of the protein encoded by the ADRM1 gene.

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