Application of ADRM1 gene / protein in preparation of medicine for treating osteosarcoma

Abstract

The invention relates to the technical field of biological medicines, and provides application of ADRM1 gene / protein in preparation of a medicine for treating osteosarcoma. When the ADRM1 gene / protein is applied to the preparation of the medicine for treating osteosarcoma, the proliferation, migration and invasion capabilities of tumor cells can be remarkably inhibited, so that the occurrence and development of osteosarcoma are delayed. The expression of the ADRM1 is knocked down through an shRNA intervention technology, the mRNA and protein level of the ADRM1 in 143b and U2OS osteosarcoma cell lines can be remarkably reduced, the short-term proliferative activity and the long-term clone forming ability of the ADRM1 are further inhibited, and the migration and invasion characteristics of cells are effectively weakened. The growth of a tumor body formed after a nude mouse is inoculated with 143b osteosarcoma cells knocking down ADRM1 is obviously slowed down, and the expression of a cell proliferation marker KI-67 in the tumor body is reduced, which indicates that the ADRM1 gene / protein also plays a key regulation role in the in-vivo growth process of osteosarcoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of ADRM1 gene / protein in the preparation of drugs for treating osteosarcoma. Background Art

[0002] Osteosarcoma (OS) is a highly malignant primary bone tumor originating from mesenchymal tissue. Its primary pathological feature is the direct production of immature bone or osteoid matrix by tumor cells. It is predominantly seen in adolescents and children, making it the most common primary malignant bone tumor in this population. Osteosarcoma typically develops in the metaphysis of long bones, most commonly in the distal femur, proximal tibia, and proximal humerus. In some cases, axial bones such as the pelvis may be involved. The disease is characterized by rapid growth, strong local invasiveness, and a high rate of early metastasis. It leads to rapid clinical progression and a poor prognosis, posing a serious threat to the life and quality of life of adolescents.

[0003] Although the current standard treatment for osteosarcoma includes neoadjuvant chemotherapy, limb-sparing surgery, and postoperative adjuvant chemotherapy, the 5-year overall survival rate for patients with localized osteosarcoma has improved. However, long-term survival remains a significant challenge for patients with lung metastasis or drug resistance, and the 5-year survival rate remains low. Furthermore, traditional chemotherapy drugs present significant toxicity and individual variability in efficacy, with some patients exhibiting significant resistance to multidrug combination regimens, limiting the effectiveness of treatment. For patients with postoperative recurrence, distant metastasis, or chemotherapy insensitivity, there is an urgent need for more effective and safe treatments. Furthermore, while signaling pathway-targeted drugs have achieved some success in other tumors (such as lung cancer and breast cancer), their application in osteosarcoma is still in the early stages of research, with no breakthroughs yet.

[0004] Therefore, in the context of limited efficacy of existing treatments and lack of targeted treatment options, developing new anti-osteosarcoma drugs with clear mechanisms of action, definite efficacy and high safety, identifying new molecular targets and translating them into clinical applications are of great significance for improving the treatment effect and long-term survival of osteosarcoma patients. This is in line with the current development trend of tumor treatment from "broad-spectrum chemotherapy" to "precision targeting" and "individualized treatment", and has significant clinical value and social needs. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of ADRM1 gene / protein in the 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 a key subunit of the 26S proteasome regulatory particle. It is widely distributed in eukaryotic cells and participates in biological processes such as protein degradation, deubiquitination regulation, cell adhesion, and proliferation. ADRM1 recognizes ubiquitin chains through its domains and recruits the deubiquitinating enzyme UCHL5, regulating protein homeostasis and playing a key role in maintaining cellular function. Previous studies have shown that ADRM1 has a role in bone metabolism-related diseases. For example, studies have shown that ADRM1 can alleviate cartilage degeneration by enhancing UCH37-mediated ALK5 deubiquitination, leading to its proposed use in the treatment of osteoarthritis. Other studies have found that inhibiting ADRM1 expression can induce osteoblast differentiation and inhibit osteoclast function, thus playing a role in regulating bone metabolism and treating osteoporosis.

[0007] However, osteosarcoma is a highly lethal malignant tumor whose pathogenesis differs significantly from bone metabolic diseases such as osteoarthritis and osteoporosis. Osteoarthritis and osteoporosis primarily involve dysfunction of chondrocytes, osteoblasts, and osteoclasts and are chronic, non-neoplastic diseases. In contrast, osteosarcoma is highly invasive and metastatic, primarily caused by the malignant transformation of bone marrow mesenchymal stem cells. Its core mechanisms involve typical molecular hallmarks of malignant tumors, such as oncogene activation, tumor suppressor gene inactivation, and chromosomal instability.

[0008] The present invention provides the use of ADRM1 gene / protein in preparing a drug for treating osteosarcoma. The drug treats osteosarcoma by inhibiting the expression of the ADRM1 gene or protein.

[0009] Furthermore, the drug inhibits the expression of ADRM1 gene or protein, thereby inhibiting the proliferation, migration and invasion of osteosarcoma cells, thereby delaying or blocking the progression of osteosarcoma.

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

[0011] Furthermore, the RNA interference molecule includes siRNA, miRNA, shRNA or dsRNA.

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

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

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

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

[0016] Furthermore, the drug also includes small molecule compounds that inhibit ADRM1 gene transcription, protein post-translational modification, or directly bind to and inhibit ADRM1 protein activity.

[0017] The present invention has the following technical effects: the application of ADRM1 gene / protein in the preparation of drugs for the treatment of osteosarcoma can significantly inhibit the proliferation, migration and invasion of tumor cells, thereby delaying the occurrence and development of osteosarcoma. Knocking down the expression of ADRM1 through shRNA intervention technology can significantly reduce the mRNA and protein levels of ADRM1 in 143b and U2OS osteosarcoma cell lines, thereby inhibiting their short-term proliferation activity and long-term clone formation ability, and effectively weakening the migration and invasion characteristics of the cells. In vivo experiments further showed that the growth of tumors formed after 143b osteosarcoma cells with ADRM1 knockdown were significantly slowed down after inoculation into nude mice, and the expression of the cell proliferation marker KI-67 in the tumor was downregulated, 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 the treatment of osteosarcoma, can be used for drug screening and research and development, and has good application prospects and translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 These are immunohistochemical staining images of the ADRM1 protein expression levels in osteosarcoma tumor tissue and adjacent normal tissue in Example 1 of the present invention; magnification ×200, scale bar: 50 μm.

[0019] Figure 2 These are the quantitative analysis results of ADRM1 protein expression levels in osteosarcoma tumor tissues and adjacent normal tissues analyzed by immunohistochemical staining in Example 1 of the present invention, where *** indicates p<0.001.

[0020] Figure 3 The figure shows the ADRM1 mRNA expression levels in the normal osteoblast cell line hFOB1.19 and different osteosarcoma cell lines (143b, MG63, and U2OS) detected by qRT-PCR in Example 2 of the present invention; compared with the osteoblast cell line hFOB1.19, *** indicates p < 0.001.

[0021] Figure 4 The following is a picture showing the expression of ADRM1 protein in the normal osteoblast cell line hFOB1.19 and different osteosarcoma cell lines (143b, MG63, and U2OS) detected by Western blotting in Example 3 of the present invention.

[0022] Figure 5 The figures are quantitative analysis images of ADRM1 protein expression in the normal osteoblast cell line hFOB1.19 and different osteosarcoma cell lines (143b, MG63, and U2OS) detected by Western blotting in Example 3 of the present invention; compared with the osteoblast cell line hFOB1.19, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0023] Figure 6 This is a picture showing the ADRM1 mRNA expression level in 143b osteosarcoma cells after stable shRNA transfection detected by qRT-PCR in Example 4 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0024] Figure 7 This is a picture showing the ADRM1 mRNA expression level in U2OS osteosarcoma cells after stable shRNA transfection detected by qRT-PCR in Example 4 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0025] Figure 8 This is a picture showing the expression of ADRM1 protein in 143b osteosarcoma cells after stable shRNA transfection detected by Western blotting in Example 4 of the present invention.

[0026] Figure 9 The results of quantitative analysis of ADRM1 protein expression in 143b osteosarcoma cells stably transfected with shRNA detected by Western blotting in Example 4 of the present invention are shown; compared with the negative control group, *** indicates p < 0.001.

[0027] Figure 10 This is a picture showing the expression of ADRM1 protein in U2OS osteosarcoma cells after stable shRNA transfection detected by Western blotting in Example 4 of the present invention.

[0028] Figure 11 The results of quantitative analysis of ADRM1 protein expression in U2OS osteosarcoma cells stably transfected with shRNA detected by Western blotting in Example 4 of the present invention are shown; compared with the negative control group, *** indicates p < 0.001.

[0029] Figure 12 The effect of knocking down ADRM1 on the short-term proliferation ability of 143b osteosarcoma cells was detected by CCK-8 cell proliferation assay in Example 5 of the present invention; compared with the negative control group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0030] Figure 13This is the effect of knocking down ADRM1 on the short-term proliferation ability of U2OS osteosarcoma cells detected by CCK-8 cell proliferation assay in Example 5 of the present invention; compared with the negative control group, * indicates p < 0.05, and *** indicates p < 0.001.

[0031] Figure 14 This is a graph showing the effect of ADRM1 knockdown on the long-term proliferation ability of 143b osteosarcoma cells detected by a plate cell clone formation experiment in Example 6 of the present invention.

[0032] Figure 15 These are the quantitative analysis results of the effect of ADRM1 knockdown on the long-term proliferation ability of 143b osteosarcoma cells detected by the plate cell clone formation assay in Example 6 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0033] Figure 16 This is a graph showing the effect of ADRM1 knockdown on the long-term proliferation ability of U2OS osteosarcoma cells detected by a plate cell clone formation experiment in Example 6 of the present invention.

[0034] Figure 17 These are the quantitative analysis results of the effect of ADRM1 knockdown on the long-term proliferation ability of U2OS osteosarcoma cells detected by the plate cell clone formation assay in Example 6 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0035] Figure 18 This is a graph showing the effect of knocking down ADRM1 on the migration ability of 143b osteosarcoma cells detected by a scratch assay in Example 7 of the present invention.

[0036] Figure 19 This is the quantitative analysis result of the effect of knocking down ADRM1 on the migration ability of 143b osteosarcoma cells detected by the scratch assay in Example 7 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0037] Figure 20 This is a graph showing the effect of knocking down ADRM1 on the migration ability of U2OS osteosarcoma cells detected by a scratch assay in Example 7 of the present invention.

[0038] Figure 21 These are the quantitative analysis results of the effect of knocking down ADRM1 on the migration ability of U2OS osteosarcoma cells detected by the scratch assay in Example 7 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0039] Figure 22 This is a graph showing the effect of knocking down ADRM1 on the invasion ability of 143b osteosarcoma cells detected by a chamber migration assay in Example 8 of the present invention.

[0040] Figure 23 This is the quantitative analysis result of the effect of knocking down ADRM1 on the invasion ability of 143b osteosarcoma cells detected by chamber migration assay in Example 8 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0041] Figure 24 This is a graph showing the effect of knocking down ADRM1 on the invasion ability of U2OS osteosarcoma cells detected by a chamber migration assay in Example 8 of the present invention.

[0042] Figure 25 These are the quantitative analysis results of the effect of knocking down ADRM1 on the invasion ability of U2OS osteosarcoma cells detected by chamber migration assay in Example 8 of the present invention; compared with the negative control group, *** indicates p < 0.001.

[0043] Figure 26 This is a graph showing the results of evaluating the effect of knocking down ADRM1 on the in vivo growth of osteosarcoma using a nude mouse osteosarcoma subcutaneous tumor model in Example 9 of the present invention.

[0044] Figure 27 These are the quantitative analysis results of the effect of knocking down ADRM1 on the in vivo growth of osteosarcoma evaluated by the nude mouse osteosarcoma subcutaneous tumor model in Example 9 of the present invention; compared with the negative control group, ** indicates p < 0.01, and *** indicates p < 0.001.

[0045] Figure 28 This is a graph showing the results of immunohistochemical staining of nude mouse tumors to detect the expression of ADRM1 and KI-67 proteins in Example 10 of the present invention; magnification ×200, scale bar: 50 μm. DETAILED DESCRIPTION

[0046] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] 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 invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0048] An embodiment of the present invention provides the use of the ADRM1 gene / protein in the preparation of a drug for treating osteosarcoma. The drug treats osteosarcoma by inhibiting the expression of the ADRM1 gene or protein.

[0049] In some embodiments, the drug delays or blocks the progression of osteosarcoma by inhibiting the expression of ADRM1 gene or protein, thereby inhibiting the proliferation, migration and invasion of osteosarcoma cells.

[0050] In some embodiments, the drug comprises an RNA interference molecule that inhibits the expression of the ADRM1 gene or protein.

[0051] In some embodiments, the RNA interference molecule comprises siRNA, miRNA, shRNA, or dsRNA.

[0052] In some embodiments, the sequence of the shRNA is shown as SEQ ID NO. 5 or SEQ ID NO. 6.

[0053] In some embodiments, shRNA is packaged using a lentiviral vector and stably transfected into osteosarcoma cells.

[0054] In some embodiments, the osteosarcoma cells are 143b cells or U2OS cells.

[0055] In some embodiments, the drug significantly reduces both the short-term and long-term proliferation capacity of osteosarcoma cells.

[0056] In some embodiments, the drug further includes small molecule compounds that inhibit ADRM1 gene transcription, protein post-translational modification, or directly bind to and inhibit ADRM1 protein activity.

[0057] Example 1: Clinical samples have shown that ADRM1 gene / protein affects the occurrence and development of osteosarcoma.

[0058] Methods: Tumor samples and clinical data were collected from 10 osteosarcoma patients who underwent first surgery and had not undergone chemoradiotherapy. All patients signed informed consent forms. The ethics review approval number was (2025)CDYFYYLK(01-058). Immunohistochemical analysis was performed on tumor tissues and adjacent normal tissues of osteosarcoma patients.

[0059] Immunohistochemical analysis Figure 1 and Figure 2 As shown, the results showed that compared with the normal tissue adjacent to the tumor, the expression level of ADRM1 protein in tumor tissue was significantly increased, suggesting that ADRM1 protein plays a key role in the occurrence and development of osteosarcoma.

[0060] Example 2: The expression levels of ADRM1 gene in normal osteoblast cell lines and different osteosarcoma cell lines were detected by qRT-PCR (quantitative reverse transcription polymerase chain reaction).

[0061] 143b, MG63, U2OS osteosarcoma cell lines and normal osteoblast cell line hFOB1.19 were selected and the above cells were cultured at 2×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates. After cells reached logarithmic phase, total RNA 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 transcript levels of the target genes were analyzed using the TB Green Premix Ex Taq II kit. The primer sequences for the ADRM1 and β-actin genes are shown in Table 1. β-actin was used as an internal reference gene, and the relative mRNA expression of the ADRM1 gene was calculated using the 2-ΔΔCt method.

[0062] Table 1 Sequences of primers before and after ADRM1 and β-actin genes

[0063] The calculation results of the relative expression of ADRM1 gene mRNA are as follows Figure 3 As shown, the results showed that compared with the osteoblast cell line hFOB1.19, the expression level of ADRM1 gene in 143b, MG63 and U2OS osteosarcoma cell lines was significantly increased.

[0064] Example 3: Western blotting was used to detect the expression level of ADRM1 protein in normal osteoblast cell lines and different osteosarcoma cell lines.

[0065] 143b, MG63, U2OS osteosarcoma cell lines and hFOB1.19 normal osteoblast cell line were used Cells were seeded at a density of 100 cells / well in a 6-well plate and grown to logarithmic phase for protein extraction. Protein extraction was performed using RIPA lysis buffer containing protease and phosphatase inhibitors. The RIPA lysis buffer was incubated on ice for 30 minutes with vortexing every 5 minutes, followed by centrifugation at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected and protein concentration was determined using a BCA protein assay kit according to the manufacturer's instructions. Protein samples (30 μg / well) were separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk overnight at 4°C and then incubated with the primary antibody for at least 8 hours. The membrane was then washed three times with TBST and incubated with a secondary antibody (1:5000) for 2 hours at room temperature. Bands on the membrane were detected using a gel imaging and western blot imaging system. Band intensities were quantified using ImageJ software, using β-actin as a loading control.

[0066] Western blotting results Figure 4 and Figure 5 As shown, the results showed that the expression level of ADRM1 protein in 143b, MG63 and U2OS osteosarcoma cell lines was significantly increased compared with the osteoblast cell line hFOB1.19.

[0067] Combined with the qRT-PCR experimental results in Example 2, this further demonstrates that the ADRM1 gene / protein may play a key role in the development and progression of osteosarcoma. Because ADRM1 protein expression levels are high in the 143b and U2OS osteosarcoma cell lines, these cell lines were used in subsequent examples for cell function studies.

[0068] Example 4: ADRM1 knockdown was constructed in 143b and U2OS osteosarcoma cell lines by stable shRNA (short hairpin RNA) transfection.

[0069] Stable knockdown of ADRM1 expression in 143b and U2OS osteosarcoma cell lines was established using lentiviral packaging and stable transfection of shRNA plasmids. In this example, osteosarcoma cells in the blank control group were untreated, while those in the negative control group were transfected with a non-targeting shRNA sequence. The osteosarcoma cells in Experimental Groups 1 and 2 were transfected with two different targeted shRNA sequences to knock down ADRM1 gene expression: shRNA1 in Experimental Group 1 and shRNA2 in Experimental Group 2. Both sequences were highly effective knockdown sequences screened in preliminary experiments. After transfection, knockdown efficiency was verified by qRT-PCR and Western blotting.

[0070] Table 2 shRNA1 and shRNA2 sequences

[0071] The results of qRT-PCR were as follows Figure 6-Figure 7 As shown, the results showed that the expression level of ADRM1 gene in 143b and U2OS osteosarcoma cell lines was successfully reduced by stable shRNA transfection.

[0072] Western blotting results were as follows Figures 8-11 As shown, the results showed that the expression level of ADRM1 protein in 143b and U2OS osteosarcoma cell lines was successfully reduced by stable shRNA transfection.

[0073] Example 5: The CCK-8 cell proliferation assay was used to examine the effect of ADRM1 knockdown on the short-term proliferation of osteosarcoma cells. In the negative control group, 143b and U2OS osteosarcoma cells were transfected with non-targeting shRNA sequences, while in the experimental group, 143b and U2OS osteosarcoma cells were transfected with targeted shRNA1 sequences to knock down ADRM1.

[0074] 143b and U2OS osteosarcoma cells in the logarithmic growth phase were selected, resuspended in complete culture medium and adjusted to a density of Inoculate 100 μL of the suspension into each well of a 96-well plate. Fill the peripheral wells with phosphate buffered saline to minimize edge effects. Place the plate in a constant humidity incubator for 18-24 hours to allow the cells to attach and enter a stable growth phase. After that, add CCK-8 working solution and continue incubation for 2.5 hours in the dark. Measure the absorbance (OD) of each well at 450 nm on a microplate reader. Repeat the readings three times for each well and take the average value.

[0075] The results of CCK-8 cell proliferation assay were as follows: Figure 12 and Figure 13 As shown, the results showed that compared with the negative control group, the short-term proliferation ability of 143b and U2OS osteosarcoma cells was significantly inhibited after knockdown of ADRM1.

[0076] Example 6: The effect of ADRM1 knockdown on the long-term proliferation of osteosarcoma cells was examined by plate-based cell colony formation assays. In the negative control group, 143b and U2OS osteosarcoma cells were transfected with non-targeting shRNA sequences, while in the experimental group, 143b and U2OS osteosarcoma cells were transfected with targeted shRNA1 sequences to knock down ADRM1.

[0077] 143b and U2OS osteosarcoma cells in the logarithmic growth phase were resuspended in complete culture medium and adjusted to a density of 500 cells / mL before being seeded into 6-well plates. After static culture for 10-14 days until colonies became visible, the cells were rinsed with phosphate buffer, fixed with 4% paraformaldehyde for 15 minutes, and stained with 0.1% crystal violet for 20 minutes. The cells were rinsed with running water and dried in a laminar flow zone at 37°C in a clean bench. High-definition images were captured with a digital camera, and colonies of ≥50 cells were counted using ImageJ software to calculate the formation rate.

[0078] The results of the plate cell clone formation experiment are as follows Figure 14-17 As shown, the results showed that compared with the negative control group, the long-term proliferation ability of 143b and U2OS osteosarcoma cells was significantly inhibited after knockdown of ADRM1.

[0079] The experimental results of Examples 5 and 6 indicate 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 cloning ability in the long term, thereby inhibiting the proliferation of osteosarcoma cells as a whole.

[0080] Example 7: The effect of ADRM1 knockdown on the migration ability of osteosarcoma cells was examined by wound healing assay. 143b and U2OS osteosarcoma cells in the negative control group were transfected with non-targeting shRNA sequences, while 143b and U2OS osteosarcoma cells in the experimental group were transfected with targeted shRNA1 to knock down ADRM1.

[0081] Take 143b and U2OS osteosarcoma cells in the logarithmic growth phase, resuspend them in complete culture medium and adjust the density to / mL. Inoculate in a 6-well plate, set the edge wells to be filled with 2mL phosphate buffer to reduce evaporation interference. Culture at 37°C and 5% CO2 saturated humidity. After the monolayer cell confluence reaches 90-95%, use a 200μL sterile pipette tip to apply force perpendicular to the bottom of the well plate to draw three parallel linear damage areas with a spacing of 8mm at a constant rate. Immediately rinse three times with pre-cooled phosphate buffer. Replace the serum-free culture medium and mark the fixed field of view at the starting end, middle end and end of the scratch. Take another picture and record it 24 hours after taking the baseline image. The calculation of the scratch healing rate is based on the measurement of changes in the scratch area using ImageJ software. The specific method is: after the scratch experiment is carried out for 24 hours, the area occupied by cell migration in the scratch area is divided by the total area of ​​the scratch area at the beginning of the experiment, and the resulting ratio is the scratch healing rate.

[0082] The scratch test results are as follows Figures 18-21 As shown, the results showed that compared with the negative control group, the scratch healing rate of 143b and U2OS osteosarcoma cells decreased and the cell migration ability was reduced after knockdown of ADRM1.

[0083] Example 8: The effect of ADRM1 knockdown on the invasion ability of osteosarcoma cells was examined by chamber migration assay. 143b and U2OS osteosarcoma cells in the negative control group were transfected with non-targeting shRNA sequences, while 143b and U2OS osteosarcoma cells in the experimental group were transfected with targeted shRNA1 to knock down ADRM1.

[0084] Take 143b and U2OS osteosarcoma cells in the logarithmic growth phase, resuspend them in complete culture medium and adjust the density to The lower chamber of the Transwell was filled with 600 μL of chemotaxis medium containing 10% fetal bovine serum, and the upper chamber was precisely loaded with 200 μL of cell suspension. Migration was allowed to continue at 37°C, 5% CO2, and saturated humidity for 24 hours. After that, the upper chamber liquid was removed and the membrane was gently rinsed three times with phosphate buffer. The upper surface of the membrane was gently wiped with a cotton swab dipped in phosphate buffer. The membrane was fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 20 minutes. The chamber was inverted on a glass slide, and the cells that had penetrated the membrane in the Transwell chamber were counted using ImageJ software. Each experiment was repeated three times independently. Five microscopic fields were randomly selected for counting in each experiment, and the average value was calculated for statistical analysis.

[0085] The results of the chamber migration experiment were as follows Figure 22-Figure 25 As shown, the results showed that compared with the negative control group, the number of invasive cells and the invasive ability of 143b and U2OS osteosarcoma cells decreased after knockdown of ADRM1.

[0086] Example 9: A nude mouse osteosarcoma subcutaneous tumor model was established to evaluate the effect of ADRM1 knockdown on osteosarcoma growth in vivo.

[0087] Four-week-old SPF-grade BALB / c-nu athymic nude mice were selected with balanced gender distribution. They were adaptively raised in an AAALAC-certified barrier environment for 7 days, during which they were given sterilized irradiated feed and acidified drinking water. The nude mice were raised in an adaptive breeding environment for 1 week and then randomly divided into four groups after adaptation, with 5 mice in each group: experimental group 1 (inoculated with 143b osteosarcoma cells that knocked down ADRM1 using the shRNA1 sequence), experimental group 2 (inoculated with 143b osteosarcoma cells that knocked down ADRM1 using the shRNA2 sequence), blank control group (inoculated with 143b osteosarcoma cells that were not treated in any way), and negative control group (inoculated with 143b osteosarcoma cells transfected with non-targeted shRNA sequences). After cell inoculation, the tumor volume was measured every 3 days. The three-dimensional diameter of the tumor was measured daily with a digital vernier caliper, and the tumor volume was measured according to the 3D diameter of the tumor. Tumor volume was calculated using the formula. When tumor volume reached or exceeded 1500 mm³, weight loss exceeded 20%, or the nude mice exhibited significant mobility impairment, the mice were euthanized by cervical dislocation and tumor tissue was collected. The experimental protocol was approved by the Animal Ethics Committee (Approval Number: CDYFY-IACUC-202404QR025).

[0088] The volume and weight of the tumor were Figure 26 and Figure 27 As shown, the results showed that compared with the negative control group, the growth of osteosarcoma in nude mice was significantly inhibited after knocking down ADRM1.

[0089] Example 10: Immunohistochemical analysis of nude mouse tumors was performed to detect important markers of osteosarcoma proliferation.

[0090] KI-67 protein is an important marker of cell proliferation, and its expression level accurately reflects cell cycle activity. In osteosarcoma, high KI-67 protein expression often indicates strong proliferation and poor clinical prognosis. Therefore, KI-67 protein is an important reference indicator of osteosarcoma biological behavior. In this example, immunohistochemical analysis was performed on the tumors of each group of nude mice described in Example 9 to detect the expression of KI-67 protein.

[0091] Immunohistochemical analysis results Figure 28 As shown, the results showed that compared with the negative control group, the expression of KI-67 protein in osteosarcoma tumors was significantly reduced after knocking down ADRM1, and the two were positively correlated, further indicating that ADRM1 plays an important role in the proliferation of osteosarcoma cells.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of the ADRM1 gene / protein in the preparation of a drug for treating osteosarcoma, characterized in that: The drug treats osteosarcoma by inhibiting the expression of ADRM1 gene or protein.

2. The use according to claim 1, characterized in that: The drug inhibits the expression of ADRM1 gene or protein, thereby inhibiting the proliferation, migration and invasion of osteosarcoma cells, thereby delaying or blocking the progression of osteosarcoma.

3. The use according to claim 2, characterized in that: The drug includes RNA interference molecules that inhibit the expression of ADRM1 gene or protein.

4. The use according to claim 3, characterized in that: The RNA interference molecules include siRNA, miRNA, shRNA or dsRNA.

5. The use according to claim 4, characterized in that: The sequence of the shRNA is shown as SEQ ID NO.5 or SEQ ID NO.

6.

6. The use according to claim 5, characterized in that: The shRNA is packaged by a lentiviral vector and stably transfected into osteosarcoma cells.

7. The use according to claim 6, characterized in that: The osteosarcoma cells are 143b cells or U2OS cells.

8. The use according to claim 7, characterized in that: The drug significantly reduces the short-term and long-term proliferation capabilities of osteosarcoma cells.

9. The use according to claim 3, characterized in that: The drugs also include small molecule compounds that inhibit ADRM1 gene transcription, protein post-translational modification, or directly bind to and inhibit ADRM1 protein activity.

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