A marker for resistance to bone sarcoma chemotherapy and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a marker for anti-osteosarcoma chemotherapy resistance and application thereof. The marker for anti-osteosarcoma chemotherapy resistance provided by the present application is SOCS1. The present application also provides application of the marker in preparation of a drug for diagnosing and / or treating anti-osteosarcoma chemotherapy resistance. The present application determines the mechanism of anti-osteosarcoma chemotherapy resistance by studying the action principle and expression mechanism of anti-osteosarcoma chemotherapy resistance, and finds that SOCS1 expression is down-regulated in anti-osteosarcoma chemotherapy resistance cell lines. Therefore, the present application takes SOCS1 as a marker for anti-osteosarcoma chemotherapy resistance, provides a new treatment strategy for osteosarcoma chemotherapy resistance, and has important academic value and good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to a biomarker for chemotherapy resistance in osteosarcoma and its application. Background Technology

[0002] Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Although it accounts for only about 5% of childhood and adolescent cancers, it has a significant impact on childhood cancer mortality. Osteosarcoma can be diagnosed in any bone, but the most common site is the metaphysis of long bones, primarily the distal femur, followed by the proximal tibia and proximal humerus. Less than 10% of pediatric cases are diagnosed in the axial skeleton. Of these, approximately 20% of patients are diagnosed with metastatic lesions at the time of diagnosis, most commonly in the lungs, followed by bone, lymph nodes, or other soft tissue lesions. The presence of metastases is a significant prognostic indicator.

[0003] In the 1970s and 1980s, the advent of neoadjuvant chemotherapy and advancements in limb-sparing surgery greatly improved the prognosis of osteosarcoma patients. The overall survival rate for osteosarcoma patients without metastatic lesions increased to 60%–70%, supporting the view that controlling micrometastases at diagnosis is essential for long-term survival. Neoadjuvant chemotherapy became the standard of care for osteosarcoma patients. However, for patients with metastatic lesions, despite aggressive resection and intensive systemic chemotherapy, the 5-year event-free survival (EFS) rate was only 20%. Relapsed patients also fared poorly, with a 10-year overall survival rate of only 20% or lower. Furthermore, approximately 40–50% of patients were insensitive to current chemotherapy regimens. In the last two or three decades, numerous studies on the pathogenesis of osteosarcoma, clinical trials of several new drugs, and studies employing intensified dosage strategies for standard chemotherapy have failed to successfully improve the prognosis of osteosarcoma patients.

[0004] To date, with standard treatment regimens of surgery and chemotherapy, the prognosis of osteosarcoma has significantly improved, with the 5-year survival rate increasing from 10%-20% to 60%-80%. Unfortunately, inherent or acquired resistance to chemotherapy, coupled with secondary metastasis, leads to treatment failure in osteosarcoma, resulting in a 5-year survival rate of approximately 20% for these patients. New evidence has revealed the molecular mechanisms underlying chemotherapy resistance in osteosarcoma, including reduced drug influx, increased drug efflux, altered drug targets, enhanced DNA repair, and resistance to cell death. Nevertheless, due to a lack of in-depth mechanistic studies and more effective chemotherapy resistance targets, osteosarcoma treatment has plateaued over the past three decades. It is evident that drug resistance is a major obstacle severely limiting the success of osteosarcoma treatment; the development of resistance and tumor metastasis leads to poor prognosis for recurrent patients, with a 5-year survival rate not exceeding 20-25%. Therefore, identifying, characterizing, and clinically validating resistance-related biomarkers, elucidating their underlying mechanisms, and searching for new potential therapeutic targets to combat chemotherapy resistance in osteosarcoma in order to utilize alternative treatment regimens are crucial and have significant clinical implications.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker for resistance to chemotherapy in osteosarcoma and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A biomarker for resistance to chemotherapy in osteosarcoma, wherein the biomarker is SOCS1.

[0009] This invention clarifies the mechanism of drug resistance to osteosarcoma by studying the mechanism of action and expression of anti-chemotherapy resistance. It was found that SOCS1 expression is downregulated in anti-chemotherapy resistant cell lines. Therefore, this invention uses SOCS1 as a marker of anti-chemotherapy resistance to osteosarcoma, providing a new treatment strategy for osteosarcoma chemotherapy resistance, which has important academic value and good clinical application prospects.

[0010] The present invention also provides the use of the aforementioned biomarkers in the preparation of medicaments for the diagnosis and / or treatment of osteosarcoma chemotherapy resistance.

[0011] Furthermore, the drug restores and enhances the sensitivity of anti-osteosarcoma drugs by upregulating SOCS1 expression.

[0012] Furthermore, the drug restores and enhances the sensitivity to anti-osteosarcoma drugs by upregulating SOCS1 expression through the inhibition of SLC7A11 expression, which promotes ferroptosis.

[0013] Furthermore, the aforementioned anti-osteosarcoma drug is an anti-osteosarcoma chemotherapy drug.

[0014] Furthermore, the aforementioned anti-osteosarcoma chemotherapy drug is a platinum-based anti-osteosarcoma chemotherapy drug.

[0015] The present invention also provides a diagnostic and / or therapeutic agent for chemotherapy resistance to osteosarcoma, wherein the diagnostic agent is an agent that overexpresses SOCS1.

[0016] Furthermore, the SOCS1 overexpression formulation restores and enhances the sensitivity of anti-osteosarcoma drugs by upregulating SOCS1 expression through increased m6A methylation of SOCS1.

[0017] Furthermore, the SOCS1 overexpression formulation is an FTO inhibitor.

[0018] Furthermore, the FTO inhibitor is FB23 / 2.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention clarifies the mechanism of drug resistance to osteosarcoma by studying the mechanism of action and expression of anti-chemotherapy resistance. It was found that SOCS1 expression is downregulated in anti-chemotherapy resistant cell lines. Therefore, this invention uses SOCS1 as a marker of anti-chemotherapy resistance to osteosarcoma, providing a new treatment strategy for osteosarcoma chemotherapy resistance, which has important academic value and good clinical application prospects. Attached Figure Description

[0021] Figure 1 To establish drug-resistant cell lines and screen SOCS1 using high-throughput sequencing;

[0022] Figure 2 This study has demonstrated in cell and animal experiments that SOCS1 enhances the chemosensitivity of osteosarcoma.

[0023] Figure 3 To analyze the expression level and significance of SOCS1 at the clinical tissue level;

[0024] Figure 4 Analysis of the classic functional mechanism of SOCS1 and identification of new mechanisms;

[0025] Figure 5 SOCS1 regulates osteosarcoma drug resistance through SLC7A11-mediated ferroptosis;

[0026] Figure 6 FTO inhibitors can relieve osteosarcoma drug resistance by upregulating SOCS1 expression. Detailed Implementation

[0027] The following are specific embodiments of the present invention. These embodiments are intended to further describe the present invention and are not intended to limit the present invention.

[0028] Experiment Example 1: Correlation between SOCS1 and resistance to anti-osteosarcoma chemotherapy

[0029] 1. Materials and Methods

[0030] 1.1 Patient Samples

[0031] A total of 29 osteosarcoma tumors and 8 adjacent normal tissue samples (including 8 pairs of samples) were obtained from osteosarcoma patients who underwent osteotomy or ultrasound-guided bone biopsy prior to chemotherapy and / or radiotherapy at Tianjin Medical University Cancer Hospital. Patient clinicopathological information was obtained from patient records. Each patient signed a written consent form post-surgery, agreeing to the use of their tissues for research purposes. All collected tissues were preserved in liquid nitrogen. The progression and prognosis of osteosarcoma patients were assessed by CT scans before and after comprehensive treatment. Chemotherapy-resistant osteosarcoma patients (11 cases) were defined as having a post-treatment tissue necrosis rate of less than 90% and significant recurrence in bone, lung, or lymph nodes. Conversely, chemotherapy-sensitive osteosarcoma patients (9 cases) had a post-treatment tissue necrosis rate greater than 90%; the determination was not made in the other 9 osteosarcoma patients. In addition, 71 samples used for immunohistochemical (IHC) staining analysis via tissue microarray (TMA) included one normal bone tissue and 70 osteosarcoma tissues from 2 stage IA patients, 31 stage IIA patients, 31 stage IIB patients, and 6 stage IVB patients (Bioaitech, cat.no.1714901; Xi'an, China). This study was authorized and supervised by the Ethics Committee of Tianjin Medical University Cancer Hospital.

[0032] 1.2 Plasmid reagents

[0033] The full-length SOCS1 (NM_003745.2; GenBank) gene fragment was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the pCDH-CMV-MCS-EF1-RFP-T2A-Puro lentiviral vector and pCDNA3.1-HA tag vector, respectively. The full-length SLC7A11 (NM_014331.4; GenBank) gene fragment was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the pEGFP-N1 and pCMV-3Xflag-tag vectors, respectively. Small hairpin RNA (shRNA) sequences targeting human SOCS1, SLC7A11, METTL3, METTL14, WTAP, FTO, YTHDF2, ALKBH5, and negative control were obtained from Qingke Biotechnology Co., Ltd. (Nanjing, China) and cloned into the pLKO.1 lentiviral vector (Sigma, USA). A stable SOCS1 knockout cell line was constructed using the lentiviral vector pLV[CRISPR]-hCas9:T2A:Puro-U6>hSOCS1[gRNA#266](VB900132-1543xcp, Vector Builder Inc, Guangzhou, China).

[0034] 1.3 Constructing stable cell lines

[0035] use Invitrogen Life Technologies (USA) used lentiviral vectors with pCDH, pLKO.1, or pLV as backgrounds, along with packaging plasmid psPAX2 (Addgene, Inc., USA) and envelope plasmid pMD2.G (Addgene, Inc., USA), to infect 293T cells. After 48 hours of culture, the supernatant was collected and filtered through a 0.45 μm PES filter. Recipient cells were then infected with the packaged lentiviral supernatant, and the medium was replaced with fresh medium 24 hours after infection. 72 hours post-infection, stable clones were screened using 2 μg / ml puromycin (A1113803, Thermo Fisher Scientific, USA). For gene overexpression and knockout, cells were screened for 10 days using puromycin. Gene transfection efficiency was verified by real-time quantitative PCR (RT-qPCR) and Western blotting. For gene knockout screening and identification, cell gradient dilution was used to screen for monoclonal mutant cells. To assess the efficiency of Cas9 / sgRNA production, Western blot analysis was performed on cell lysates.

[0036] 1.4 Xenograft Tumor Mouse Model

[0037] Nude mice (5 weeks old, male, BALB / C-nu / nu, SPF Biotechnology Co., Ltd., Beijing, China) were randomly selected for ectopic tumor construction. Lentiviral vectors carrying different genes, including human SOCS1 gene (OESOCS1), SLC7A11 gene (OESOCS1), SOCS1 shRNA, or their negative control cells (OENC or shNC) (5 × 10⁻⁶ cells) were used. 6 Administer the indicated drug ( / 100μL). Starting from day 10, administer PBS or cisplatin (MedChemExpress, 15663-27-1, dissolved in physiological saline, 3mg / kg) intraperitoneally every 5 days for a total of 4 times. Monitor tumor volume and body weight every 5 days or at each injection. Then measure tumor volume (V) using the standard formula V = L*W² / 2 (L = length, W = width). Sacrifice mice by cervical dislocation, remove xenograft tumors, photograph and cut into two parts. One half is fixed and embedded in paraffin for immunohistochemistry (IHC), and the other half is rapidly frozen in liquid nitrogen for later use. For nude mouse experiments, all animal operations and procedures were authorized by the Animal Protection Committee of Tianjin Medical University Cancer Hospital.

[0038] 1.5 Cell Culture

[0039] Human osteosarcoma cell lines 143B (ATCC, CRL-8303) and U2OS (ATCC, HTB-96), and human embryonic kidney cell line 293T (ATCC and CRL-1573) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell lines were cultured using DMEM medium (143B and 293T) or McCoy 5a modified medium (U2OS), supplemented with 10% heat-inactivated fetal bovine serum (Gibco, 16000044) and 1% penicillin / streptomycin (10000 mg / mL / 10000 U / mL, Gibco, 15140-122).

[0040] 1.6 Construction of cisplatin-resistant cell lines

[0041] U2OS and 143B cells were treated with cisplatin (MedChemExpress, 15663-27-1) at concentrations increasing from 250 ng / ml to 2.5 μg / ml for at least 6 months. Cisplatin-resistant cells were named U2OS-DDPr and 143B-DDPr. The resistant cells were cultured at a cisplatin concentration of 1 μg / ml, and incubated in drug-free medium for 3 days prior to the experiment.

[0042] 1.7. Generation and Infection of Lentivirals

[0043] To establish stable cell lines, lentiviruses were generated from 293T cells using the second-generation viral packaging system psPAX2 and the PMD2.G plasmid. Lentiviral viruses collected from the cell supernatant were used to infect osteosarcoma cells with 10 mg / mL polybrene (Sigma-Aldrich, St. Louis, MO) for 48 hours. Infected cells were then selected with 2 μg / mL puromycin for 7–10 days. Infection efficiency was verified by RT-qPCR or Western blot analysis.

[0044] 1.8 Cell proliferation experiment

[0045] Cell proliferation was analyzed using the CCK8 assay kit (Yeasen, 40203ES80, Shanghai, China). 1×10⁶ cells were used. 3 Cells were seeded in 96-well plates with five time gradients and five accessory wells per well. On days 0, 1, 2, 3, and 4, CCK-8 reagent was added to the culture medium in each well at a ratio of 1:10. After incubation at 37°C for 1 hour, the OD450 value of the cells was measured using a microplate reader (BioTek Instruments, Wino Osteosarcomaki, USA).

[0046] 1.9 Colony formation

[0047] Cells were seeded into six-well plates at a density of 500 osteosarcoma cells per well and cultured at 37°C and 5% CO2. Cells were treated with cisplatin (1 μg / mL) or DMSO for 10–14 days until colonies formed. Cells were washed three times with PBS, fixed with methanol for 20 minutes, and stained with 0.1% crystal violet for 20 minutes at room temperature. After rinsing with PBS, cell colonies were photographed and counted.

[0048] 1.10. Half-maximal inhibitory concentration (IC50)

[0049] IC50 values ​​were used to analyze the drug sensitivity of osteosarcoma cells. Osteosarcoma cells were seeded into 96-well plates at 5000 cells per well, replicated 5 times. After adhesion, complete culture medium containing different concentrations of the drug was added, and the cells were incubated at 37°C. After 24 hours, 100 μL of DMEM containing 10 μL of CCK-8 reagent was added to each well. After incubation at 37°C for 1 hour, OD450 values ​​were measured using a microplate reader (BioTek Instruments, Wino Osteosarcomaki, USA), and the IC50 values ​​were calculated using GraphPadPrism software.

[0050] 1.11 RNA sequencing (RNA-seq)

[0051] U2OS-DDPr and its parental cells were selected, and total RNA was extracted and sent to Beijing Novogene Technology Co., Ltd. for RNA-seq analysis to screen for genes associated with osteosarcoma resistance. The criteria for screening differentially expressed genes (DEGs) were |log2(FoldChange)|>1 and adjusted p-value <0.05. KEGG pathway and GO analyses were performed on DEGs to analyze potential molecular mechanisms and biological functions.

[0052] 1.12. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)

[0053] Total RNA was extracted from osteosarcoma cells or tissues using TRIeasy reagent (Yeasen, Shanghai, China), and then followed the instructions. First-strand cDNA was synthesized using Synthesis SuperMix (Yeasen, Shanghai, China). RT-qPCR was then performed using... The IIIOne Step RT-qPCR-SYBRGreen Kit (Yeasen, Shanghai, China) was used with a CFX Connect real-time PCR detection system (Bio-Rad, CA, USA). The relative expression of target RNA was calculated using the 2–ΔΔCt method, with GAPDH used as an internal control.

[0054] 1.13. Western Blot

[0055] Total protein was extracted from osteosarcoma cultured cells or tissues using RIPA lysis buffer (Solarbio, Beijing, China) and boiled at 100°C for 10 minutes with 5×SDS loading buffer. Equal volumes of denatured proteins were first electrophoresed by SDS-PAGE and transferred to a 0.45 μm polyvinylidene fluoride membrane. After blocking in 10% skim milk for 1.5 hours, the membrane was incubated overnight at 4°C with a specific primary antibody. The next day, the membrane was incubated with a secondary antibody at room temperature for 1 hour and then detected using ECL Western blot reagent (Yeasen, Shanghai, China). β-actin was used as an endogenous protein for normalization.

[0056] 1.14 Immunohistochemistry (IHC)

[0057] 4 μm tissue sections were dewaxed sequentially with xylene and rehydrated in a gradient of ethanol, followed by antigen retrieval using citrate antigen retrieval buffer (pH 6.0). The sections were then placed in 3% hydrogen peroxide solution to block endogenous peroxidase activity. After blocking with serum for 30 minutes at room temperature, the sections were incubated overnight at 4°C with the corresponding primary antibody. The next day, the tissue sections were incubated with secondary antibody at room temperature for 1 hour, followed by 3,3'-diaminobenzidine (ZSGB bio, Beijing, China) staining and counterstaining with Mayer hematoxylin. The semi-quantitative evaluation method for immunohistochemical results is as follows:

[0058] 1) Percentage score: Based on the percentage of antibody-positive stained cells, the lowest score is 0 and the highest score is 4.

[0059] 0 points: No positive stained cells were found.

[0060] 1 point: Percentage of positively stained cells <10%;

[0061] 2 points: 10% ≤ percentage of positively stained cells < 50%;

[0062] 3 points: 50% ≤ percentage of positively stained cells < 80%;

[0063] 4 points: Percentage of positively stained cells >80%.

[0064] 2) Intensity score: Based on the intensity score of positively stained cells, the lowest score is 0 and the highest score is 3.

[0065] 0 points: No positive staining cells;

[0066] 1 point: Weakly positive staining cells;

[0067] 2 points: moderately positive in stained cells;

[0068] 3 points: Strongly positive staining cells.

[0069] Finally, the final staining score was calculated by multiplying the percentage score and the intensity score (five fields of view were selected for each slice, and the average staining score was taken). The samples were then categorized as low expression (final score ≤ 6) and high expression (score > 6).

[0070] 1.15 Statistical Analysis

[0071] The trials were repeated at least three times. All values ​​are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPadPrism 8.0.2 (GraphPad Software, La Jolla, CA, USA) and the Mann-Whitney U test, followed by unpaired Student's t-test or ANOVA. Statistical significance was set at P < 0.05. Overall survival (osteosarcoma) and progression-free survival (PFS) of patients in the TCGA database were analyzed using Kaplan-Meier survival curves and log-rank tests.

[0072] 2. Results

[0073] 2.1 SOCS1 is negatively correlated with chemotherapy resistance in osteosarcoma.

[0074] Using a gradient drug concentration induction method, the inventors established two drug-resistant osteosarcoma cell lines (U2OS-DDPr and 143B-DDPr). The IC50 values ​​of these cell lines after treatment with different chemotherapeutic drugs were analyzed using the CCK-8 assay, and their resistance indices were calculated. The results showed that compared with the parental cells, these drug-resistant cell lines exhibited strong resistance to cisplatin, with resistance indices (RI) > 5-fold. The resistance index of U2OS-DDPr was 16.08 times that of the parental cells. Furthermore, these drug-resistant cell lines also showed varying degrees of cross-resistance to other chemotherapeutic drugs, demonstrating multidrug resistance.

[0075] To explore novel targets for chemosensitivity in osteosarcoma, this invention selected U2OS-DDPr and its parental cell lines for RNA-Seq analysis. For example... Figure 1 As shown in Figure A, 1539 differentially expressed genes were identified in chemotherapy-resistant cells, of which 879 genes were upregulated and 660 genes were downregulated. KEGG pathway analysis indicated that alterations in tumor signaling pathways, cytokine-cytokine receptors, and the PI3K-Akt signaling pathway were the most significant. Figure 1 B). Then, this invention used the TCGA database to obtain a set of genes associated with the survival of osteosarcoma patients. Then, this invention took the intersection of the above 1539 differentially expressed genes with survival-related genes to obtain 33 candidate genes (B). Figure 1 C and 1D). This invention validated the screening results using RT-qPCR analysis, showing that SOCS1 mRNA was most significantly downregulated in U2OS-DDPr cells (C and 1D). Figure 1 E). Similarly, this invention further validates that SOCS1 protein is significantly downregulated in drug-resistant osteosarcoma cells (E). Figure 1 F). Furthermore, this invention further demonstrates that cisplatin can inhibit SOCS1 protein expression in osteosarcoma cells in a dose- and time-dependent manner. Figure 1 (G and H). In summary, SOCS1 is significantly underexpressed in chemotherapeutic osteosarcoma cells.

[0076] 2.2 Overexpression of SOCS1 enhances the sensitivity of osteosarcoma cells to chemotherapeutic drugs.

[0077] This invention uses a lentiviral infection system to construct osteosarcoma cell lines with stable SOCS1 knockdown and overexpression, and verifies the expression efficiency using RT-qPCR and Western blot methods. Figure 2 (A and 2B). This invention uses the CCK8 method to demonstrate that SOCS1 knockdown in osteosarcoma cells significantly reduces the sensitivity of tumor cells to cisplatin. Figure 2 C and 2D). Conversely, overexpression of SOCS1 enhances the chemosensitivity of osteosarcoma cells to cisplatin (C and 2D). Figure 2 The results (E and 2F) suggest that SOCS1 plays a key role in regulating the sensitivity of osteosarcoma cells to chemotherapeutic drugs.

[0078] Plate colony formation assays also showed that overexpression of SOCS1 enhanced the chemosensitivity of osteosarcoma cells to cisplatin, while knockdown of SOCS1 had the opposite effect. Figure 2 (G and 2H). This invention further utilizes a nude mouse subcutaneous tumorigenesis model to evaluate the in vivo effect of SOCS1 on the sensitivity of osteosarcoma cells to cisplatin treatment. This invention reveals that SOCS1 overexpression inhibits the growth of xenograft tumors. Furthermore, compared to cisplatin treatment alone or SOCS1 overexpression, the combination of SOCS1 overexpression and cisplatin treatment produced a more significant growth-inhibiting effect on osteosarcoma xenograft tumors. Figure 2 In summary, SOCS1 can enhance the chemosensitivity of osteosarcoma cells to cisplatin.

[0079] 2.3 SOCS1 is significantly associated with survival and chemotherapy resistance in clinical osteosarcoma patients.

[0080] This invention explored the clinical relevance of SOCS1 to osteosarcoma. Using the TARGET public database, this invention analyzed the relationship between SOCS1 and the prognosis of osteosarcoma patients, suggesting that low SOCS1 expression is associated with poor patient prognosis. Figure 3 A). This invention further performed SOCS1 immunohistochemistry on osteosarcoma tissue microarrays, determining that SOCS1 expression is closely related to osteosarcoma stage; the higher the stage, the lower the expression. Figure 3 B).

[0081] This invention collected 20 osteosarcoma tissue samples from our center, including 11 clinically diagnosed drug-resistant osteosarcoma and 9 chemotherapy-sensitive osteosarcoma. Both qPCR and immunohistochemical results indicated significantly low SOCS1 expression in drug-resistant patients and high SOCS1 expression in sensitive patients. Figure 3(C and 3D). In summary, this invention reveals that low SOCS1 expression is significantly associated with poor survival and chemotherapy resistance in clinical osteosarcoma, and SOCS1 can serve as a potential biomarker for predicting the chemosensitivity of osteosarcoma.

[0082] 2.4 SOCS1 activates ferrodeath by downregulating SLC7A11.

[0083] This invention, by detecting SLC7A11 expression at the cellular level through knockdown, knockout, or overexpression of SOCS1, confirms that downregulating SOCS1 can upregulate SLC7A11 protein expression, and overexpressing SOCS1 downregulates SLC7A11 protein expression. Figure 4 A), further immunohistochemistry of clinical tissues confirmed a negative correlation between the expression of SOCS1 and SLC7A11. Figure 4 (B and 4C) In summary, SOCS1 downregulates the expression of SLC7A11.

[0084] This invention further demonstrated through rescue and ferroptosis phenotype experiments that SOCS1 overexpression inhibited cystine uptake and increased erastin-induced ROS, and the restoration of SLC7A11 significantly reversed ferroptosis activated by SOCS1 overexpression. Figure 4 (D-4F). In summary, SOCS1 mainly promotes osteosarcoma ferroptosis by inhibiting the expression of SLC7A11.

[0085] 2.5 SOCS1 sensitizes osteosarcoma with cisplatin via SLC7A11-mediated ferroptosis.

[0086] This invention identifies that SOCS1 sensitizes osteosarcoma to cisplatin via SLC7A11-mediated ferroptosis. Through rescue experiments, by overexpressing SLC7A11 or adding a ferroptosis inhibitor to SOCS1 overexpression, the corresponding drug resistance phenotypes—CCK8, colony formation, and cell death—were detected. The results indicate that SLC7A11 overexpression or a ferroptosis inhibitor can reverse cisplatin sensitivity induced by SOCS1 overexpression. Figure 5 A-5D). Further subcutaneous tumor formation in nude mice in vivo yielded the same conclusion. Figure 5 In summary, SOCS1 can drive ferroptosis by regulating the expression of SLC7A11, thereby enhancing the sensitivity of osteosarcoma to cisplatin.

[0087] Experimental Example 2: The effect of FB23-2 on chemotherapy resistance in osteosarcoma

[0088] 1. Materials and Methods

[0089] 1.1 Analysis of m6A levels using dot blot hybridization

[0090] Total RNA was extracted from cells using Trizol reagent (Invitrogen, USA), and mRNA was purified from the total RNA using the Gen Elute mRNA Miniprep kit (Promega, TM021) according to the manufacturer's instructions. The concentration of mRNA was measured using a NanoDrop2000 (Thermo, USA). The mRNA isolated from osteosarcoma cells was mixed into three volumes of RNA incubation buffer and denatured by heating at 65°C for 5 minutes. RNA samples dissolved in pre-chilled 20×SSC buffer were then spotted onto an Amersham Hybond-N+ membrane (GE Healthcare, USA) and UV crosslinked for 10 minutes. Next, the membrane was stained with 0.02% methylene blue (Solarbio, China) at room temperature for 5 minutes and scanned to indicate the input RNA content. After washing with TBST for 15 minutes, the membrane was blocked with 10% skim milk for 1 hour and then incubated with m6A antibody (1:1000, Abcam, USA) at 4°C. Finally, the membrane was incubated with HRP-conjugated anti-mouse IgG (1:3000, Proteintech, USA) at room temperature for 1 hour and detected with ECL Western blot reagent.

[0091] 1.2 MeRIP-RT-qPCR

[0092] Two 10cm cell culture plates were washed with pre-chilled PBS, and then lysed on ice with 400mL IP lysis buffer for 30 minutes. Clear lysates were collected after centrifugation at 12000g for 10 minutes. 4mL of m6A antibody or IgG (NEB, USA) was premixed on magnetic beads for 2 hours. The antibody-coated magnetic beads were incubated with the clear cell lysates overnight at 4°C. Proteinase K was added to digest the proteins in the immunoprecipitated RNA-protein complex, and then the precipitated RNA was extracted with TRIzol. The RNA of interest was detected by RT-qPCR and normalized to the input.

[0093] 1.3 Cell Heat Transfer Assay (CETSA)

[0094] U2OS-DDPr cells were collected and incubated in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2 mM DTT). 20 μM FB23-2 or DMSO was added to the supernatant, and the cells were incubated at room temperature for 30 minutes. After denaturation for 5 minutes at different indicated temperatures, the samples were centrifuged, and the supernatant was analyzed by Western blotting. All experiments were performed in triplicate.

[0095] 1.4 mRNA stability measurement

[0096] Osteosarcoma cell lines were incubated with 200 μM actinomycin D for 30 minutes and collected as the 0-hour sample. Samples were collected at 1, 2, 3, and 4 hours for total RNA extraction. The 1st Strand cDNA Synthesis SuperMix (Yeasen, Shanghai, China) used oligo-d(T) primers to generate cDNA. mRNA levels were quantified by RT-qPCR.

[0097] 1.5 Nude Mouse Xenograft Experiment

[0098] Nude mice (5 weeks old, male, BALB / C-nu / nu, SPF Biotechnology Co., Ltd., Beijing, China) were randomly selected for ectopic tumor construction. Mice were randomly assigned to three treatment groups. Starting from day 9, mice were intraperitoneally injected with FB23-2 (Selleck, S8837, dissolved in physiological saline, 2 mg / kg) and cisplatin every 3 days, 4 times each. Tumor volume and body weight were monitored every 5 days or at each injection. Tumor volume (V) was then measured using the standard formula V = L * W² / 2 (L = length, W = width). Mice were euthanized by cervical dislocation, xenograft tumors were removed, photographed, and cut into two halves. One half was fixed and embedded in paraffin for immunohistochemistry (IHC), and the other half was rapidly frozen in liquid nitrogen for later use. All animal operations and procedures for nude mouse experiments were authorized by the Animal Protection Committee of Tianjin Medical University Cancer Hospital.

[0099] 2. Results

[0100] 2.1 FB23 / 2 is an inhibitor of FTO, which rescues SOCS1 expression by increasing m6A methylation, thereby enhancing chemosensitivity in osteosarcoma.

[0101] Since SOCS1 restoration can enhance the chemosensitivity of OS, this invention identifies the potential mechanism by which SOCS1 is inhibited in chemosensitized OS cells and reveals that FB23 / 2 is a small molecule drug that upregulates SOCS1 expression to promote chemosensitization.

[0102] This invention, through m6A-related experiments such as m6A dot blot, m6A RIP qPCR, and Western blotting, screened for FTO demethylases that can downregulate m6A modification and thus downregulate SOCS1 expression. Figure 6A-6D). This invention confirms that FB23-2 and FTO inhibitors can restore SOCS1 expression and thus enhance chemosensitivity by inhibiting FTO-mediated m6A demethylation. This invention reveals that with FB23-2 treatment, SOCS1 expression is significantly upregulated at both the mRNA and protein levels, accompanied by increased m6A enrichment, significantly increasing SOCS1 mRNA stability. Figure 6 E-6H) indicates that FB23-2, as an inhibitor of FTO, can restore SOCS1 levels in chemotherapeutic osteosarcoma cells by promoting M6A.

[0103] This invention further explores how FB23-2 reverses chemoresistance by regulating SOCS1 and its associated ferroptosis. In vitro and in vivo experiments—including CCK8, colony formation, subcutaneous tumor formation in nude mice, and rescue experiments—confirmed that FB23-2 treatment significantly restores cisplatin sensitivity in cisplatin-resistant osteosarcoma cells, and that this can be reversed by co-treatment with SOCS1 knockdown or the ferroptosis inhibitor ferr-1. Figure 6 In summary, FB23-2, as an inhibitor of FTO, can increase the m6A modification level of SOCS1, upregulate SOCS1, and promote chemosensitization in osteosarcoma.

Claims

1. Application of SOCS1 overexpression reagent in the preparation of drugs that enhance the sensitivity of osteosarcoma cells to cisplatin chemotherapy.

2. Application of FB23-2 in the preparation of drugs that enhance the sensitivity of osteosarcoma cells to cisplatin chemotherapy.

Images