{"paper_id":"ece4ff25-3855-43d2-a955-a7f0c72ca6a0","body_text":"The existing evidence confirms that long non-coding RNAs (lncRNAs) can function as competitive endogenous RNAs (ceRNAs) by modulating microRNAs (miRNAs), thereby influencing tumor cell migration and proliferation [ 1 ]. Long intergenic non-protein coding  RNA 02381  ( LINC02381 ), a member of the lincRNA family, has emerged a crucial regulator in oncogenesis. Recent studies have indicated that lincRNAs are actively involved in transcriptional reprogramming and play essential roles in malignant progression [ 1 ]. Aberrant  LINC02381  expression has been documented in various malignancies. For instance, it is significantly overexpressed in esophageal squamous cell carcinoma tissues compared to normal esophageal mucosa [ 2 ], and elevated levels have also been observed in glioma [ 3 ] and cervical carcinoma cells, where it exerts oncogenic functions contributing to tumor progression [ 1 ]. Notably, Xia Bian et al. reported that  LINC02381  knockdown suppresses osteosarcoma (OS) cell invasion, proliferation, and migration [ 4 ]. However, the precise molecular mechanisms through which  LINC02381  functions in OS remain largely undefined.\nTo explore its downstream regulatory network, we focused on miRNAs potentially interacting with  LINC02381 . Among them,  microRNA-let-7g-5p  ( let-7g-5p ) attracted our attention due to its established tumor-suppressive role. As a member of the let-7 family,  let-7g-5p  is known for its low expression in cancers such as nasopharyngeal carcinoma and cholangiocarcinoma [ 5 , 6 ]. Meanwhile, reduced  let-7g-5p  levels have been detected in osteoporotic fractures [ 7 ], although its role in OS remains poorly understood. Emerging evidence suggests that  let-7g-5p  may target oncogenes like  HMGA2  and  MAP3K1  [ 8 , 9 ], indicating its potential involvement in OS progression [ 10 ]. Bioinformatic predictions reveal potential binding sites between  let-7g-5p  and Thrombospondin-1 ( THBS1 ), a stromal glycoprotein involved in tumor-associated processes such as cell–matrix interactions, extracellular matrix remodeling, and angiogenesis [ 11 , 12 ].  THBS1  is frequently overexpressed in gastric and colorectal cancers and is related to poor prognosis [ 13 , 14 ]. Furthermore, elevated  THBS1  levels have been linked to osteoporosis [ 15 ], and its upregulation in OS has been correlated with aggressive tumor behavior and poor clinical outcomes [ 16 , 17 , 18 ]. Mechanistically,  THBS1  is believed to promote tumor progression by modulating the extracellular matrix, stimulating angiogenesis, and activating the  transforming growth factor beta  ( TGF-β ) signaling pathway [ 19 ]. However, the direct regulatory relationship between  THBS1  and  let-7g-5p  has not yet been established.\nBased on these molecular insights, we propose that  LINC02381 ,  let-7g-5p , and  THBS1  may constitute a cascade regulatory network in OS. Supported by bioinformatic predictions and preliminary experimental data, we hypothesize that  LINC02381  modulates  let-7g-5p  activity via a ceRNA mechanism, thereby regulating  THBS1  expression and contributing to OS pathogenesis. Accordingly, this study aims to investigate the role of the  LINC02381 / let-7g-5p / THBS1  axis in OS cell viability and tumor progression.\n\nAll experimental procedures were authorized by the Research Ethics Committee of Shanghai Changzheng Hospital, Navy Medical University in compliance with international ethical guidelines (approval number: 2017052, ethics approval data: 20170523). Written informed consent was obtained from all patients. All animal procedures were authorized by the Animal Ethics Review Board of the same institution (approval number: 2017122, ethics approval data: 20171225) and conducted following the NIH Guide for the Care and Use of Laboratory Animals as well as relevant local animal welfare regulations.\nA total of 71 patients with OS who underwent surgical resection at Shanghai Changzheng Hospital, Navy Medical University from July 2017 to July 2022 were included. Paired OS tumor tissues and paracancerous tissues were collected. Clinicopathological variables such as gender (male/female), age (≥20 vs. <20 years), tumor size (≥3 cm vs. <3 cm), TNM stage (I–II vs. III–IV), and lymph node metastasis (yes/no) were systematically recorded. Detailed information is presented in  Table 1 . All clinical definitions adhered to the  AJCC Cancer Staging Manual, 8th Edition , and institutional diagnostic criteria established by the pathology and radiology departments. Data were independently reviewed and validated by two researchers. None of the enrolled subjects had received prior radiotherapy or chemotherapy. Collected tissues were labeled and cryopreserved at −70 °C for subsequent experiments.\nTo further investigate the correlation between molecular expression and treatment response, follow-up was conducted for all 71 patients who received postoperative adjuvant chemotherapy. According to RECIST criteria, patients were categorized into chemotherapy-sensitive (CR/PR) and chemotherapy-insensitive (SD/PD) groups based on radiological evaluations.\nHuman OS cell lines (143B, U-2OS, Saos-2, MNNG-HOS, MG-63) and the human normal osteoblast cell line hFOB1.19 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). 143B and MNNG-HOS cells were cultured in RPMI-1640; Saos-2 in α-minimum Eagle’s medium (α-MEM); MG-63 in MEM; and U-2OS and hFOB1.19 in Dulbecco’s modified Eagle medium (DMEM), all supplied by Gibco (Grand Island, NY, USA). Media were supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution (100 U/mL penicillin and 100 U/mL streptomycin). Cells were maintained in a 37 °C incubator with 5% CO 2 , with medium changes every two days. Subculturing was performed when cell confluence reached 80–90%.\nCells in the logarithmic growth phase after 2–3 stable passages were used for experiments. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (acceptable A260/A280 ratio: 1.8–2.1). The integrity of 28S/18S ribosomal RNA was confirmed by agarose gel electrophoresis to ensure RNA quality for downstream reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. The mRNA expression levels of  LINC02381 ,  let-7g-5p , and  THBS1  were detected by RT-qPCR, and  THBS1  protein levels were assessed by Western blotting. Based on relative expression differences, U-2OS and Saos-2 cells—both exhibiting the greatest divergence from the normal osteoblast hFOB1.19—were selected for subsequent functional experiments.\nU-2OS and SaOS-2 cells in the logarithmic growth phase were seeded into 6-well plates at a density of 2 × 10 5  cells per well. Transfection was initiated when cell confluence reached approximately 80%. Lipofectamine™ 2000 (Catalog No.: 11668-027, Invitrogen, Carlsbad, CA, USA) was used for transfection, strictly following the manufacturer’s protocol. Briefly, each oligonucleotide (final concentration: 50 nM; all synthesized by Shanghai GenePharma Co., Ltd., Shanghai, China) was diluted in 250 μL of serum-free DMEM and incubated at room temperature for 5 min. Separately, 5 μL of Lipofectamine™ 2000 was diluted in 250 μL of serum-free DMEM and incubated for another 5 min. The two solutions were then combined and allowed to form complexes at room temperature for 20 min before being added to the wells. Cells were incubated at 37 °C in a humidified incubator with 5% CO 2  for 6 h. Afterward, the transfection medium was replaced with DMEM containing 10% FBS, and cells were cultured further for downstream applications.\nCells were divided into the following eight groups, with three replicates per group. Transfection efficiency was verified by RT-qPCR and/or Western blot prior to functional assays: short hairpin RNA (sh)-negative control (NC) group (transfected with the negative control of  LINC02381  knockdown vector), sh- LINC02381  group (transfected with  LINC02381  knockdown vector), mimic-NC group (transfected with the negative control of  let-7g-5p  mimic),  let-7g-5p  mimic group (transfected with  let-7g-5p  mimic), sh- LINC02381  + inhibitor-NC group (transfected with  sh-LINC02381  background with negative control miRNA inhibitor), sh- LINC02381  +  let-7g-5p  inhibitor group (transfected with  sh-LINC02381  background with  let-7g-5p  inhibitor), sh- LINC02381  + overexpression (oe)-NC group (co-transfected with the negative control of  THBS1  overexpression vector and  LINC02381  knockdown vector), and sh- LINC02381  + oe- THBS1  group (co-transfected with the  THBS1  overexpression plasmid and  LINC02381  knockdown vector).\nThe proliferative capacity of U-2OS and SaOS-2 cells was assessed using a CCK-8 assay kit (Dojindo Molecular Technologies, Kumamoto, Japan). Cells from each group were seeded into 96-well plates at a density of 1 × 10 4  cells per well, with three replicates per group and one blank well as an NC. Cells were incubated at 37 °C in a 5% CO 2  atmosphere for 24, 48, and 72 h. At each time point, 10 μL of CCK-8 working solution was added directly to each well (without medium replacement), followed by a 4 h incubation under the same conditions. The optical density (OD) at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell proliferation curves were plotted accordingly. All procedures strictly adhered to the manufacturer’s instructions, and all experiments were independently repeated at least three times to ensure data reliability and reproducibility.\nA plate colony formation assay was performed to evaluate the clonogenic potential of U-2OS and SaOS-2 cells in each experimental group. Cells in the logarithmic growth phase were seeded into 6 cm culture dishes at a density of 200 cells per dish (1 × 10 3 /mL, in 200 μL of cell suspension). The dishes were gently swirled to evenly distribute the cells and then incubated at 37 °C in a humidified incubator with 5% CO 2  for 10 days.\nOnce visible colonies (each > 50 μm in diameter) were observed by the naked eye, the culture was terminated. The supernatant was carefully aspirated, and cells were washed twice with phosphate-buffered saline (PBS). Colonies were fixed with 96% ethanol for 10 min, stained with 1% crystal violet solution for 5 min, rinsed gently with tap water, and air-dried for approximately 1 h. The dishes were then inverted, and colonies were counted using a transparent grid film and an inverted microscope (Olympus Corporation, Tokyo, Japan). Each group included three replicates, and all experiments were repeated at least three times. The average number of colonies and colony formation rate (number of colonies/number of seeded cells × 100%) were recorded.\nTranswell chambers with an 8.0 μm pore size (Corning, Corning, USA) were used to assess the migratory ability of U-2OS and SaOS-2 cells. At 24 h post-transfection, cells were digested, resuspended, and adjusted to a concentration of 1 × 10 5 /mL. A 200 μL serum-free cell suspension was added to the upper chamber, while 600 μL of complete medium containing 10% FBS was added to the lower chamber as a chemoattractant. The cells were incubated at 37 °C with 5% CO 2  for 24 h.\nAfter incubation, non-migrated cells on the upper surface of the membrane were carefully removed with a cotton swab. The inserts were then fixed with 4% paraformaldehyde for 30 min, washed twice with PBS, and stained with 0.5% crystal violet for 20 min. Following staining, the membranes were rinsed thoroughly and air-dried. Five randomly selected non-overlapping fields per chamber were imaged and counted under an inverted microscope (×200 magnification, Olympus, Tokyo, Japan). Each group included three parallel replicates, and the experiments were independently repeated at least three times. The mean number of migrated cells was used for statistical analysis.\nTotal RNA was extracted from tissues and cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with an A260/A280 ratio between 1.8 and 2.1 considered acceptable. RNA integrity was confirmed by 1% agarose gel electrophoresis through visualization of intact 28S and 18S rRNA bands and evaluation of the 28S:18S ratio.\nComplementary DNA (cDNA) was synthesized using the PrimeScript RT Master Mix Perfect Real Time kit (Takara, Kyoto, Japan). Each 10 μL reaction contained 500 ng of total RNA. The reaction conditions included denaturation at 70 °C for 5 min to eliminate secondary structures, cooling on ice for 3 min, reverse transcription at 37 °C for 60 min, and enzyme inactivation at 95 °C for 10 min. The resulting cDNA was stored at −20 °C for further analysis.\nQuantitative real-time PCR was performed using SYBR Green chemistry. Each 20 μL reaction mixture consisted of 10 μL of 2× Taq MasterMix, 0.4 μL each of forward and reverse primers (5 μM), 1 μL of cDNA template, 0.4 μL of ROX Reference Dye, and nuclease-free water. Primer sequences were synthesized by Shanghai GenePharma Co., Ltd., Shanghai, China ( Table 2 ).\nThe qPCR protocol was as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 45 s, with a final extension at 72 °C for 5 min. Amplification specificity was verified using melting curve analysis. GAPDH was used as the internal control for  LINC02381  and  THBS1 , and U6 for  let-7g-5p . Each sample was analyzed in triplicate. Relative gene expression was calculated using the 2 ^ΔΔCt  method. A no-template control was included in each run to monitor for contamination.\nTissues and cells were lysed using RIPA lysis buffer (Cat# P0013C, Beyotime Biotechnology, Jiangsu, China), and total protein was collected by centrifugation at 12,000×  g  for 15 min at 4 °C. Protein concentrations were determined with a BCA protein assay kit (Beyotime, Jiangsu, China). For each sample, 30 μg of protein in 20 μL loading buffer was separated on 15% SDS-PAGE gels under the following conditions: pre-electrophoresis at 80 V for 40 min, followed by separation at 120 V for 70 min.\nProteins were transferred onto PVDF membranes (Millipore, Burlington, MA, USA) using a Bio-Rad (Hercules, CA, USA) electrotransfer system at 100 V for 90 min. The membranes were blocked in 5% non-fat milk (prepared in TBST buffer) at room temperature for 1 h, then incubated overnight at 4 °C with the following primary antibodies: anti-THBS1 (ab267388, Abcam, Cambridge, UK; 1:1000) and anti-GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000). After three 10 min washes with TBST, membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (Goat anti-Rabbit IgG-HRP, Bio-Rad, Hercules, CA, USA; 1:5000).\nProtein bands were visualized using an enhanced chemiluminescence (ECL) kit (Millipore, Burlington, MA, USA) and exposed to X-ray film (Kodak, Rochester, NY, USA). Band intensities were analyzed using ImageJ software (version 1.53k, NIH, Bethesda, MD, USA), and target protein levels were normalized to GAPDH.\nThe binding sites between  LINC02381  and  let-7g-5p  were predicted using the Starbase database ( https://starbase.sysu.edu.cn/agoClipRNA.php ). The wild-type (Wt) sequence of  LINC02381  containing the predicted binding site for  let-7g-5p  was synthesized and cloned into the psiCHECK2 luciferase reporter vector (Promega, WI, USA), generating the LINC02381-Wt construct. A mutant version (LINC02381-Mut) was created using the QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). U-2OS and SaOS-2 cells at 70–80% confluence were co-transfected with either  let-7g-5p  mimic or mimic negative control (mimic-NC) along with LINC02381-Wt or LINC02381-Mut using Lipofectamine TM  2000 (Invitrogen, Carlsbad, CA, USA). After 48 h, luciferase activity was measured using a Dual-Luciferase ®  Reporter Assay System (Promega, Madison, WI, USA).\nThe same method was employed to verify the targeting relationship between  let-7g-5p  and the 3′UTR of  THBS1 .\nTo validate the direct interaction between  let-7g-5p  and  LINC02381 , biotin-labeled wild-type (Wt) and mutant (Mut) probes of  let-7g-5p  were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) U-2OS and SaOS-2 cells (2 × 10 5  cells/well in 6-well plates) were transfected with these probes using Lipofectamine™ 2000. After 48 h of incubation, cells were lysed with RNA pull-down lysis buffer (Ambion, Austin, TX, USA) on ice for 10 min. Lysates were centrifuged, and supernatants were incubated with streptavidin-coated magnetic beads (M-280 Dynabeads, Sigma-Aldrich, Saint Louis, MO, USA) pre-blocked with 0.1 mg/mL yeast tRNA and 0.1 mg/mL RNase-free BSA for 30 min. Bead-lysate mixtures were rotated at 4 °C for 3 h.\nThe beads were then washed sequentially: twice with cold lysis buffer, three times with low-salt buffer (50 mM NaCl), and once with high-salt buffer (500 mM NaCl) to eliminate nonspecific binding. The unlabeled Mut probe served as the negative control. RNA bound to the beads was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA), reverse transcribed (PrimeScript RT Master Mix, Takara, Kyoto, Japan), and LINC02381 enrichment was quantified via qPCR. Each group was tested in triplicate, and experiments were independently repeated three times.\nMale BALB/c nude mice (18–20 g and 4–5 weeks old) were maintained under specific pathogen-free conditions. U-2OS and SaOS-2 cells stably transfected with sh-NC, sh- LINC02381 , mimic-NC, or  let-7g-5p  mimic were subcutaneously injected into the flanks (2 × 10 6  cells per mouse, with 7 nude mice in each group). Tumor volumes were measured every 7 days and calculated using the formula: (length × width 2 )/2. On day 28, mice were euthanized, tumors were harvested, excised, and weighed. Power analysis using G*Power v3.1 software (α = 0.05, effect size f = 0.8) confirmed that 7 mice per group would achieve >85% power for detecting differences in tumor volume and weight via one-way analysis of variance (ANOVA).\nData analysis was carried out using SPSS v21.0 (IBM, Armonk, NY, USA). Measurement data following a normal distribution were presented as mean ± standard deviation. Differences between two groups were analyzed using the independent samples  t -test, while multiple-group comparisons were performed via one-way ANOVA with Tukey’s post-hoc test. Fisher’s exact test was used to assess the relationships between  LINC02381 / let-7g-5p  expression and clinicopathological characteristics. Quality control measures included assessment of data normality using the Shapiro–Wilk test, homogeneity of variance via the Levene test, and outlier detection using the Grubbs test (α = 0.05). Abnormal results due to technical error were excluded and experiments repeated. Animal grouping was randomized, and both tumor measurements and endpoint evaluations were conducted in a blinded manner. Meanwhile, in vitro experiments such as cell proliferation, migration, and dual-luciferase assays were also implemented and interpreted independently to minimize bias. A  p -value below 0.05 was considered statistically significant.\n\nLINC02381  and  let-7g-5p  expression are linked to lymph node metastasis (LNM) and tumor node metastasis (TNM) staging of patients with OS\nInitially,  LINC02381 ,  let-7g-5p , and  THBS1  expression levels in adjacent normal tissues and OS tissues were measured.  LINC02381  was upregulated in OS tissues (fold change ≈ 3.77,  p  < 0.001,  Figure 1 A), while  let-7g-5p  was downregulated (fold change ≈ 0.38,  p  ≤ 0.001,  Figure 1 B), suggesting a potential regulatory interaction.\nFurthermore,  THBS1 , a predicted target of  let-7g-5p , was significantly upregulated at both the mRNA (fold change ≈ 2.77,  p  < 0.001,  Figure 1 C) and protein level (fold change ≈ 3.03,  p  < 0.001,  Figure 1 D) in OS tissues. Kaplan–Meier survival analysis revealed that high  LINC02381  expression was associated with shorter overall survival (Log-rank  p  = 0.0448,  Figure 1 E).\nPatients with OS were stratified into low- and high-expression groups based on the mean levels of  LINC02381  or  let-7g-5p . High  LINC02381  or low  let-7g-5p  expression was significantly associated with advanced TNM stage (III–IV) and the presence of LNM (all  p  < 0.05), whereas no significant correlation was observed with gender, tumor size, or age (all  p  > 0.05) ( Table 1 ).\nTo assess the relationship between molecular expression and therapeutic response, follow-up analysis was performed in 71 OS patients who underwent postoperative adjuvant chemotherapy. Based on RECIST criteria, patients were divided into chemotherapy-sensitive (CR/PR) and chemotherapy-insensitive (SD/PD) groups. Notably, the proportion of chemotherapy-insensitive patients was higher in the high  LINC02381  expression group ( p  = 0.008), while elevated  let-7g-5p  expression was associated with improved chemotherapy response ( Table 3 ).\nTo further validate these findings, we assessed the expression of  LINC02381 ,  let-7g-5p , and  THBS1  in human normal osteoblasts (hFOB1.19) and five OS cell lines (143B, U-2OS, SaOS-2, MNNG-HOS, and MG63) using RT-qPCR and Western blot. Compared to hFOB1.19,  LINC02381  was significantly upregulated in all OS cell lines ( p  < 0.001,  p  < 0.001,  p  < 0.001,  p  = 0.001,  p  < 0.001), while  let-7g-5p  was consistently downregulated ( p  < 0.001,  p  < 0.001,  p  = 0.005,  p  < 0.001,  p  < 0.001). Correspondingly,  THBS1  expression was increased across all OS cell lines ( p  < 0.001,  p  < 0.001,  p  < 0.001,  p  < 0.001,  p  < 0.001) ( Figure 2 A).\nAt the protein level, the expression of  THBS1  was markedly higher in U-2OS cells ( p  = 0.009), 143B, SAOS-2, and MNNG-HOS cells ( p  < 0.001) ( Figure 2 B,C).\nBased on these results, U-2OS and SAOS-2 cells with a high expression of  LINC02381  and  THBS1  and a low expression of  let-7g-5p  were finally selected for subsequent functional experiments.\nPrevious studies have demonstrated that  LINC02381  regulates the process of cancer cells by interacting with miRNAs. For example, it promotes the viability and migration of cervical cancer cells by targeting  miR-133b  [ 1 ]. Based on this evidence, we speculated that  LINC02381  may exert regulatory effects in OS by regulating  let-7g-5p .\nBioinformatic analysis predicted potential binding sites between  LINC02381  and  let-7g-5p  ( Figure 3 A). Dual-luciferase reporter assays in U-2OS and SaOS-2 cells confirmed this interaction: co-transfection with LINC02381-Wt and let-7g-5p mimic significantly reduced luciferase activity compared to the mimic-NC group (U-2OS:  p  = 0.002; SaOS-2:  p  < 0.001), whereas no significant change was observed in the LINC02381-Mut group ( p  > 0.05) ( Figure 3 B). These findings indicate specific binding between  let-7g-5p  and  LINC02381 . The interaction was further validated via RNA pull-down assays, which showed significant enrichment of  LINC02381  by the biotin-labeled  let-7g-5p  probe (U-2OS:  p  < 0.001; SaOS-2:  p  < 0.001) ( Figure 3 C).\nTarget prediction tools identified  THBS1  as a putative downstream target of  let-7g-5p  ( Figure 3 D). This relationship was confirmed by dual-luciferase assays: co-transfection of THBS1-Wt with the let-7g-5p mimic led to reduced luciferase activity (U-2OS:  p  = 0.002; SaOS-2:  p  = 0.002), whereas THBS1-Mut showed no significant change ( p  > 0.05), indicating that  THBS1  is a direct target of  let-7g-5p  ( Figure 3 E).\nSubsequent RT-qPCR and Western blot analyses demonstrated that either knockdown of  LINC02381  or overexpression of  let-7g-5p  significantly downregulated THBS1 expression (all  p  < 0.01,  Figure 3 F,G). In addition, sh- LINC02381  elevated  let-7g-5p  levels, an effect that was reversed by co-transfection with the  let-7g-5p  inhibitor ( p  < 0.01), further proving that  LINC02381  can promote the expression of  THBS1  by acting as a molecular sponge for  let-7g-5p .\nTo investigate the functional consequences of the  LINC02381/let-7g-5p  axis in OS, we performed CCK-8 and colony formation assays. In both U-2OS and SaOS-2 cells, knockdown of  LINC02381  or overexpression of  let-7g-5p  significantly inhibited cell proliferation (U-2OS:  p  < 0.001; SaOS-2:  p  < 0.001) and reduced colony formation (U-2OS:  p  < 0.001; SaOS-2:  p  = 0.001). Notably, co-treatment with the  let-7g-5p  inhibitor reversed the suppressive effects of sh-LINC02381 on proliferation and colony formation (U-2OS:  p  < 0.001; SaOS-2:  p  = 0.001) ( Figure 4 A–C).\nCell migration capacity, assessed using Transwell migration assays, revealed similar trends. Silencing  LINC02381  or overexpressing  let-7g-5p  markedly inhibited migration in both cell lines (U-2OS:  p  = 0.002 and  p  < 0.001; SaOS-2:  p  < 0.001 and  p  = 0.001), while co-transfection with the  let-7g-5p  inhibitor rescued this migratory defect (U-2OS:  p  < 0.001; SaOS-2:  p  = 0.005) ( Figure 4 D).\nCollectively, these findings suggest that  LINC02381  regulates  THBS1  by targeting  let-7g-5p , thereby affecting the proliferation and migration ability of OS cells.\nTo clarify whether  THBS1  can mediate the regulatory effects of the  LINC02381 / let-7g-5p  axis on the biological functions of OS cells, rescue experiments were conducted in U-2OS and SaOS-2 cells by co-transfecting sh- LINC02381  with either oe-NC or oe- THBS1 .\nqPCR results showed that compared with the sh- LINC02381  + oe-NC group, the expression of  THBS1  in the sh- LINC02381  + oe- THBS1  group was increased (U-2OS:  p  < 0.001; SaOS-2:  p  = 0.001) ( Figure 5 A).\nFunctional assays showed that  THBS1  overexpression significantly reversed the inhibitory effects of  LINC02381  silencing. CCK-8 and colony formation assays revealed restored proliferative capacity (U-2OS:  p  < 0.001; SaOS-2:  p  < 0.001) (U-2OS:  p  = 0.002; SaOS-2:  p  = 0.002) ( Figure 5 B,C). Transwell migration assays further demonstrated that  THBS1  overexpression significantly rescued the migration ability suppressed by  LINC02381  knockdown (U-2OS:  p  = 0.002; SaOS-2:  p  = 0.001) ( Figure 5 D).\nThese findings indicate that  THBS1 , as a downstream effector, plays a crucial role in mediating the pro-tumorigenic functions of the  LINC02381/let-7g-5p  axis in OS cells.\nTo further validate the role of  LINC02381  and  let-7g-5p  in vivo, U-2OS cells from the sh-NC, sh- LINC02381 , agomir-NC, and  let-7g-5p  agomir groups (2 × 10 6  cells/mouse) were subcutaneously injected into the flanks of 4–5-week-old male nude mice. Tumor dimensions were measured every 7 days to calculate volume and plot growth curves.\nTumors began to visibly grow by day 7 post-inoculation in all groups. From day 14 onward, tumor volumes in both the sh- LINC02381  group and the  let-7g-5p  agomir group were notably smaller than their respective controls. On day 28, tumor volume was significantly reduced in the sh- LINC02381  group compared to the sh-NC group ( p  < 0.001), and similarly in the  let-7g-5p  agomir group versus the agomir-NC group ( p  < 0.001) ( Figure 6 A).\nFollowing euthanasia on day 28, tumor weights were recorded. Both  LINC02381  knockdown and  let-7g-5p  overexpression significantly reduced tumor weight compared to their controls ( p  < 0.001) ( Figure 6 B).\nCollectively, these in vivo findings confirm that both  LINC02381  silencing and  let-7g-5p  upregulation effectively inhibit OS tumor growth, reinforcing their critical roles in OS progression and potential as therapeutic target.\n\nOS is a prevalent primary malignant bone tumor, predominantly affecting adolescents [ 20 ]. In this study, we demonstrated that  LINC02381  is significantly involved in the pathogenesis of OS through modulation of the  let-7g-5p / THBS1  axis. Mechanistically,  LINC02381  acts as a molecular sponge for  let-7g-5p  that sequesters  THBS1  thereby relieving its suppressive effect on  THBS1 , and consequently enhancing OS cell proliferation, migration, and invasion in vitro and in vivo.\nOur study revealed upregulated  LINC02381  and  THBS1  expression, alongside downregulated  let-7g-5p  levels in OS tissues compared to normal controls. Furthermore,  LINC02381  and  let-7g-5p  expression levels correlated with TNM staging and LNM in OS patients, indicating their clinical relevance in OS progression. These findings are consistent with prior reports showing that  LINC02381  is aberrantly expressed in various malignancies [ 2 , 3 ], with its overexpression linked to advanced disease stages and poor outcomes [ 4 ]. Additionally,  let-7g-5p  downregulation has been observed in multiple cancers, including cholangiocarcinoma and glioblastoma [ 6 , 9 ], and its re-expression can inhibit cancer cell proliferation, migration, and invasion [ 10 ]. The overexpression of  THBS1  in OS tissues, as previously reported [ 17 ], further reinforces its oncogenic role.\nThrough dual-luciferase reporter and RNA pull-down assays, we validated the direct interaction between  let-7g-5p  and the 3′UTRs of both  LINC02381  and  THBS1 , confirming a ceRNA regulatory mechanism. Functional assays demonstrated that  INC02381  could regulate  THBS1  expression by targeting  let-7g-5p , thereby influencing OS cell proliferation and migration. Importantly, overexpression of  THBS1  mitigated the inhibitory effects of  LINC02381  knockdown, highlighting  THBS1  as a critical downstream effector of this axis. These findings align with existing evidence that lncRNAs can function as ceRNAs in tumorigenesis. For instance,  LINC02381  has been shown to promote cervical cancer progression through sponging  miR-133b  [ 1 ], and to upregulate  CTNNB1  in endometriosis via  miR-27b-3p  [ 21 ]. In OS specifically,  LINC02381  was reported to promote malignancy by downregulating  CDCA4  via sponging  miR-503-5p  [ 4 ]. Together, these data suggest that  LINC02381  may operate through multiple ceRNA pathways, possibly exhibiting functional redundancy across different oncogenic contexts.\nNotably,  THBS1  is a multifunctional matricellular protein that relies on downstream signaling pathways for its tumor-promoting activities. The literature indicates that  THBS1  regulates cell migration by binding to integrin receptors, as well as interacting with  CD47  and  CD36 , which modulate integrin-mediated signaling [ 22 ].  THBS1  is also known to activate latent TGF-β1, contributing to bone metastasis in prostate cancer by facilitating TGF-β signaling [ 23 ]. These mechanisms are highly relevant to OS, particularly in the context of lung metastasis, as integrin β3 has been implicated in metastatic OS cell lines, and its silencing significantly impairs invasion and metastasis [ 24 ]. Moreover,  TGF-β  pathway overactivation has been associated with chemoresistance in OS [ 25 ]. Based on these insights, future research should incorporate phosphoproteomic profiling to assess downstream signaling alterations following  THBS1  overexpression and apply pharmacological inhibitors of integrin or  TGF-β  receptors to further dissect  THBS1 -mediated pathways.\n\nIn summary, this study confirms that  LINC02381  promotes OS progression by functioning as a molecular sponge for  let-7g-5p , thereby upregulating  THBS1  expression and promoting OS cell proliferation, migration, and invasion both in vitro and in vivo ( Figure 7 ). This axis represents a novel regulatory pathway in OS and offers potential targets for future therapeutic strategies. However, several limitations must be acknowledged. Specifically, the downstream signaling pathways of  THBS1  remain incompletely characterized, and clinical translation of RNA-based therapeutics faces significant challenges, including oligonucleotide instability, tissue-specific delivery, and off-target effects. Therefore, future studies should incorporate advanced delivery systems and mechanistic pathway analyses to enhance the feasibility of targeted interventions in OS.","source_license":"CC-BY-4.0","license_restricted":false}