Sixty paired osteosarcoma tissue samples and adjacent non-tumorous tissues were archived in the sample repository of Fujian Orthopedic Research Institute, with pathological confirmation. All cases underwent systematic follow-up, during which patient survival outcomes were meticulously documented. This study was granted ethical approval by the Institutional Ethics Committee (Ethics Approval Number: MRCTA, ECFAH of FMU [2021] 156), and all participants provided written informed consent after full disclosure of study objectives and procedures.

To explore the roles of differentially expressed miRNAs in KEGG pathways, the differential miRNAs were uploaded into the online analysis tool DIANA TOOLS mirpathe (http://diana.imis.athena-innovation.gr/DianaTools/index.php), and miR-495 with high functional pathway enrichment was screened out.

The miRNAs were uploaded into the Sarcoma-microRNA Expression Database (S-MED, https://www.oncomir.umn.edu/SMED/index.php) to analyze the expression profiles of miR-495 in normal tissues and various sarcoma subtypes.

Subsequently, the osteosarcoma tissues from 60 patients were stratified into high- and low-expression groups based on miR-495 expression levels. Survival analysis was performed by integrating patient survival time and status, and Kaplan-Meier curves were plotted to visualize the results.

The osteoblastic cell line hFOB 1.19 and osteosarcoma cell lines MG63, U2OS, 143B, HOS, and SaOS2 were obtained from the Cell Bank of the Chinese Academy of Sciences Typical Culture Collection Committee (Shanghai, China). The culture conditions were as follows:

hFOB 1.19 cells were maintained in DMEM/F12 medium (Gibco, Carlsbad, CA, United States) supplemented with 0.3 mg/mL G418 (Gibco, Carlsbad, CA, United States), 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States), 100 mg/mL penicillin (Gibco, Carlsbad, CA, United States), and 100 mg/mL streptomycin (Gibco, Carlsbad, CA, United States).

MG63, U2OS, 143B, and HOS cells were cultured in DMEM containing 10% FBS (Gibco, Carlsbad, CA, United States), 100 U/mL penicillin, and 100 mg/mL streptomycin.

SaOS2 cells were grown in RPMI 1640 medium (Gibco, Carlsbad, CA, United States) supplemented with 10% FBS, 100 mg/mL penicillin, and 100 mg/mL streptomycin.

All cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO₂.

The miR-495 mimic, miR-495 negative control (NC), miR-495 inhibitor, and miR-495 inhibitor NC (inhibitor NC) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) (Table 1). Cell transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions.

Total RNA was extracted from osteosarcoma cells using TRIzol reagent (Invitrogen), and reverse-transcribed into cDNA with a reverse transcription kit from Tiangen Biotech (Beijing, China). Quantitative detection was performed using the Tiangen real-time fluorescence quantitative qPCR kit (Beijing, China) and the miRcute Enhanced miRNA Fluorescence Quantitative Detection Kit (Beijing, China) according to the manufacturer’s instructions. U6 served as an internal reference. Primers for miR-495, U6, and RUNX3 were synthesized by Sangon Biotech Co., Ltd (Shanghai, China) (Tables 1, 2). The relative expression levels of RUNX3 and miR-495 were calculated using the 2^−ΔΔCt method.

The miR-495 stable transfection plasmid and its control plasmid miR-495 NC (Normal Control) were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). The miR-495 overexpression plasmid was constructed on the GV514 vector backbone (CMV-EGFP-MCS-SV40-Neomycin), with the target sequence inserted at the XhoI/BamHI cloning sites. The corresponding negative control plasmid was Catalog No. CON107, with no exogenous insert sequence. After transfecting osteosarcoma 143B cells with Lipofectamine 3000 (Invitrogen) for 48 h, the transfection efficiency was observed under a fluorescence inverted microscope. The cells were then screened and expanded in DMEM complete medium containing 0.8 mg/mL G418 (Promega, Madison, WI, United States). RT-PCR was used to verify the changes in miR-495 expression levels in the stably transfected 143B osteosarcoma cell line. The 143B cells stably transfected with the miR-495 plasmid were named 143B-495 cells, while those transfected with miR-495 NC were named 143B-NC cells.

The osteosarcoma 143B-495 and 143B-NC cells were routinely cultured in 10-cm culture dishes. When the cell confluence reached 80%–90%, the culture medium was discarded, and the cells were washed twice with PBS. After removing the PBS completely, 1 mL of Trizol Reagent was added to each dish. Three biological replicates were set for each group. The total RNA was then sent to Shanghai Geneuine Biotechnology Co., Ltd. for stranded transcriptome sequencing. Transcriptome sequencing was performed on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, United States) with paired-end 150 bp reads. Differentially expressed genes (DEGs) were identified using DESeq2 with a threshold of |log₂FC(Fold Change)| ≥ 1 and adjusted p-value <0.05. The RNA sequencing data generated in this study have been deposited in the National Genomics Data Center (NGDC) database under accession number PRJCA058457.

The potential target genes of miR-495 were predicted by querying the miRNA target prediction databases miRDB, TarBase, TargetScan, and miRTarBase with miR-495.

Differentially downregulated genes identified from transcriptome sequencing of osteosarcoma 143B-495 and 143B-NC cells were cross-referenced with the bioinformatics-predicted target genes from miRDB, TarBase, TargetScan, and miRTarBase. The intersection of these gene sets was further selected to obtain the preliminary identified target genes.

The wild-type (RUNX3 3′UTR) and mutant (RUNX3 3′UTR mut) sequences of the RUNX3 mRNA 3′-untranslated region (3′UTR) were designed based on the complementary sequences between miR-495 and RUNX3. The gene fragments were amplified by PCR, digested with restriction enzymes, and ligated into the GV272 vector. 293T cells were transfected using Lipofectamine 3000 (Invitrogen) and divided into six groups: ①

miR-495 + GV272 CON,

Relative luciferase activity was measured using the Dual-Luciferase® Reporter Assay System E1910 (Promega, Madison, WI, United States) according to the manufacturer’s instructions.

Osteosarcoma 143B cells were transfected with miR-495 NC, mimic, mimic + CON, or mimic + RUNX3, and designated as four groups: ① NC, ② mimic, ③ mimic + CON, ④ mimic + RUNX3.

Total RNA and protein were extracted 48 h post-transfection for subsequent analyses. miR-495 expression levels were quantified by reverse transcription and qRT-PCR. RUNX3 protein levels were detected by Western blotting.

Forty-eight hours after transfection, the following assays were performed to assess changes in proliferation, colony formation, invasion, migration, and apoptosis: CCK-8 assay for cell proliferation, Colony formation assay, Transwell invasion assay, Wound healing assay for cell migration, Flow cytometry for apoptosis analysis.

Osteosarcoma 143B-495 and 143B-NC cells in logarithmic growth phase were digested with trypsin, centrifuged, and collected.

For subcutaneous xenograft models: Ten 6–8-week-old SPF-grade BALB/c-nu mice were randomly divided into two groups (n = 5 per group). Cells (2.0 × 10⁶ in 100 μL PBS) were subcutaneously injected into the dorsal region above the forelimb. Tumor length and width were measured twice weekly using calipers, and tumor volume was calculated using the formula: V = (length × width²)/2. Mice were sacrificed after 8 weeks, and tumors were excised for weighing and further analysis.

For lung metastasis models: Under aseptic conditions (75% ethanol disinfection), 2.0 × 10⁶ cells in 100 μL PBS were injected into the tail vein of each mouse (n = 5 per group). Four weeks post-injection, mice were euthanized, and lungs were harvested, fixed in 4% paraformaldehyde, and processed for H&E staining to assess metastatic nodules.

Total protein was extracted from osteosarcoma tissues and cells using RIPA Lysis Buffer (Beyotime, China). Samples were lysed on a horizontal shaker at 30 rpm for 30 min at 4 °C, sonicated, and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected, and protein concentration was determined using a BCA Protein Assay Kit (Beyotime, China).

Protein samples (50 μg) were separated by SDS-PAGE and transferred to PVDF membranes at 400 mA for 60 min at 4 °C.

Membranes were blocked with 5% non-fat milk for 60 min and incubated overnight at 4 °C with primary antibodies against RUNX3 (1:1000), PI3K (1:1000), Akt (1:1000), p-Akt (1:1000), Bax (1:1000), Bcl-2 (1:1000), c-casp3 (1:1000), c-casp9 (1:1000), and GAPDH (1:1000) (Cell Signaling Technology, Beverly, MA, United States).

After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:50,000, CST) for 60 min at room temperature in the dark.

Protein bands were visualized using ECL Chemiluminescent Substrate (Beyotime, China) and imaged with a FlourChem Imaging System (United States). Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD).

Data was analyzed using SPSS 25.0 software (SPSS Inc., Chicago, IL, United States). Measurement data are presented as mean ± standard deviation (SD). Prior to parametric testing, data normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated using Levene’s test. For comparisons between two groups, Student’s t-test was applied when assumptions were satisfied. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test when variances were homogeneous. When variance heterogeneity was detected, Welch’s ANOVA with Games-Howell post hoc correction was used. For RNA sequencing analysis, differentially expressed genes were identified using DESeq2, and multiple testing correction was applied using the Benjamini–Hochberg false discovery rate (FDR) method. A p-value <0.05 was considered statistically significant.

Differential expression analysis of miRNA data from recurrent and non-recurrent osteosarcoma samples in the GSE39058 dataset identified 43 downregulated and 47 upregulated miRs (Figures 1A,B; Tables 3, 4). Analysis using the S-MED database (https://www.oncomir.umn.edu/SMED/) revealed that miR-495 expression was significantly lower in osteosarcoma tissues than in normal bone tissues (Figure 1C; Table 5). Functional pathway enrichment analysis of differentially expressed miRNAs was performed using the DIANA TOOLS miRPath online tool (http://diana.imis.athena-innovation.gr/DianaTools/index.php). Heatmap visualization showed that miR-495 was significantly enriched in cancer-related functional pathways (Figure 1D).

qRT-PCR analysis showed that miR-495 expression was significantly lower in osteosarcoma cells than in normal osteoblastic cells hFOB1.19 (p < 0.05). Among the osteosarcoma cell lines examined, miR-495 expression followed the order: U2OS > MG63 > SaOS2 > HOS > 143B (Figure 1E).

Consistently, miR-495 expression was significantly reduced in 60 pairs of osteosarcoma tissues compared with adjacent normal tissues (Figure 1F). Kaplan-Meier survival analysis revealed that osteosarcoma patients with low miR-495 expression had a median survival time of 42.22 months (95% CI: 32.38–52.06), while those with high miR-495 expression had a median survival time of 54.22 months (95% CI: 49.01–59.43). A Log-Rank test (p = 0.024) indicated that miR-495 expression levels were positively correlated with patient cumulative survival rate (Figure 1G).

Transfection with miR-495 mimics significantly increased miR-495 expression in 143B, HOS, and SaOS2 cells, while the inhibitor group showed lower levels compared to the inhibitor NC group (Figure 2A). Increased miR-495 expression was accompanied by a marked reduction in RUNX3 expression, while miR-495 inhibition resulted in RUNX3 upregulation (Figure 2B).

Functional assays demonstrated that miR-495 overexpression significantly suppressed cell proliferation, colony formation, invasion, and migration in all three osteosarcoma cell lines, as assessed by CCK-8, colony formation, Transwell invasion, and wound healing assays, respectively (Figures 2C–F). In contrast, miR-495 overexpression markedly increased apoptotic rates compared with control cells, as determined by flow cytometry (Figure 2G).

RNA sequencing (RNA-seq) analysis of 143B cells transfected with miR-495 mimic versus NC identified 126 upregulated and 45 downregulated genes (Figure 3A). KEGG pathway enrichment analysis of differentially expressed genes was performed using the EnrichKEGG function in the R package clusterprofiler (Yu et al., 2012), identifying significant alterations in multiple cancer-related signaling pathways (Figure 3B). Target genes of miR-495 predicted by miRDB, TarBase, TargetScan, and miRTarBase were intersected and visualized by a Venn diagram, with common predicted targets including DDIT4, MTA3, HSP90AA1, CNBP, BMI1, ZBTB18, BTF3L4, RUNX3, and CD164. Integration of RNA-seq and bioinformatics predictions identified RUNX3 as a target gene of miR-495 (Figure 3C). Western blot analysis further confirmed that RUNX3 protein expression was significantly higher in 12 pairs of osteosarcoma tissues (OS) than in adjacent normal tissues (N) (Figure 3D).

Dual-luciferase reporter assays showed that co-transfection of RUNX3 mRNA 3′UTR wild-type (wt) and miR-495 mimic in 293T cells resulted in a relative luciferase activity of 0.45 ± 0.06, significantly lower than the control group (p < 0.05), indicating direct binding and luciferase activity inhibition. In contrast, co-transfection of RUNX3 mRNA 3′UTR mutant (mut) and miR-495 mimic showed no significant difference in luciferase activity compared to the control (p > 0.05), confirming failed binding. These results demonstrate that miR-495 directly recognizes and binds to the RUNX3 mRNA 3′UTR, suppressing RUNX3 mRNA translation and protein expression, thus validating RUNX3 as a target gene of miR-495 (Figure 3E).

qRT-PCR result showed that miR-495 levels in cells transfected with miR-495 mimic were significantly higher than those in the NC group, while miR-495 levels remained unchanged in the co-transfection of miR-495 and RUNX3 (Figure 4A, p < 0.05).

WB analysis revealed that RUNX3 protein expression was significantly reduced in the miR-495 mimic group, with a statistically significant difference compared to the NC group (p < 0.05). Co-transfection of miR-495 and the RUNX3 overexpression vector restored RUNX3 expression levels compared to the mimic + CON group (Figure 4B), indicating that RUNX3 transfection reversed the inhibitory effect of miR-495 on RUNX3.

CCK-8 assay results showed that the restoration of RUNX3 expression reversed the inhibitory effect of miR - 495 on the proliferative capacity of osteosarcoma 143B cells and promoted cell viability (Figure 4C).

Colony formation assays indicated that increased miR-495 levels suppressed the colony forming ability of osteosarcoma cells, while the simultaneous overexpression of RUNX3 restored colony formation (Figure 4D).

Regarding the invasion ability (Figure 4E), the number of 143B osteosarcoma cells penetrating Matrigel in the mimic group was 25 ± 3.3, which was significantly lower than that in the NC control group (62 ± 5.1, p < 0.05). Co-transfection with mimic and RUNX3 increased the number of invasive cells to 55 ± 3.1, compared to 24 ± 2.1 in the mimic + CON group (p < 0.05).

Wound healing assays showed that the scratch repair rate in the mimic group was significantly lower than that in the NC control group after 24 h (Figure 4F, p < 0.05). No significant difference in the scratch repair rate was observed between the mimic group and the mimic + CON group (p > 0.05), but the mimic + RUNX3 group exhibited a significantly higher repair rate than the mimic + CON group (p < 0.05).

Flow cytometry analysis of apoptosis in osteosarcoma 143B cells revealed that the apoptosis rate was 9.2% ± 0.15% in the miR - 495 NC group, while the overexpression of miR - 495 in the mimic group significantly increased apoptosis to 21.5% ± 0.19% (Figure 4G, p < 0.05), indicating that miR-495 promotes apoptosis. Co-transfection with miR - 495 mimic and RUNX3 reduced the apoptosis rate to 9.8% ± 0.17%, whereas co - transfection with miR-495 mimic and control plasmid CON showed no significant change (20.7% ± 0.16%, p > 0.05). Similar results were observed when comparing the mimic group and the mimic + CON group (p > 0.05). These findings suggest that upregulating RUNX3 protein expression counteracts the pro - apoptotic effect of miR-495 in osteosarcoma 143B cells.

Subcutaneous xenograft models of osteosarcoma were established using 143B-495 and 143B-NC cells in nude mice. Results showed that miR-495 significantly inhibited tumor growth in vivo (Figure 4H). Additionally, lung metastasis models established via tail vein injection confirmed that the number of lung metastatic nodules in the 143B-495 cell group was significantly reduced compared to the 143B-NC group 4 weeks after injection (Figure 4I, p < 0.05). These findings indicate that miR-495 suppresses both the growth and metastasis of osteosarcoma cells in animal models.

WB analysis was performed to detect changes in PI3K/Akt pathway-related molecules in osteosarcoma 143B cells transfected with: ① miR-495 NC (NC), ② miR-495 mimic (mimic), ③ miR-495 mimic + RUNX3 CON (mimic + CON), or ④ miR-495 mimic + RUNX3 (mimic + RUNX3). High miR-495 expression significantly downregulated the levels of PI3K, Akt, p-Akt (Ser473), mTOR, p70S6K, and Bcl-2, inhibiting PI3K/Akt pathway activity. Concurrently, the expressions of Bax, cleaved-caspase-3 (c-casp3), and cleaved-caspase-9 (c-casp9) increased compared to the control group, promoting tumor cell apoptosis.

Co-transfection with miR-495 and RUNX3 revealed that restoration of RUNX3 protein expression rescued the levels of key molecules including PI3K, Akt, p-Akt(Ser473), mTOR, p70S6K, and Bcl-2. Correspondingly, the expressions of Bax, c-casp3, and c-casp9 were partially suppressed, alleviating the inhibition of the PI3K/Akt pathway (Figure 5). These results indicate that miR-495 overexpression suppresses the levels of PI3K, Akt, p-Akt(Ser473), mTOR, p70S6K, and Bcl-2, which are rescued by increased RUNX3 protein expression, with opposite effects observed for Bax, c-casp3, and c-casp9. No significant differences in these molecular changes were detected between the miR-495 mimic group and the miR-495 mimic + CON group (p > 0.05). Collectively, these findings demonstrate that miR-495 inhibits PI3K/Akt pathway activity by targeting RUNX3.