Osteosarcoma (OS) is the most prevalent primary sarcoma of bone, accounting for 35% of all primary malignant bone tumors and ranking among the most common causes of cancer-related death in pediatric patients. With the application of neoadjuvant chemotherapy and advances in surgical techniques, the overall 5-year survival rate has improved from less than 20% to 65 to 70%. However, this survival improvement has plateaued over the past 20 years, and new therapeutic targets are urgently needed.
Ribosomal proteins perform housekeeping functions in ribosome biogenesis, but a growing body of evidence shows they also have important extraribosomal functions in proliferation, differentiation, apoptosis, DNA repair, and chemoresistance. Dysregulated expression of specific ribosomal proteins has been observed in several cancers: RPS6 in non-small cell lung cancer (NSCLC) and non-Hodgkin lymphoma, RPS7, RPS15A, and RPS20 in colorectal cancer, RPL5 and RPL10 in T-cell acute lymphoblastic leukemia, and RPS14 in myelodysplastic syndrome.
Ribosomal protein L34 (RPL34) belongs to the L34E family and is located in the cytoplasm. The gene is mapped to chromosome 4q25 and spans approximately 10 kb with 6 exons. RPL34 (molecular weight approximately 13 kDa) is a component of the 60S large ribosomal subunit. Prior work in gastric cancer and NSCLC showed that RPL34 is overexpressed in tumor tissues and its knockdown inhibits proliferation, but its role in osteosarcoma had not been investigated.
Eleven pairs of OS tissues and adjacent normal tissues were collected from patients at the First Affiliated Hospital of Guangxi Medical University for RT-qPCR mRNA level analysis. A separate set of 155 formalin-fixed paraffin-embedded (FFPE) specimens from the pathology department, including 95 OS tissues and 60 normal bone tissues, were used for immunohistochemical (IHC) staining and survival analysis. All 95 OS patients had received no prior radiotherapy or chemotherapy, ensuring clean prognostic data uncomplicated by treatment effects.
Patients were followed up every 3 months within the first 2 years after surgery and semiannually thereafter, with the follow-up deadline set at October 1, 2015. Survival status was confirmed through outpatient re-examination and telephone follow-up. Written informed consent was obtained from all participants, and the study was approved by the research ethics committee.
Five osteosarcoma cell lines (U2OS, MG-63, HOS, and Saos-2) and the normal human osteoblast cell line hFOB1.19 were used for in vitro experiments. All cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI-1640 or DMEM medium with 10% fetal bovine serum. The Saos-2 cell line was selected for lentivirus-mediated knockdown experiments based on its RPL34 expression levels.
RT-qPCR analysis of 11 paired OS and adjacent tissue samples showed that RPL34 mRNA was upregulated in 7 of 11 (63.64%) OS tissues at more than 3-fold higher levels than adjacent normal tissue. Across all 11 pairs, RPL34 mRNA was significantly elevated in OS compared with adjacent tissue (p = 0.015).
IHC of 95 OS and 60 normal bone tissue sections revealed that RPL34 protein was localized to the cytoplasm, consistent with its 60S subunit localization. Using a 12-point immunoreactive scoring system (0 to 12), 75 of 95 (78.95%) OS tissues showed strongly positive staining (score greater than 4), while the other 20 of 95 (21.05%) showed weakly positive staining (score greater than 0 to 4). In contrast, all 60 normal bone tissues were weakly stained. The difference in RPL34 protein expression between OS and normal bone was highly significant (p = 0.000).
Expression of RPL34 mRNA was also elevated in all four human OS cell lines (U2OS, MG-63, HOS, Saos-2) compared with the normal osteoblast cell line hFOB1.19. The consistency of elevated RPL34 expression across fresh tissue pairs, FFPE specimens, and multiple cell lines establishes that RPL34 overexpression is a reproducible feature of osteosarcoma biology rather than an artifact of a specific sample type or assay.
Among the 95 OS patients, the average immunoreactive score for RPL34 was 7.1, which was used as the threshold to divide patients into high-expression (score greater than 7.1, n=48) and low-expression (score 7.1 or below, n=47) groups. Kaplan-Meier survival analysis with log-rank test showed a significant difference in overall 3-year survival rates between the two groups.
The 3-year overall survival rate was 35.42% (17 of 48) in the high-expression group compared with 61.70% (29 of 47) in the low-expression group (log-rank p = 0.004). This nearly 26-percentage-point gap in 3-year survival confirms that high RPL34 expression at diagnosis independently predicts a worse prognosis. This is consistent with prior findings in gastric cancer (where RPL34 knockdown reduced proliferation) and NSCLC (where RPL34 overexpression correlated with poor outcomes).
The prognostic significance of RPL34 was evaluated in patients who had received no prior treatment, making the survival differences attributable to biological tumor characteristics rather than treatment heterogeneity. The relatively short 3-year endpoint reflects the aggressive natural history of osteosarcoma, particularly the risk of pulmonary metastasis within the first 2 to 3 years after diagnosis.
To functionally characterize RPL34's role in OS cells, the authors used a lentivirus-mediated siRNA system targeting the RPL34 transcript (GenBank NM_000995.4). The siRNA sequence CACAGAGTCAGAAAGCTAA was inserted into the lentiviral vector GV115 carrying a GFP reporter gene, enabling fluorescence-based tracking of transduced cells. A scramble sequence (TTCTCCGAACGTGTCACGT) served as the negative control (NC-siRNA) lentivirus. Co-transfection with helper plasmids pHelper 1.0 and 2.0 in 293T cells produced lentiviral particles, which were then used to transduce Saos-2 cells at the appropriate multiplicity of infection (MOI).
Knockdown efficiency was confirmed by both RT-qPCR and Western blot 5 days after transduction. RT-qPCR showed that RPL34 mRNA in RPL34-siRNA-treated Saos-2 cells was reduced by approximately 64% (p = 0.0008) relative to NC-siRNA-treated cells. Western blot confirmed significant protein-level reduction. Fluorescence microscopy showed that more than 80% of Saos-2 cells expressed GFP 3 days after transduction, confirming efficient lentiviral infection.
Cell proliferation was assessed by two complementary methods: high content screening (HCS) using the Cellomics ArrayScan VTI reader (automated daily cell counting over 5 days in 96-well plates) and the MTT assay (at 10 days post-transduction). Colony formation was assessed by plating 600 cells per well in 6-well plates and quantifying colonies after 10 to 14 days. Cell cycle distribution and apoptosis were analyzed by flow cytometry (FCM) using propidium iodide staining and Annexin V-APC staining respectively.
Both HCS and MTT assay confirmed that RPL34 knockdown significantly inhibited Saos-2 cell proliferation relative to NC-siRNA controls over the observation periods. Colony formation assay showed a dramatic effect: after 14 days, the RPL34-siRNA group formed only 2 plus or minus 1 colonies compared with 84 plus or minus 7 colonies in the NC-siRNA group (p = 0.0026). The colonies in the RPL34-siRNA group also contained fewer cells per colony, indicating impaired clonogenic capacity.
Flow cytometry cell cycle analysis revealed a shift in cell cycle distribution upon RPL34 knockdown. In NC-siRNA cells: G0/G1 = 35.63 plus or minus 0.76%, S = 55.56 plus or minus 0.99%, G2/M = 8.82 plus or minus 0.47%. In RPL34-siRNA cells: G0/G1 = 36.81 plus or minus 0.74%, S = 49.20 plus or minus 0.66%, G2/M = 13.99 plus or minus 0.08%. RPL34 knockdown caused a significant decrease in S phase (p = 0.0014) and a significant increase in G2/M phase (p = 0.0022), indicating G2/M phase arrest.
Annexin V-APC apoptosis analysis showed that the percentage of apoptotic cells increased significantly in the RPL34-siRNA group (6.73 plus or minus 0.63%) compared with the NC-siRNA group (5.64 plus or minus 0.14%) (p = 0.042). While the absolute increase in apoptosis is modest, the G2/M arrest and proliferation inhibition together suggest that RPL34 knockdown imposes multiple cellular constraints on OS cell growth, consistent with its role in maintaining the translational capacity needed for rapid tumor cell proliferation.
To explore the upstream regulation of RPL34, the UCSC ENCODE Genome Browser was queried for human transcription factor (TF) to gene-pair data. Eleven transcription factors were predicted to regulate RPL34, with particular emphasis on MYC and MYC-associated factor X (MAX). MYC is a well-characterized oncogene that functions as a transcriptional activator via MYC-MAX heterodimers. MYC amplification is reported in osteosarcoma and correlates with oncogenesis, proliferation, and metastasis. MYC is also known to transcriptionally regulate ribosomal protein genes broadly, making its identification as an RPL34 regulator biologically plausible.
A PPI network for proteins interacting with RPL34 was constructed using the STRING database (combined score threshold greater than 0.9 for high-confidence interactions), yielding a network of 112 nodes and 4,669 interactions. GO and KEGG enrichment analysis of the 112 interacting proteins using DAVID identified the ribosome KEGG pathway (hsa03010) as the top enriched pathway (85 genes, p = 2.04 x 10^-171) and structural constituent of ribosome as the top GO Molecular Function term (88 genes, p = 6.82 x 10^-162).
Three specific interacting proteins of particular interest were EIF3A, EIF3G, and EIF3F, subunits of the eukaryotic translation initiation factor 3 (eIF3) complex. eIF3 is the largest translation initiation factor, comprising 13 non-identical subunits (eIF3a through eIF3m) essential for multiple steps in the initiation phase of protein synthesis. Individual eIF3 subunits are frequently dysregulated in cancer: overexpression of subunits 3a, 3b, 3c, 3h, 3i, and 3m leads to malignant transformation, while reduced expression of 3e and 3f causes a similar outcome.
The study demonstrates that RPL34 knockdown suppresses OS cell proliferation and induces G2/M arrest, but the detailed molecular mechanism through which RPL34 mediates these effects remains unknown. The links to MYC transcriptional regulation and eIF3 interactions are inferred computationally from the UCSC TF database and STRING PPI data and have not been directly demonstrated experimentally (e.g., via ChIP-seq for MYC binding at the RPL34 promoter or co-immunoprecipitation to confirm RPL34-eIF3 physical interaction).
The functional experiments were conducted exclusively in one OS cell line (Saos-2), which may not represent the full biological heterogeneity of osteosarcoma. Saos-2 cells are p53-null, which could affect cell cycle arrest responses. Whether RPL34 knockdown has similar anti-proliferative effects in other OS cell lines (U2OS, MG-63, HOS) or in primary OS cultures was not tested.
The survival analysis is univariate and does not account for established prognostic variables in OS such as tumor site, tumor size, pathologic necrosis after chemotherapy, presence of metastasis at diagnosis, and surgical margin status. A multivariate Cox regression analysis incorporating RPL34 expression alongside these clinical variables would be necessary to confirm that RPL34 expression provides prognostic information independently of known risk factors.
RPL34 represents a potential therapeutic target in osteosarcoma because it is broadly overexpressed across OS tissues and cell lines, its suppression inhibits proliferation without apparent effects on normal osteoblast biology, and its upstream regulator MYC is already a recognized target in osteosarcoma. RNA-based therapies (siRNA, antisense oligonucleotides, or shRNA delivered via nanoparticles or viral vectors) targeting RPL34 could be evaluated in OS xenograft models.
Mechanistically, the most important open question is whether MYC directly transcribes RPL34 by binding its promoter, and whether RPL34 physically interacts with eIF3 subunits to selectively promote translation of growth-promoting mRNAs such as RRM2 (ribonucleotide reductase M2), NDRG1, and CDKN1B. ChIP-seq of MYC in OS cells and co-immunoprecipitation followed by mass spectrometry of RPL34 interaction partners would address these questions directly.
The observation that RPL34 may act through translational control of growth-promoting proteins connects to a broader theme in cancer biology: tumor cells achieve accelerated growth in part through selective upregulation of the translation machinery for specific mRNA programs. Understanding which mRNAs are preferentially translated in high-RPL34 OS cells (via ribosome profiling or polysome sequencing) could reveal additional downstream effectors that are cotargeted with RPL34 for combinatorial therapeutic strategies.