m6A-related metabolism molecular classification with distinct prognosis and immunotherapy response in soft tissue sarcoma.

N6-methyladenosine (m6A) methylation, one of the most crucial RNA modifications, has been proven to play a key role that affect prognosis of soft tissue sarcoma (STS). However, m6A methylation potential role in STS metabolic processes remains unknown. We comprehensively estimated the m6A metabolic molecular subtypes and corresponding survival, immunity, genomic and stemness characteristics based on 568 STS samples and m6A related metabolic pathways. Then, to quantify the m6A metabolic subtypes, machine learning algorithms were used to develop the m6A-metabolic Scores of individual patients. Finally, two distinct m6A metabolic subtypes (Cluster A and Cluster B) among the STS patients were identified. Compared to Cluster B subtype, the Cluster A subtype was mainly characterized by better survival advantages, activated anti-tumor immune microenvironment, lower gene mutation frequency and higher anti-PD-1 immunotherapy response rates. We also found that the m6A-metabolic Scores could accurately predict the molecular subtype of STS, prognosis, the abundance of immune cell infiltration, tumor metastasis status, sensitivity to chemotherapeutics and immunotherapy response. In general, this study revealed that m6A-regulated tumor metabolism processes played a key role in terms of prognosis of STS, tumor progression, and immune microenvironment. The identification of metabolic molecular subtypes and the construction of m6A-metabolic Score will help to more effectively guide immunotherapy, metabolic therapy and chemotherapy in STS.

Integrated Analysis of Nine Prognostic RNA-Binding Proteins in Soft Tissue Sarcoma.

RNA-binding proteins (RBPs) have been shown to be dysregulated in cancer transcription and translation, but few studies have investigated their mechanism of action in soft tissue sarcoma (STS). Here, The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to identify differentially expressed RBPs in STS and normal tissues. Through a series of biological information analyses, 329 differentially expressed RBPs were identified. Functional enrichment analysis showed that differentially expressed RBPs were mainly involved in RNA transport, RNA splicing, mRNA monitoring pathways, ribosome biogenesis and translation regulation. Through Cox regression analyses, 9 RBPs (BYSL, IGF2BP3, DNMT3B, TERT, CD3EAP, SRSF12, TLR7, TRIM21 and MEX3A) were all up-regulated in STS as prognosis-related genes, and a prognostic model was established. The model calculated a risk score based on the expression of 9 hub RBPs. The risk score could be used for risk stratification of patients and had a high prognostic value based on the receiver operating characteristic (ROC) curve. We also established a nomogram containing risk scores and 9 key RBPs to predict the 1-year, 3-year, and 5-year survival rates of patients in STS. Afterwards, methylation analysis showed significant changes in the methylation degree of BYSL, CD3EAP and MEX2A. Furthermore, the expression of 9 hub RBPs was closely related to immune infiltration rather than tumor purity. Based on the above studies, these findings may provide new insights into the pathogenesis of STS and will provide candidate biomarkers for the prognosis of STS.

m6A Modification Patterns With Distinct Immunity, Metabolism, and Stemness Characteristics in Soft Tissue Sarcoma.

N6-methyladenosine (m6A) RNA methylation has been shown to have prognostic value in cancer. Nonetheless, its potential role regarding immunity, metabolism, and stemness in soft tissue sarcoma (STS) remains unknown. We comprehensively estimated the m6A modification patterns and corresponding immunity, metabolism, and stemness characteristics based on 568 STS samples and 21 m6A regulators. The m6Ascore was constructed to quantify m6A modification patterns in individuals using machine learning algorithms. Two distinct m6A modification patterns among the STS patients were identified, which exhibited differences in prognosis, immune cell infiltration, metabolic pathways, stemness, somatic mutation, and copy number variation. Thereafter, immunity-, metabolism-, and stemness phenotype-related genes associated with m6A modification were identified. Furthermore, patients with lower m6Ascores had increased antitumor immune responses, survival benefit under immunotherapy, tumor mutation burden, immunogenicity, and response to anti-PD-1/L1 immunotherapy. Immunotherapy sensitivity was validated using the IMvigor210 dataset. STS patients with lower m6Ascore might be more sensitive to docetaxel and gemcitabine. Finally, pan-cancer analysis illustrated the significant correlations of m6Ascore with clinical outcomes, immune cell infiltration, metabolism, and stemness. This study revealed that m6A modification plays an important role in immunity, metabolism, and stemness in STS. Evaluating the m6A modification pattern and development of m6Ascore may help to guide more effective immunotherapy and chemotherapy strategies.

Molecular Subtypes Based on Cell Differentiation Trajectories in Head and Neck Squamous Cell Carcinoma: Differential Prognosis and Immunotherapeutic Responses.

Objective: Head and neck squamous cell carcinoma (HNSCC) is one of the most common and lethal malignant tumors. We aimed to investigate the HNSCC cell differentiation trajectories and the corresponding clinical relevance. Methods: Based on HNSCC cell differentiation-related genes (HDRGs) identified by single-cell sequencing analysis, the molecular subtypes and corresponding immunity, metabolism, and stemness characteristics of 866 HNSCC cases were comprehensively analyzed. Machine-learning strategies were used to develop a HNSCC cell differentiation score (HCDscore) in order to quantify the unique heterogeneity of individual samples. We also assessed the prognostic value and biological characteristics of HCDscore using the multi-omics data. Results: HNSCCs were stratified into three distinct molecular subtypes based on HDRGs: active stroma (Cluster-A), active metabolism (Cluster-B), and active immune (Cluster-C) types. The three molecular subtypes had different characteristics in terms of biological phenotype, genome and epigenetics, prognosis, immunotherapy and chemotherapy responses. We then demonstrated the correlations between HCDscore and the immune microenvironment, subtypes, carcinogenic biological processes, genetic variation, and prognosis. The low-HCDscore group was characterized by activation of immunity, enhanced response to anti-PD-1/PD-L1 immunotherapy, and better survival compared to the high-HCDscore group. Finally, by integrating the HCDscore with prognostic clinicopathological characteristics, a nomogram with strong predictive performance and high accuracy was constructed. Conclusions: This study revealed that the cell differentiation trajectories in HNSCC played a nonnegligible role in patient prognosis, biological characteristics, and immune responses. Evaluating cancer cell differentiation will help to develop more effective immunotherapy, metabolic therapy, and chemotherapy strategies.

The Potential of Five Immune-Related Prognostic Genes to Predict Survival and Response to Immune Checkpoint Inhibitors for Soft Tissue Sarcomas Based on Multi-Omic Study.

Low response rates to immunotherapy have been reported in soft tissue sarcoma (STS). There are few predictive biomarkers of response, and the tumor immune microenvironment associated with progression and prognosis remains unclear in STS. Gene expression data from the Cancer Genome Atlas were used to identify the immune-related prognostic genes (IRPGs) and construct the immune gene-related prognostic model (IGRPM). The tumor immune microenvironment was characterized to reveal differences between patients with different prognoses. Furthermore, somatic mutation data and DNA methylation data were analyzed to understand the underlying mechanism leading to different prognoses. The IGRPM was constructed using five IRPGs (IFIH1, CTSG, STC2, SECTM1, and BIRC5). Two groups (high- and low-risk patients) were identified based on the risk score. Low-risk patients with higher overall survival time had higher immune scores, more immune cell infiltration (e.g., CD8 T cell and activated natural killer cells), higher expression of immune-stimulating molecules, higher stimulating cytokines and corresponding receptors, higher innate immunity molecules, and stronger antigen-presenting capacity. However, inhibition of immunity was observed in low-risk patients owing to the higher expression of immune checkpoint molecules and inhibiting cytokines. High-risk patients had high tumor mutation burden, which did not significantly influence survival. Gene set enrichment analysis further revealed that pathways of cell cycle and cancers were activated in high-risk patients. DNA methylation analysis indicated that relative high methylation was associated with better overall survival. Finally, the age, mitotic counts, and risk scores were independent prognostic factors for STS. Five IRPGs performed well in risk stratification of patients and are candidate biomarkers for predicting response to immunotherapy. Differences observed through the multi-omic study of patients with different prognoses may reveal the underlying mechanism of the development and progression of STS, and thereby improve treatment.

Risk score based on expression of five novel genes predicts survival in soft tissue sarcoma.

In this study, The Cancer Genome Atlas and Genotype-Tissue Expression databases were used to identify potential biomarkers of soft tissue sarcoma (STS) and construct a prognostic model. The model was used to calculate risk scores based on the expression of five key genes, among which MYBL2 and FBN2 were upregulated and TSPAN7, GCSH, and DDX39B were downregulated in STS patients. We also examined gene signatures associated with the key genes and evaluated the model's clinical utility. The key genes were found to be involved in the cell cycle, DNA replication, and various cancer pathways, and gene alterations were associated with a poor prognosis. According to the prognostic model, risk scores negatively correlated with infiltration of six types of immune cells. Furthermore, age, margin status, presence of metastasis, and risk score were independent prognostic factors for STS patients. A nomogram that incorporated the risk score and other independent prognostic factors accurately predicted survival in STS patients. These findings may help to improve prognostic prediction and aid in the identification of effective treatments for STS patients.

Identification of the Biomarkers and Pathological Process of Osteoarthritis: Weighted Gene Co-expression Network Analysis.

Osteoarthritis (OA) is a joint disease resulting in high rates of disability and low quality of life. The initial site of OA (bone or cartilage) is uncertain. The aim of the current study was to explore biomarkers and pathological processes in subchondral bone samples. The gene expression profile GSE51588 was downloaded from the Gene Expression Omnibus database. Fifty subchondral bone [knee lateral tibial (LT) and medial tibial (MT)] samples from 40 OA and 10 non-OA subjects were analyzed. After data preprocessing, 5439 genes were obtained for weighted gene co-expression network analysis. Highly correlated genes were divided into 19 modules. The yellow module was found to be highly correlated with OA (r = 0.71, p = 1e-08) and the brown module was most associated with the differences between the LT and MT regions (r = 0.77, p = 1e-10). Gene ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment indicated that the yellow module was enriched in a variety of components including proteinaceous extracellular matrix and collagen trimers, involved in protein digestion and absorption, axon guidance, ECM-receptor interaction, and the PI3K-Akt signaling pathway. In addition, the brown module suggests that the differences between the early (LT) and end (MT) stage of OA are associated with extracellular processes and lipid metabolism. Finally, 45 hub genes in the yellow module (COL24A1, COL5A2, COL3A1, MMP2, COL6A1, etc.) and 72 hub genes in the brown module (LIPE, LPL, LEP, SLC2A4, FABP4, ADH1B, ALDH4A1, ADIPOQ, etc.) were identified. Hub genes were validated using samples from cartilage (GSE57218). In summary, 45 hub genes and 72 hub genes in two modules are associated with OA. These hub genes could provide new biomarkers and drug targets in OA. Further studies focusing on subchondral bone are required to validate these hub genes and better understand the pathological process of OA.

[Protective effect of Ligustri Lucidi Ait Polysaccharide against lipopolysaccharide-induced inflammatory injury of Sertoli cells in rats].

OBJECTIVE: To explore the effect of Ligustri Lucidi Ait Polysaccharide (LLP) on lipopolysaccharide (LPS)-induced inflammatory injury of Sertoli cells. METHODS: Rat Sertoli cells were isolated and cultured in vitro and then divided into five groups, blank control, LPS, LPS + low-dose LLP, LPS + medium-dose LLP, and LPS + high-dose LLP. After 48 hours of treatment, the proliferation of the cells was detected by CCK-8, their apoptosis determined by FMC, and the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) in the supernatant of the cell culture medium measured by ultraviolet spectrophotometry. The contents of IL-1α, IL-6 and TGF-ß in the culture medium were detected by ELISA before and after removal of LPS. RESULTS: The proliferation of the cells showed statistically significant differences among different groups (F = 153.93, P < 0.01), markedly reduced in the LPS group as compared with the blank control (P < 0.01), but remarkably increased in the high- and medium-dose LLP groups in comparison with the LPS group (both P < 0.01), and so did the apoptosis of the cells (F = 64.06, P < 0.01), significantly increased in the LPS group as compared with the blank control (P < 0.05), but markedly decreased in the high- and medium-dose LLP groups in comparison with the LPS group (both P < 0.01). Statistically significant differences were also observed among different groups in the levels of SOD (F = 56.07, P < 0.01), CAT (F = 41.57, P < 0.01), GSH-Px activity (F = 238.46, P < 0.01), and MDA (F = 285.31, P < 0.01), with decreased SOD, CAT and GSH-Px activity (P < 0.01) and increased MDA (P < 0.01) in the LPS group as compared with the control, but elevated SOD and CAT in the high- and medium-dose LLP groups and increased GSH-Px activity and decreased MDA concentration in all the three LLP groups in comparison with the LPS group (P < 0.01). Before the removal of LPS, the contents of IL-1α, IL-6 and TGF-ß in the culture medium were markedly higher in the LPS than in the control group (all P < 0.01), that of IL-1α was increased significantly in the high- and medium-dose LLP groups (P < 0.01 and P < 0.05) while those of IL-6 and TGF-ß showed no statistically significant differences in the three LPS groups as compared with the LLP group (P > 0.05). After the removal of LPS, the contents of IL-1α and IL-6 were remarkably reduced (t = 25.26 and 61.43, P < 0.01) and that of TGF-ß increased (t = －18.16, P < 0.01), even more significantly in the LLP+LPS groups (P < 0.01). CONCLUSIONS: Ligustri Lucidi Ait Polysaccharide plays a protective role in LPS-induced inflammatory injury of Sertoli cells by reducing cell apoptosis and regulating the contents of IL-1α, IL-6 and TGF-ß from Sertoli cells in inflammation.

[Hepatitis B virus infection increases the incidence of immune infertility in males].

OBJECTIVE: To investigate the relationship between hepatitis B virus (HBV) infection and the incidence of male immune infertility. METHODS: Based on the levels of serum HBsAg, 3 124 infertile men were classified into an HBV-positive and an HBV-negative group and, according to the results of IBT tests, those with immune infertility were further divided into an HBV-positive and an HBV-negative group. Statistical analyses were made on the incidence rate of immune infertility and seminal parameters in the immune infertility patients of the HBV-positive and HBV-negative groups, the correlation of the number of HBV DNA copies in the serum with that in the seminal plasma of the HBV-positive patients, the association of the numbers of HBV DNA copies in the serum and seminal plasma with semen parameters, and the relationship of the number of HBV DNA copies in the seminal plasma with the incidence of immune infertility. Sperm concentration and the percentage of progressively motile sperm (PMS) were measured by computer-aided sperm analysis, sperm morphology determined by Diff-Quik staining, the level of HBsAg detected by ELISA, and the numbers of HBV DNA copies in the serum and seminal plasma calculated by RT-PCR. RESULTS: The incidence rate of immune infertility was significantly higher in the HBV-positive than in the HBV-negative group (20.3 vs 3.3%, χ2 = 187.5, P <0.01), and the percentage of morphologically normal sperm (MNS) was markedly lower in the HBV-positive than in the HBV-negative infertility patients (ï¼»3.9 ± 1.7ï¼½ vs ï¼»6.3 ± 2.2ï¼½%, P <0.05), but no statistically significant differences were observed between the two groups of infertile males in the semen volume, sperm concentration, or PMS (P >0.05). The number of HBV DNA copies in the serum was positively correlated with that in the seminal plasma (rs = 0.86, P <0.01) while both the number of HBV DNA copies in the serum and that in the seminal plasma were negatively correlated with PMS (r = －0.233 and －0.465, P <0.01) and MNS (r = －0.250 and －0.508, P <0.01). The incidence rate of immune infertility showed no statistically significant differences among the groups with different numbers of HBV DNA copies in the seminal plasma (P >0.05). CONCLUSIONS: HBV infection can increase the incidence rate of immune infertility in men and is correlated with the low quality of sperm.

[Role of TGF-ß1 in Sertoli cells and tight junction].

OBJECTIVE: To explore the role of TGF-ß1 in the proliferation and apoptosis of Sertoli cells and its effect on the expressions of tight junction-related proteins and genes in rats. METHODS: Rat Sertoli cells were isolated in vitro, primarily cultured, and divided into groups A (blank control), B (TGF-ß1 receptor blocker), C (TGF-ß1), and D (TGF-ß1 + receptor blocker). The proliferation and apoptosis of the cells were detected by CCK-8 and flow cytometry, respectively. After establishment of the dual-chamber model for the primary culture of Sertoli cells, the trans-epithelia electrical resistance (TER) value was measured and the relative expressions of Occludin, ZO-1 and Claudin Ⅱ determined by RT-PCR and Western blot. RESULTS: The OD value of the proliferation of the Sertoli cells was markedly higher in group C than in groups A and D (0.79 ± 0.04 vs 0.66 ± 0.05 and 0.68 ± 0.02, P<0.05), with statistically significant differences among the four groups (F = 5.05, P <0.05). However, no remarkable difference with found among the four groups in the apoptosis rate of the cells (F = 1.13, P >0.05). The TER value was dramatically decreased in group C as compared with groups A and D (ï¼»176.37 ± 16.61ï¼½ vs ï¼»281.42 ± 9.83ï¼½ and ï¼»254.37 ± 13.55ï¼½ /cm2, P<0.01), with statistically significant differences among the four groups (F = 38.99, P<0.01). There were no remarkable differences among the four groups in the mRNA expressions of ZO-1 and Claudin Ⅱ (F = 0.49 and 0.93, P>0.05) or their protein expressions (F = 0.28 and 1.31, P>0.05). Both the mRNA and protein expressions of Occludin were markedly lower in group C than in A and D (P<0.01 and P<0.05), with statistically significant differences among the four groups (F = 6.86 and 6.87, P<0.01). CONCLUSIONS: TGF-ß1 can promote the proliferation of Sertoli cells in rats and act on the tight junction of the cells by regulating the expression of Occludin.

[Type of sperm DNA strand breaks in infertile men and its clinical implication].

OBJECTIVE: To observe the characteristics of sperm single-stranded DNA breaks (SSB) and double-stranded DNA breaks (DSB) in infertile men, explore the association of DSB with male infertility, and provide a new observation index and idea for the diagnosis and treatment of the disease. METHODS: This study involved 60 infertile men (infertility group) and 30 normal healthy males with infertile wives (control group). We comparatively analyzed the seminal parameters of the two groups, determined sperm concentration and viability using the computer aided sperm analysis system, measured the sperm survival rate by hypoosmotic swelling (HOS) test, examined sperm morphology by Diff-Quick staining, and detected sperm DNA damage by two-tail comet assay. RESULTS: Nine two-tail comet models were established for detecting sperm DNA integrity. Comparisons between the fertility and control groups showed that the sperm DNA fragmentation index (DFI) was (33.8 ± 13.1) vs (16.3 ± 7.9)% (P < 0.01), the SSB-DFI was (19.2 ± 11.4) vs (14.9 ± 7.6)% (P > 0.05), the SSB-DFI/DFI was (56.8 ± 32.4) vs (91.4 ± 27.8)% (P < 0.01), the DSB-DFI was (23.9 +13.4) vs (6.1 ± 2.7)% (P < 0.01), and the DSB-DFI/DFI was (70.8 ± 19.5) vs (37.4 ± 11.3)% (P < 0.01). The optimal cut-off value of DSB-DFI/DFI in the diagnosis of male infertility was 39.5%, with the AUG, sensitivity, and specificity of 0.969, 98.3%, and 90%; that of DSB-DFI was 15.85%, with the AUC, sensitivity, and specificity of 0.912, 86.7%, and 80%; and that of DFI was 18.65%; with the AUC, sensitivity, and specificity of 0.861, 90%, 70%, respectively. In the infertile men, neither SSB-DFI nor SSB-DFI/DFI exhibited any correlation with semen parameters (P > 0.05); DFI was correlated negatively with the percentage of progressively motile sperm, sperm survival rate, and the percentage of morphologically normal sperm (P < 0.05 or P < 0.01), but not correlated with sperm concentration (P > 0.05); both DSB-DFI and DSB-DFI/DFI showed a negative correlation with sperm concentration, sperm survival rate, and the percentages of progressively motile sperm and morphologically normal sperm (P < 0.05 or P < 0.01). CONCLUSION: Double-stranded, rather than single-stranded DNA breaks, may be a factor inducing male infertility. The type of sperm DNA strand damage is of much reference value for the assessment of male fertility.

[IL-6 and sICAM-1 in seminal plasma relate to male immune infertility].

OBJECTIVE: To detect the expressions of interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sI- CAM-1) in the seminal plasma of infertile men and explore the role of inflammatory cytokines in male immune infertility. METHODS: Based on the results of the sperm-bound antibody immunobead test, 123 males with clinically suspected infertility were divided into an immune infertility group (n = 41), other infertility group A (n = 37), and other infertility group B (n = 45). The immune infertility patients were further subdivided into a leukocyte-positive and a leukocyte-negative group according to the results of leukocyte peroxidase staining. The control group included 31 males confirmed to be fertilein the clinic. Statistical analyses were conducted on the differences in inflammatory cytokines expressions and main parameters in the seminal plasma among different groups. The seminal liquefaction time was measured by visual and microscopic observation, sperm concentration and motility detected using the computer-assisted sperm analysis system, sperm viability determined by hypotonic swelling assay, and the expression levels of IL-6 and sICAM-1 meas- ured by ELISA. RESULTS: The infertility groups showed significantly lowers perm viability (P < 0.05) and progressive motility (P < 0.01) than the fertile control, but no remarkable differences from the latter in sperm concentration (P > 0.05) and semen liquefaction time (P > 0.05). No statistically significant differences were observed in seminal parameters between the immune infertility group and other infertility groups (P > 0.05). The IL-6 and sICAM-1 levels in the seminal plasma were extremely significantly higher in the im- mune infertility group ([37.92 ± 17.01] ng/L and [89.15 ± 41.82] ng/ml), other infertility group A ([22.23 ± 13.77] ng/L and [67.81 ± 33.24] ng/ml), and other infertility group B ([18.75 ± 14.32] ng/L and [53.25 ± 27.09] ng/ml) than in the normal control group ([9.47 ± 5.76] ng/L and [19.46 ± 9.77] ng/ml) (P <0.01) , with remarkable differences between the immune infertility group and the other two infertility groups (P < 0.05). The leukocyte-positive patients showed significantly increased levels of IL-6 ([49.25 ± 21.46] ng/L) and sICAM-1 ([104.36 ± 46.41] ng/ml) as compared with the leukocyte-negative ones ([31.38 ± 15.54] ng/Land [80.38 ± 35.52] ng/ml) (both P < 0.05). CONCLUSION: IL-6 and sICAM-1 in the seminalplasma are involved in male immune infertility.

Tanshinone IIA induces apoptosis and inhibits the proliferation, migration, and invasion of the osteosarcoma MG-63 cell line in vitro.

Tanshinone IIA (Tan IIA) is an active ingredient extracted from the widely used Danshen root (Salvia miltiorrhiza Bunge), a traditional Chinese medicine. Recent studies have indicated that Tan IIA may play important roles in anticancer treatment. However, its effects on the most common primary malignant bone tumor, osteosarcoma (OS), are unknown. Here, we report that Tan IIA may be an efficacious anti-OS drug as it could induce cell apoptosis and inhibit proliferation, migration, and invasion in vitro. Furthermore, we detected possible molecular mechanisms for Tan IIA activity by examining the levels of Bcl-2, Bax expression, and caspase-3, caspase-8, and caspase-9 activities that regulate apoptosis, matrix metalloproteinase (MMP)-2, and MMP-9 involved in regulating migration and invasion. In this study, we find that Tan IIA inhibits proliferation and induces apoptosis in the human OS cell line MG-63 in a time-dependent and dose-dependent manner. In addition, Tan IIA displays inhibitory activity on OS cell migration and invasion. Mechanistic studies have shown that Tan IIA activity is mediated by caspase activation. Tan IIA was also shown to reduce antiapoptotic Bcl-2, MMP-2, and MMP-9 levels, whereas it increased proapoptotic Bax levels. These data suggest that Tan IIA may be a novel, efficient candidate agent for OS treatment.

Different expression of alternative lengthening of telomere (ALT)-associated proteins/mRNAs in osteosarcoma cell lines.

Tumors, including osteosarcoma (OS), are capable of evading senescence and cell death, which is caused by telomere loss with cell division. Alternative lengthening of telomeres (ALT) is considered as the main telomere maintenance mechanism in OS. In this study, we investigated the expression of ALT-associated proteins and mRNAs in human OS cell lines. Western blotting was used to detect the protein expression in OS cell lines, while the expression of mRNA was determined by reverse-transcriptase PCR and quantitative real-time PCR analysis. Whole-genome expression arrays were used to analyze the expression of all the mRNAs involved in telomere maintenance mechanisms (TMMs) including human telomerase reverse transcriptase, promyelocytic leukemia proteins and other related proteins. OS and normal cell lines do not express telomerase reverse transcriptase (hTERT) as a key subunit of telomerase, although they show varying levels of ALT-associated proteins and mRNAs such as PML, Rad52, MRE11 and FEN1 by Western blotting and quantitative real-time PCR analysis. A number of mRNAs that play essential roles in ALT are expressed more in OS cell lines than in the osteoblast cell line, as shown by whole-genome expression arrays. In conclusion, OS cell lines maintain their telomere length primarily through the ALT mechanism. There are numerous other proteins that regulate this process in OS; therefore, anti-ALT therapy may be a more effective method to treat OS than anti-telomerase therapy.