Single-cell RNA-seq reveals T cell exhaustion and immune response landscape in osteosarcoma.

Background: T cell exhaustion in the tumor microenvironment has been demonstrated as a substantial contributor to tumor immunosuppression and progression. However, the correlation between T cell exhaustion and osteosarcoma (OS) remains unclear. Methods: In our present study, single-cell RNA-seq data for OS from the GEO database was analysed to identify CD8+ T cells and discern CD8+ T cell subsets objectively. Subgroup differentiation trajectory was then used to pinpoint genes altered in response to T cell exhaustion. Subsequently, six machine learning algorithms were applied to develop a prognostic model linked with T cell exhaustion. This model was subsequently validated in the TARGETs and Meta cohorts. Finally, we examined disparities in immune cell infiltration, immune checkpoints, immune-related pathways, and the efficacy of immunotherapy between high and low TEX score groups. Results: The findings unveiled differential exhaustion in CD8+ T cells within the OS microenvironment. Three genes related to T cell exhaustion (RAD23A, SAC3D1, PSIP1) were identified and employed to formulate a T cell exhaustion model. This model exhibited robust predictive capabilities for OS prognosis, with patients in the low TEX score group demonstrating a more favorable prognosis, increased immune cell infiltration, and heightened responsiveness to treatment compared to those in the high TEX score group. Conclusion: In summary, our research elucidates the role of T cell exhaustion in the immunotherapy and progression of OS, the prognostic model constructed based on T cell exhaustion-related genes holds promise as a potential method for prognostication in the management and treatment of OS patients.

Clinical efficacy of different surgical approaches in the treatment of thoracolumbar tuberculosis: a multicenter retrospective case-control study with a minimum 10-year follow-up.

OBJECTIVE: To evaluate the long-term clinical efficacy of three different surgical approaches in treating thoracolumbar tuberculosis. METHODS: A total of 176 patients with thoracolumbar tuberculosis, treated with open surgery at two hospitals, were retrospectively analyzed. Patients were stratified into three groups based on the surgical approach: anterior-only (AO), posterior-only (PO), and anterior-posterior combined (AP) approaches. Collected data encompassed operative duration, intraoperative blood loss, hospital stay length, complications, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Visual Analog Scale (VAS) score, Oswestry Disability Index (ODI), American Spinal Injury Association (ASIA) classification, and radiographic measurements of segmental lordotic Cobb angles, correction angles, and correction rates. RESULTS: The minimum duration of follow-up among all patients was 10 years. Postoperatively, all patients experienced a reduction in ESR and CRP, with normalization occurring within 3 months and sustained normal at the last follow-up. The AP group had a longer operative duration and higher intraoperative blood loss than the other two groups. The Cobb correction rates for AO, PO, and AP were (56.33±6.62)%, (72.82±5.66)%, and (74.45±5.78)%, respectively. The correction loss of Cobb angles for AO, PO, and AP were (2.85±1.01)°, (1.42±0.97)°, and (1.19±0.89)°, respectively. Patients in all groups showed significant improvement in VAS scores and ODI postoperatively, with no notable intergroup differences. The neurological recovery rates for the AO, PO, and AP groups were 84.62, 87.10, and 83.72%, respectively, while the complication rates were 12.73, 16.98, and 22.06%, respectively. CONCLUSION: An anterior-only approach is recommended for cases with localized lesions and smaller angular deformities. For patients with multisegmental lesions and larger angular deformities, a posterior-only or anterior-posterior combined approach is advised, with a preference for the posterior-only approach.

Can a Nomogram Predict Survival After Treatment for an Ankylosing Spondylitis Cervical Fracture in a Patient With Neurologic Impairment? A National, Multicenter Study.

BACKGROUND: Ankylosing spondylitis-related cervical spine fracture with neurologic impairment (ASCF-NI) is a rare but often lethal injury. Factors independently associated with survival after treatment remain poorly defined, and identifying patients who are likely to survive the injury remains challenging. QUESTIONS/PURPOSES: (1) What factors are independently associated with survival after treatment among patients with ASCF-NI? (2) Can a nomogram be developed that is sufficiently simple for clinicians to use that can identify patients who are the most likely to survive after injury? METHODS: This retrospective study was conducted based on a multi-institutional group of patients admitted and treated at one of 29 tertiary hospitals in China between March 1, 2003, and July 31, 2019. A total of 363 patients with a mean age of 53 ± 12 years were eventually included, 343 of whom were male. According to the National Household Registration Management System, 17% (61 of 363) died within 5 years of injury. Patients were treated using nonsurgical treatment or surgery, including procedures using the anterior approach, posterior approach, or combined anterior and posterior approaches. Indications for surgery included three-column injury, unstable fracture displacement, neurologic impairment or continuous progress, and intervertebral disc incarceration. By contrast, patients generally received nonsurgical treatment when they had a relatively stable fracture or medical conditions that did not tolerate surgery. Demographic, clinical, and treatment data were collected. The primary study goal was to identify which factors are independently associated with death within 5 years of injury, and the secondary goal was the development of a clinically applicable nomogram. We developed a multivariable Cox hazards regression model, and independent risk factors were defined by backward stepwise selection with the Akaike information criterion. We used these factors to create a nomogram using a multivariate Cox proportional hazards regression analysis. RESULTS: After controlling for potentially confounding variables, we found the following factors were independently associated with a lower likelihood of survival after injury: lower fracture site, more-severe peri-injury complications, poorer American Spinal Injury Association (ASIA) Impairment Scale, and treatment methods. We found that a C5 to C7 or T1 fracture (ref: C1 to C4 and 5; hazard ratio 1.7 [95% confidence interval 0.9 to 3.5]; p = 0.12), moderate peri-injury complications (ref: absence of or mild complications; HR 6.0 [95% CI 2.3 to 16.0]; p < 0.001), severe peri-injury complications (ref: absence of or mild complications; HR 30.0 [95% CI 11.5 to 78.3]; p < 0.001), ASIA Grade A (ref: ASIA Grade D; HR 2.8 [95% CI 1.1 to 7.0]; p = 0.03), anterior approach (ref: nonsurgical treatment; HR 0.5 [95% CI 0.2 to 1.0]; p = 0.04), posterior approach (ref: nonsurgical treatment; HR 0.4 [95% CI 0.2 to 0.8]; p = 0.006), and combined anterior and posterior approach (ref: nonsurgical treatment; HR 0.4 [95% CI 0.2 to 0.9]; p = 0.02) were associated with survival. Based on these factors, a nomogram was developed to predict the survival of patients with ASCF-NI after treatment. Tests revealed that the developed nomogram had good performance (C statistic of 0.91). CONCLUSION: The nomogram developed in this study will allow us to classify patients with different mortality risk levels into groups. This, coupled with the factors we identified, was independently associated with survival, and can be used to guide more appropriate treatment and care strategies for patients with ASCF-NI. LEVEL OF EVIDENCE: Level III, therapeutic study.

Instantaneous death risk, conditional survival and optimal surgery timing in cervical fracture patients with ankylosing spondylitis: A national multicentre retrospective study.

Background: The mortality rate in patients with ankylosing spondylitis (AS) and cervical fracture is relatively high. Objectives: This study aimed to investigate the instantaneous death risk and conditional survival (CS) in patients with AS and cervical fracture. We also studied the relationship between surgical timing and the incidence of complications. Methods: This national multicentre retrospective study included 459 patients with AS and cervical fractures between 2003 and 2019. The hazard function was used to determine the risk of instantaneous death. The five-year CS was calculated to show the dynamic changes in prognosis. Results: The instantaneous death risk was relatively high in the first 6 months and gradually decreased over time in patients with AS and cervical fracture. For patients who did not undergo surgery, the instantaneous risk of death was relatively high in the first 15 months and gradually decreased over time. For patients with American Spinal Injury Association impairment scale (ASIA) A and B, the 5-year CS was 55.3% at baseline, and improved steadily to 88.4% at 2 years. Odds ratios (ORs) for pneumonia, electrolyte disturbance, respiratory insufficiency, and phlebothrombosis decreased as the surgery timing increased. Conclusion: Deaths occurred mainly in the first 6 months after injury and gradually decreased over time. Our study highlights the need for continued surveillance and care in patients with AS with cervical fractures and provides useful survival estimates for both surgeons and patients. We also observed that early surgery can significantly increase functional recovery, and decrease the incidence of complications and rehospitalisation.

IL-1ß secreted by macrophage M2 promotes metastasis of osteosarcoma via NF-κB/miR-181α-5p/RASSF1A/Wnt pathway.

BACKGROUND: Ras-associated domain family protein1 isoform A (RASSF1A) was significantly absent in clinical samples and many osteosarcoma (OS) cell lines. Overexpression of RASSF1A could suppress OS metastasis, which may be mediated by tumor-associated macrophages polarized M2 (M2-TAMs). However, the relationship between IL-1ß secreted by M2-TAMs and RASSF1A remains unknown. METHODS: The expression levels of M2-TAMs markers CD68 and CD204 were measured by flow cytometry, and arginase-1 (Arg-1) and interleukin-1ß (IL-1ß) secreted by M2-TAMs were examined by real-time quantitative PCR (RT-qPCR). MTT assay was employed to determine the proliferation of OS cells, while scratch wound healing assay and Transwell assay were used to evaluate their migration and invasion, respectively. The level of miR-181α-5p was measured by RT-qPCR, while the levels of RASSF1A, GSK-3ß, p-GSK-3ß, ß-catenin, MMP-2 and MMP-9 were evaluated by Western blot. The direct binding of miR-181α-5p and RASSF1A was identified using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: The levels of CD68, CD204, Arg-1 and IL-1ß were elevated in M2-TAMs compared with control group. Overexpression of RASSF1A and knockdown of miR-181α-5p could both suppress invasion and migration of OS cells through Wnt pathway. IL-1ß secreted by M2-TAMs facilitated the OS metastasis via RASSF1A/Wnt pathway, which could be targeted by miR-181α-5p and affected by nuclear factor-kappa B (NF-κB). CONCLUSIONS: IL-1ß secreted by M2-TAMs contributed to OS metastasis, which could be suppressed by knockdown of miR-181α-5p or overexpression of RASSF1A through NF-κB/miR-181α-5p/RASSF1A/Wnt pathway. These findings can guide new target discovery for drug development in OS treatment.

Interleukin-1ß/nuclear factor-κB signaling promotes osteosarcoma cell growth through the microRNA-181b/phosphatase and tensin homolog axis.

So far, microRNA has attracted plenty of interest due to its role in tumorigenesis. Reportedly, miR-181b may be involved in the tumorigenesis of osteosarcoma (OS). In the current study, we attempted to investigate the detailed function and mechanism of miR-181b in OS carcinogenesis. Herein, miR-181a, miR-181b, miR-181c, and miR-181d expressions in OS tissues were higher than that in nontumor tissue samples as examined real-time polymerase chain reaction. Via direct targeting, miR-181b negatively regulated the expression of phosphatase and tensin homolog (PTEN), a well-known tumor suppressor. Furthermore, a small interfering RNA strategy was used to find that interleukin (IL)-1B and nuclear factor-κB (NF-κB) regulate miR-181b and PTEN expression. Consequently, the repression of PTEN by miR-181b promotes OS cell proliferation. In summary, our data support a critical role for NF-κB-dependent upregulation of miR-181b, which further inhibited PTEN expression and promoted the cell proliferation of OS cell lines. The above findings represent a new pathway for the repression of PTEN and the promotion of cell proliferation upon IL-1ß induction.

miR-194 Suppresses Proliferation and Migration and Promotes Apoptosis of Osteosarcoma Cells by Targeting CDH2.

BACKGROUND/AIMS: Studies have shown that miR-194 functions as a tumour suppressor and is associated with tumour growth and metastasis. This study intends to uncover the mechanism of tumour suppression by miR-194. The expression of miR-194 in osteosarcoma cell lines and tissues were monitored by real-time PCR. METHODS: The proliferation ability was examined by MTT assay. Migration and apoptosis of cells were monitored by migration assay and flow cytometry, respectively. The regulation of miR-194 on CDH2 was determined by luciferase assays and western blot assays. RESULTS: The results showed that miR-194 was significantly reduced in osteosarcoma compared with that in normal bone tissue. Overexpression of miR-194 significantly attenuated the proliferation and migration and induced the apoptosis of osteosarcoma cells. Furthermore, we demonstrated that miR-194 has inhibited the malignant behaviour of osteosarcoma by downregulating CDH2 expression. CONCLUSIONS: These findings suggested that miR-194 may act as a tumour suppressor in osteosarcoma. miR-194/CDH2 may be a novel therapeutic target in the treatment of osteosarcoma.

[miR-181a promotes the proliferation and metastasis of osteosarcoma cells by targeting RASSF1A].

OBJECTIVE: To investigate the role of miR-181a in promoting the proliferation and metastasis of osteosarcoma cells by targeting RASSF1A.
 METHODS: The level of miR-181a in 30 human osteosarcoma tissues and corresponding bone tissues was detected by real-time PCR, and the correlation between the level of miR-181a and clinicopathological characteristics of osteosarcoma was analyzed. Osteosarcoma cells MG-63 were transfected with chemically-synthesized miR-181a mimics and inhibitors, and the proliferation, migration and invasion of MG-63 cells were detected by MTT and Transwell assay. The specific binding ability of miR-181a to RASSF1A 3'-UTR was theoretically predicted and detected by the dual luciferase reporter gene assay.
 RESULTS: The level of miR-181a in osteosarcoma tissues was statistically higher than that in the corresponding bone tissues (P<0.001). However, the level of miR-181a was not correlated with gender, age, tumor size and stage (P>0.05). MTT and Transwell assays showed that the growth rate, migration and invasion ability of MG-63 cells with up-regulation of miR-181a was significantly increased compared with negative control (P<0.05), while the growth rate, migration and invasion ability of MG-63 with down-regulated miR-181a was significantly decreased compared with negative control (P<0.05). Luciferase reporter gene assay showed that miR-181a targeted the 3'-UTR of RASSF1A and regulated the expression of RASSF1A.
 CONCLUSION: MiR-181a promotes the proliferation and metastasis of osteosarcoma cells through specifically binding to RASSF1A 3'-UTR and subsequent down-regulation of RASSF1A.

Study of breakthrough cancer pain in an animal model induced by endothelin-1.

Cancer patients with bone metastases often suffer breakthrough pain. However, little progress has been made in the treatment of breakthrough pain and its associated mechanism(s) in the patient with cancer due to lacking of resembling and predictive animal models. We previously have demonstrated that endothelin-1 plays an important role in breakthrough cancer pain. In the present study, we have established an animal model of breakthrough cancer pain induced by endothelin-1. The animal model of breakthrough cancer pain is strictly followed the definition and meets the characteristics of breakthrough pain. The model is reliable, reproducible and easy to be produced. To our knowledge, this is the first report for establishing such an animal model. In addition, we also found that a selective ETA receptor antagonist BQ-123 could reverse endothelin-1 induced breakthrough pain. We further studied the characteristics of pain behaviors such as hind limb use score and voluntary wheel running as well as the electrophysiology of sciatic nerve fibers with the model. The murine model shows high resemblance compared to the breakthrough cancer pain in the patients with cancer clinically. It provides a platform for further study of the pathogenesis of breakthrough cancer pain and targeted intervention.

The tumor suppressive role of RASSF1A in osteosarcoma through the Wnt signaling pathway.

Ras-association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene and its expression is lost in numerous types of cancer cells, including primary osteosarcoma cells. However, its functional significance in osteosarcoma has not been well defined. The messenger RNA (mRNA) expression of RASSF1A in osteosarcoma tissues and corresponding non-tumoral tissues was measured by real-time PCR. Overexpression of RASSF1A was established by an adenoviral vector expressing RASSF1A. Cell migration and invasion were analyzed in transwells. Apoptosis and cell cycle were analyzed using flow cytometry. Wnt/ß-catenin activity was measured by TCF reporter dual-luciferase assay. Cell viability was measured by MTT assay. Protein expression was detected by Western blot. RASSF1A mRNA expression was significantly lower in osteosarcoma tissues than that in the corresponding non-tumoral tissues. The lowered RASSF1A expression correlated with the clinical severity of osteosarcoma. rAd-RASSF1A injection significantly inhibited the growth of xenograft MNNG/HOS tumors in mice. Overexpression of RASSF1A resulted in significant inhibition of the proliferation, migration, and invasion; induced apoptosis; and arrested cell cycle at G0/G1 phase in both the MNNG/HOS and SaOS2 cells. Overexpression of RASSF1A inhibited the Wnt/ß-catenin activity, decreased phosphorylation of Akt/glycogen synthase kinase-3-ß (GSK3-ß), and increased phosphorylation of mammalian sterile 20-like kinase 1 (MST1). Overexpression of RASSF1A downregulated the cyclin D1, c-Myc, and matrix metalloproteinase-7 (MMP-7) protein levels. RASSF1A functions as a tumor suppressor in osteosarcoma and exerts anti-cancer roles through regulating Akt/GSK-3-Wnt/ß-catenin signaling.

[Surgical correction of post-traumatic thoracolumbar kyphotic deformity with posterior transpedicular limited osteotomy technique].

OBJECTIVE: To evaluate the clinical outcome of surgical management for post-traumatic thoracolumbar kyphotic deformity with single-stage posterior transpedicularlimited osteotomies. METHODS: From March 2007 to May 2010, 17 patients with post-traumatic thoracolumbar kyphotic deformity treated with posterior limited transpedicular osteotomy were admitted. The preoperative Cobb angle was 41°-62°(52.5° ±6.4°). Sagittal balance was evaluated by the standing lateral films measuring the C7 plumb line distance (C7 PLD) from the posterior superior corner of S1. The C7 PLD was 18-58 (41.2 ±12.4) mm in the sagittal plane. The preoperative oswestry disability index (ODI) was 42-50 (45.7 ±2.7), and the average preoperative visual analogue scale (VAS) was 8-10 (8.8 ±0.7). The American Spinal Injury Association (ASIA) impairment scale was used to assess the neurological deficits, and grade C in 1 patient, grade D in 7 and grade E in 9 patients. The operation time, blood loss, complications, post-operative Cobb angle, ODI and VAS score at the follow-up were collected and analyzed. RESULTS: The average duration of postoperative follow-up was 24-53 (34.5 ±7.1) months. The operation time was 180-400 (287.1 ±65.9) min, with an blood loss of 350-1 300 (838.2 ±276.4) mL. The postoperative kyphotic angle was 3°-12° (6.1° ±3.0°), and it was 7.5° ±2.6° at the final follow-up evaluation. The postoperative C7PLD was (3.6 ±3.9) mm and it was (3.4 ±2.3) mm at the final follow-up evaluation. Postoperatively, the ASIA impairment scale was grade D in 4 and grade E in 13 patients. At the final follow-up ODI and VAS were reduced to an average of 5.2 ±2.4 and 2.4 ±1.0, respectively. Cerebrospinal fluid leakage was found in 2 patients, deep wound infection in 1, and intercostal neuralgia in 2. All the complications were relieved after conservative medical therapy. One patient received additional surgery at postoperative 12 weeks due to breakage of posterior implants. Another screw pullout case was treated with reinsertion of larger screws at postoperative 4 months. Solid fusion was confirmed by plain film and CT scan in all patients within 1 year after the surgery. CONCLUSION: Single-staged posterior transpedicular limited osteotomies is safe and effective to correct post-traumatic thoracolumbar kyphotic deformity.

Ezrin promotes invasion and migration of the MG63 osteosarcoma cell.

BACKGROUND: Evidence shows that ezrin plays an important role in the development of some human malignancies. But the mechanism by which ezrin may affect tumor cell invasion and metastasis remains unclear. METHODS: In this study, the expression of ezrin was verified in osteosarcoma (OS) cells and tissues by comparison with normal bone cells and tissues using Western blotting. OS-MG63 were transfected with pcDNA3.1-ezrin or pGenesil-1/shRNA-ezrin and the stably transfected cells were selected with G418 to yield the ezrin cell line. The OS-MG63 tumor cells were delivered by tail vein to female BALB/c to develop pulmonary metastasis model in vivo. Ezrin was identified as a direct target of miR-183 via a luciferase reporter carrying the 3'-untranslated region of ezrin. Migration assays and invasion assays were done with the transwells. Signaling pathway was studied by Western blotting and/or inhibitor. RESULTS: Ectopic overexpression of ezrin in OS cell line MG63 promoted tumor cell invasion and migration. Consistent with this, knockdown of ezrin inhibited tumor cell invasion and migration. Similar results were obtained in the experimental metastasis model in vivo. We identified ezrin as a direct target of miR-183. What is more, ectopic expression of ezrin could induce the expression of N-cadherin and enhance the activity of extracellular signal-regulated kinase (ERK) signaling. CONCLUSION: Collectively, these results suggest that ezrin as a direct target of miR-183 promotes the aggressiveness of OS via increased N-cadherin and activating ERK signaling.

miR-33a is up-regulated in chemoresistant osteosarcoma and promotes osteosarcoma cell resistance to cisplatin by down-regulating TWIST.

BACKGROUND: miRNAs are involved in osteosarcoma (OS) chemoresistance, and TWIST reportedly enhances cisplatin-induced OS cell apoptosis by inhibiting multiple signaling pathways. In this study, we profiled miRNAs differentially expressed in chemoresistant OS, with a focus to identify miRNAs that regulate TWIST expression and OS chemoresistance. METHODS: OS patients who showed <90% tumor necrosis after neochemotherapy were defined as poor responders (chemoresistant), and those who showed ≥90% tumor necrosis were defined as good responders (control). miRNA microarray analysis was carried out with a discovery cohort (n = 12) of age-, sex- and tumor stage-matched chemoresistant and control OS patients. RESULTS: Among the up-regulated miRNAs in chemoresistant OS samples, miR-33a was verified to down-regulate TWIST expression, which was supported by an inverse miRNA-33a/TWIST expression trend in the validation cohort (n = 70), target-sequence-specific inhibition of TWIST-3' untranslated region-luciferase reporter activity by miR-33a, and alteration of TWIST expression by overexpression or inhibition of miR-33a in human OS cell lines. In Saos-2 cells treated with cisplatin, inhibition of miR-33a by antagomir-33a markedly increased cell apoptosis, which was enhanced by overexpression of TWIST. The apoptosis-inducing effect of TWIST overexpression was reversed by overexpression of miR-33a. In MG-63 cells, overexpression of miR-33a significantly decreased cisplatin-induced cell apoptosis, which was enhanced by knockdown of TWIST. Antagomir-33a significantly increased cisplatin-induced cell apoptosis, which was reversed by knockdown of TWIST. CONCLUSIONS: We have demonstrated in this study that miR-33a is up-regulated in chemoresistant OS and that the miR-33a level is negatively correlated with the TWIST protein level in OS. Our in vitro data indicate that miR-33a promotes OS cell resistance to cisplatin by down-regulating TWIST; on the other hand, inhibition of miR-33a by antagomir-33a enhances cisplatin-induced apoptosis in OS cells by up-regulating TWIST expression. The findings suggest that inhibition of miR-33a/TWIST signaling could be a potential new strategy to enhance neoadjuvant chemotherapy for OS.

Endothelin-1 single nucleotide polymorphisms and risk of pulmonary metastatic osteosarcoma.

Pulmonary metastases are the major cause of death of osteosarcoma (OS) patients. Endothelin-1 (ET-1) reportedly plays an important role in OS metastasis. In the present study, we for the first time explored the association of ET-1 SNPs with the risk of pulmonary metastatic OS. We genotyped three SNPs (rs1800541, rs2070699 and rs5370) in the ET-1 gene in a case-control study, using 260 pairs of age-, sex-, residence area- and tumor location-matched subjects. Patients with pulmonary metastatic OS and patients with localized high-grade (stage IIB) OS were enrolled as cases and controls, respectively. The G allele at rs1800541 was found associated with reduced risk of pulmonary metastatic OS after adjustment for body mass index, systolic blood pressure, diastolic blood pressure and the plasma ET-1 level (P=10(-4); adjusted OR, 0.55; 95% CI, 0.42-0.70), while the G allele at rs2070699 was not significantly associated with the risk of pulmonary metastatic OS (P=0.15; adjusted OR, 1.15; 95% CI, 0.87-1.50). The mRNA and the secreted protein levels of ET-1 in primary OS cell cultures (POCCs) established from surgically resected primary OS in the rs1800541 TT homozygotes were higher than those from the TG heterozygotes (P<0.05), who in turn showed higher ET-1 mRNA and secreted ET-1 levels than the GG homozygotes (P<0.05). In the control subjects, the rs1800541 TT homozygotes showed an 18.4% relapse rate, significantly higher than that of the GG homozygotes (0%) (P<0.01). On the other hand, the GG homozygotes showed a 71.4% complete recovery rate, significantly higher than that of the TG heterozygotes (7.3%) and the TT homozygotes (0%) (P<0.01). This study provides the first evidence of an association between the ET-1 gene SNPs and the risk of pulmonary metastatic OS.

TWIST interacts with endothelin-1/endothelin A receptor signaling in osteosarcoma cell survival against cisplatin.

Both TWIST and the endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling are important in osteosarcoma (OS) progression. In the present study, the interaction between TWIST and ET-1/ETAR signaling in OS cells was investigated, and the impact of the functional interaction on OS cell survival against chemotherapy agent-induced apoptosis was assessed. TWIST was overexpressed and knocked down in Saos-2 and MG-63 OS cells, respectively. In Saos-2 cells, overexpression of TWIST significantly decreased ET-1 mRNA and protein expression levels, cell survival against cisplatin and phosphorylation of Akt at serine 473 (ser473), which was abolished by the selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or the selective ETAR inhibitor, BQ123. In MG-63 cells, knockdown of TWIST significantly increased ET-1 expression, cell survival against cisplatin and phosphorylation of Akt at ser473. However, exogenous ET-1 only partially rescued cell survival against cisplatin-induced apoptosis in the cells in which TWIST had been knocked down in the presence of LY294002. In conclusion, we have demonstrated that TWIST significantly, although only partially, decreases OS cell survival against cisplatin by downregulating ET-1/ETAR signaling via inhibition of the PI3K/Akt pathway. To the best of our knowledge, the present study has provided the first evidence of a functional interaction between TWIST and ET-1/ETAR signaling in OS cells. This finding adds novel insights into the molecular mechanisms underlying OS progression, cell survival and chemoresistance.