Integration of single-cell and bulk RNA sequencing data reveals that CYTOR is a potential prognostic and immunotherapeutic response marker for skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is a highly malignant tumor that is prone to immune escape and distant metastasis. Immunotherapy is considered to be the best treatment for patients with SKCM. However, not all patients benefit from it. We observed a significant differential expression of the lncRNA CYTOR in patients with SKCM based on single-cell and bulk RNA sequencing data mining results. The results showed that compared to normal tissue lncRNA CYTOR expression was significantly upregulated in SKCM tissue. Subsequently, we validated this finding in clinical samples, and we also found that the expression of lncRNA CYTOR in SKCM was higher as it progressed. lncRNA CYTOR was differentially expressed in patients who responded to immunotherapy, suggesting that it may serve as a biomarker to predict the efficacy of SKCM immunotherapy. In-depth analysis revealed that lncRNA CYTOR expression was strongly correlated with immune cell infiltration, immune response, and immune checkpoint expression. Meanwhile, our experiments revealed that CYTOR affects SKCM cell invasion and clone formation and is associated with the activation of the EMT pathway. In summary, our findings illustrate, for the first time, the value of CYTOR as a potential prognostic and immunotherapeutic response marker in SKCM.

TNFSF10, an autophagy related gene, was a prognostic and immune infiltration marker in skin cutaneous melanoma.

Autophagy exerts a pivotal effect on skin cutaneous melanoma (SKCM). This study was aimed to investigate the expression of autophagy related genes (ARGs) in SKCM as well as its clinical value. Differentially expressed (DE) ARGs were downloaded from the intersection of SKCM data in GEPIA2 database and ARGs in Human Autophagy Database (HADB) database, and were verified in SKCM datasets GSE46517 and GSE15605. DE ARGs were enriched by Metascape online tools. According to GEPIA2 database, tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10) was identified as a closely related factor and prognostic marker of SKCM. Then the correlation analysis of clinicopathological characteristics between TNFSF10 and SKCM was completed by several online tools such as TISCH, HPA, BEST and qRT-PCR. Subsequently, we investigated TNFSF10 related functions and signal pathways with LinkedOmics online tool, and immune infiltration using Assistant for Clinical Bioinformatics online tool. Furthermore, correlation analysis between TNFSF10 expression and immunotherapy response was performed by TIDE algorithm and BEST online tool. And Kaplan-Meier Plotter was used to assessing the prognosis of SKCM patients receiving immunotherapy. Finally, the correlation analysis among TNFSF10 methylation, TNFSF10 expression and patient prognosis was completed by the DiseaseMeth version 2.0, UCSC XENA and qRT-PCR. ARGs are DE in SKCM and participate in the ERBB signaling pathway, as well as the processing and presentation of antigens. Moreover, TNFSF10's expression along with methylation expression were significantly associated with the prognosis. Low expression of TNFSF10 was associated with malignant clinicopathological features, lower immune signal activity and lower immunocytes abundance in patients with SKCM. As an ARG, TNFSF10 has a potential capacity in predicting the prognosis of SKCM patients, meanwhile, may be a novel immunotherapy marker for SKCM.

Albiflorin alleviation efficacy in osteoarthritis injury using in-vivo and in-vitro models.

OBJECTIVES: Osteoarthritis seriously affects the daily life of people. Albiflorin (AF) has anti-inflammatory and antioxidant functions in various human diseases. This study aimed to clarify the function and mechanism of AF in osteoarthritis. METHODS: The functions of AF on rat chondrocyte proliferation and apoptosis, inflammatory response, oxidative stress and extracellular matrix (ECM) degradation in rat chondrocytes induced by interleukin-1beta (IL-1ß) were evaluated by Western blot, immunofluorescence, flow cytometry and enzyme-linked immunosorbent assay. The mechanism of AF on the IL-1ß induced rat chondrocyte injury was investigated by multiple experiments in vitro. Meanwhile, the AF function in vivo was assessed using haematoxylin-eosin staining, Alcian blue, Safranin O/Fast green staining, immunohistochemical analysis and TUNEL assay. KEY FINDINGS: Functionally, AF accelerated the rat chondrocyte proliferation and repressed cell apoptosis. Meanwhile, AF reduced the inflammatory response, oxidative stress and ECM degradation in rat chondrocytes caused by IL-1ß. Mechanistically, the receptor activator of the NF-kappaB ligand (RANKL), an activator for the NF-κB signalling pathway, partially reversed the alleviating effect of AF on IL-1ß-induced chondrocyte injury. Furthermore, the in-vitro results confirmed that AF exerted protective properties against osteoarthritis injury in vivo. CONCLUSION: Albiflorin relieved osteoarthritis injury in rats by inactivating the NF-κB pathway.

METTL14 promotes fibroblast-like synoviocytes activation via the LASP1/SRC/AKT axis in rheumatoid arthritis.

The objective of this study is to explore the specific roles of a crucial N6-methyladenosine (m6A) methyltransferase, methyltransferase-like 14 (METTL14), in fibroblast-like synoviocytes (FLSs) activation of rheumatoid arthritis (RA). RA rat model was induced by administering intraperitoneally collagen antibody alcohol. Primary fibroblast-like synoviocytes (FLSs) were isolated from joint synovium tissues in rats. shRNA transfection tools were used to downregulate METTL14 expression in vivo and vitro. The injury of joint synovium was shown by hematoxylin and eosin (HE) staining. The cell apoptosis of FLSs was determined by flow cytometry. The levels of IL-6, IL-18, and C-X-C motif chemokine ligand (CXCL)10 in serum and culture supernatants were measured by ELISA kits. The expressions of LIM and SH3 domain protein 1 (LASP1), p-SRC/SRC, and p-AKT/AKT in FLSs and joint synovium tissues were determined by Western blots. The expression of METTL14 was greatly induced in the synovium tissues of RA rats compared with normal control rats. Compared with sh-NC-treated FLSs, METTL14 knockdown significantly increased cell apoptosis, inhibited cell migration and invasion, and suppressed the production of IL-6, IL-18, and CXCL10 induced by TNF-α. METTL14 silencing suppresses the expression of LASP1 and the activation of Src/AKT axis induced by TNF-α in FLSs. METTL14 improves the mRNA stability of LASP1 through m6A modification. In contrast, these were reversed by LASP1 overexpression. Moreover, METTL14 silencing clearly alleviates FLSs activation and inflammation in a RA rat model. These results suggested that METTL14 promotes FLSs activation and related inflammatory response via the LASP1/SRC/AKT signaling pathway and identified METTL14 as a potential target for treating RA.

SMG7-AS1 as a prognostic biomarker and predictor of immunotherapy responses for skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is the most life-threatening skin cancer and lacks early detection and effective treatment strategies. Many long noncoding RNAs are associated with the development of tumors and may serve as potential immunotherapeutic targets. In this study, microarray analysis was performed to screen for differentially expressed lncRNAs between SKCM and normal tissues, and SMG7-AS1 was identified as an upregulated lncRNA in SKCM. Subsequently, bioinformatic analysis revealed that dysregulation of SMG7-AS1 influences metastasis and immune infiltration. qRT-PCR of clinical samples demonstrated that the expression of SMG7-AS1 was higher in melanoma tissues. Flow cytometry showed that SMG7-AS1 plays a vital role in the cell cycle. Additionally, SMG7-AS1 was found to be associated with immunotherapy responses. To the best of our knowledge, this study is the first to report that SMG7-AS1 is associated with SKCM and may serve as a prognostic biomarker and predictor of immunotherapy responses in SKCM.

Single Nuclear RNA Sequencing Highlights Intra-Tumoral Heterogeneity and Tumor Microenvironment Complexity in Testicular Embryonic Rhabdomyosarcoma.

BACKGROUND: Testicular embryonic rhabdomyosarcoma (ERMS) is a rare soft tissue tumor in children featured with high intra-tumoral heterogeneity. In this study, we aimed to comprehensively delineate the testicular ERMS intra-tumoral heterogeneity and tumor microenvironment. METHODS: Cell types and the corresponding marker genes were identified by single-nuclear RNA sequencing (snRNA-seq). Functional states of different clusters were evaluated by uniform manifold approximation and projection and differentially expressed genes. Kaplan-Meier curve analysis was constructed according to the gene expression profile to determine the correlation between candidate marker genes and the overall survival and disease-free survival of patients with osteosarcoma from TCGA. RESULTS: A total of 8868 tumor cells and 10,147 normal cells were obtained from testicular ERMS tissues. The heterogeneous malignant subtype was composed of six subgroups (C1-C6) with differential proliferative and migratory potentials. Cell trajectory analysis revealed the C1 subgroup might be the starting cells of the tumor and transform into two different types of malignant cells, C2 and C5/6, during the development of RMS. The differentially expressed genes were closely related to cell adhesion and extracellular matrix signaling pathways. Furthermore, the interaction analysis between cell subgroups (macrophages and tumor cells, endothelial cells and tumor cells) demonstrated that collagen-related gene COL6A1 plays a key role from the initiation of ERMS to the entire process of malignant transformation. CONCLUSION: Our findings provide a new insight in the understanding of the initiation and progression of testicular ERMS and have potential value in the development of markers for the diagnosis and stratification of testicular ERMS.

Efficient and Consistent Orthotopic Osteosarcoma Model by Cell Sheet Transplantation in the Nude Mice for Drug Testing.

Osteosarcoma is a big challenge on clinical treatment. The breakthrough associated with osteosarcoma in basic research and translational research depends on the reliable establishment of an animal model, whereby mice are frequently used. However, a traditional animal modeling technique like tumor cell suspension injection causes batch dynamics and large mice consumption. Here, we suggested a novel approach in establishing an orthotropic osteosarcoma model in nude mice rapidly by cell sheet culture and transplantation. Our findings demonstrated that the 143b osteosarcoma cell sheet orthotopically implanted into the nude mice could form a visible mass within 10 days, whereas it took over 15 days for a similar amount of cell suspension injection to form a visible tumor mass. Living animal imaging results showed that a tumor formation rate was 100% in the cell sheet implantation group, while it was 67% in the cell suspension injection group. The formed tumor masses were highly consistent in both growth rate and tumor size. Massive bone destruction and soft tissue mass formation were observed from the micro CT analysis, suggesting the presence of osteosarcoma. The histopathological analysis demonstrated that the orthotropic osteosarcoma model mimicked the tumor bone growth, bone destruction, and the lung metastasis. These findings imply that such a cell sheet technology could be an appropriate approach to rapidly establish a sustainable orthotropic osteosarcoma model for tumor research and reduce mice consumption.

LncRNA SNHG16 promotes epithelial-mesenchymal transition by upregulating ITGA6 through miR-488 inhibition in osteosarcoma.

BACKGROUND: Osteosarcoma is a primary cause of cancer-associated death in children and adolescents worldwide. Long non-coding RNAs SNHG16 (lncRNA SNHG16) and integrin subunit-a 6 (ITGA6) are recently reported to be involved in the tumorigenesis of osteosarcoma by multiple mechanisms. However, the correlation between SNHG16 and ITGA6 in osteosarcoma remains undetermined. METHODS: Expression of miR-488, SNHG16 and ITGA6, as well as epithelial-mesenchymal transition (EMT) associated markers in osteosarcoma tissues and cell lines were examined by qRT-PCR or Western blotting. Effects of miR-488, SNHG16 and ITGA6 on cell migration, invasion were evaluated by wound-healing assay and transwell assay. Bioinformatics analysis and dual-luciferase reported assays were applied to assess the interaction among miR-488, SNHG16 and ITGA6. RNA immunoprecipitation (RIP) was also used to verify SNHG16 and miR-488 interaction. Finally, animal study was used to detect the effect of SNHG16 on osteosarcoma in vivo. RESULTS: SNHG16 and ITGA6 were significantly increased while miR-488 was decreased in osteosarcoma. ITGA6 was screened as a target gene of miR-488, and SNHG16 was sponged by miR-488 in osteosarcoma cells. MiR-488 overexpression and SNHG16 knockdown suppressed migration, invasion and EMT of osteosarcoma cells. Moreover, rescue assays proved that the influences of SNHG16 on osteosarcoma cells migration, invasion and EMT were dependent on miR-488 and ITGA6. In addition, the promotive effects of SNHG16 on osteosarcoma tumor growth and metastasis were further supported by xenograft tumor growth assay. CONCLUSION: SNHG16 promoted migration, invasion and EMT of osteosarcoma by sponging miR-488 to release ITGA6.

LncRNA HOTTIP facilitates cell proliferation, invasion, and migration in osteosarcoma by interaction with PTBP1 to promote KHSRP level.

This study designs to investigate the role and potential mechanism of lncNRA HOTTIP in OS progression in vitro and in vivo. HOTTIP, PTBP1, and KHSRP expression levels were tested through qRT-PCR and western blot in OS tissues or cell lines. Cell proliferation was examined via CCK-8 and colony formation. Cell cycle and apoptosis were analyzed via flow cytometry analysis. The invasive and migratory abilities of OS cells were evaluated by transwell and wound-healing assays. The localization of HOTTIP in OS cells was determined by subcellular fractionation assay. RNA pull down and RNA immunoprecipitation were allowed to assess the interaction between HOTTIP and PTBP1. Xenograft tumor growth assay was employed to test the role of HOTTIP and KHSRP in OS progression. Our data demonstrated HOTTIP was upregulated in OS tissues. HOTTIP knockdown resulted in a suppression of OS cell proliferation, invasion and migration, as well as a promotion of OS cell apoptosis, while HOTTIP overexpression exhibited opposite effects. In mechanism, PTBP1 and KHSRP highly expressed in OS and HOTTIP was identified to interact with PTBP1 to promote KHSRP expression. Meanwhile, we found that overexpression of KHSRP or PTBP1, individually, can partially remove the repression of HOTTIP suppression for OS cell progression. Moreover, xenograft tumor growth assay revealed that HOTTIP knockdown significantly inhibited tumor growth, and this inhibitory effect was abolished by KHSRP overexpression. Collectively, these findings confirmed that HOTTIP facilitates OS cell proliferation, invasion and migration by binding to PTBP1 to promote KHSRP level. Abbreviation: LncRNA: long noncoding RNA; HOTTIP: HOXA distal transcript antisense RNA; KHSRP: KH-Type Splicing Regulatory Protein; qRT-PCR: quantitative real-time PCR; OS: osteosarcoma; OST: osteosarcoma tissues; ANT: adjacent normal tissue.

LncRNA GAS5 Regulates Osteosarcoma Cell Proliferation, Migration, and Invasion by Regulating RHOB via Sponging miR-663a.

INTRODUCTION: Emerging evidence has revealed the importance of long non-coding RNAs (lncRNAs) in carcinogenesis. The aim of this work was to investigate the roles of lncRNA growth arrest specific 5 (GAS5) in osteosarcoma (OS) progression. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to explore GAS5, microRNA-663a (miR-663a), and ras homolog family member B (RHOB) expression levels in OS tissues and cells. Moreover, cell counting kit-8 assay, wound-healing assay, and transwell invasion assay were conducted to investigate biological roles of GAS5 in OS progression. In addition, mechanisms underlying the functions of GAS5 in OS were investigated by bioinformatic analysis, luciferase activity reporter assay, and rescue experiments. RESULTS: The GAS5 expression level was significantly decreased in OS tissues and cells compared with normal tissues and cells, and could negatively regulate miR-663a expression. Moreover, we found RHOB expression can be negatively regulated by miR-663a. Overexpression of GAS5 and RHOB suppresses, while overexpression of miR-663a stimulates, OS cell proliferation, migration, and invasion in vitro. In summary, we revealed lncRNA GAS5 was a downregulated lncRNA in OS and impaired OS malignant behaviors. In addition, this regulation relied on miR-663a and its target gene, RHOB. DISCUSSION: To sum up, we showed lncRNA GAS5 regulates OS progression via regulating the miR-663a/RHOB axis.

Characterizations of Gene Alterations in Melanoma Patients from Chinese Population.

Melanoma is a human skin malignant tumor with high invasion and poor prognosis. The limited understanding of genomic alterations in melanomas in China impedes the diagnosis and therapeutic strategy selection. We conducted comprehensive genomic profiling of melanomas from 39 primary and metastatic formalin-fixed paraffin-embedded (FFPE) samples from 27 patients in China based on an NGS panel of 223 genes. No significant difference in gene alterations was found between primary and metastasis melanomas. The status of germline mutation, CNV, and somatic mutation in our cohort was quite different from that reported in Western populations. We further delineated the mutation patterns of 4 molecular subgroups (BRAF, RAS, NF1, and Triple-WT) of melanoma in our cohort. BRAF mutations were more frequently identified in melanomas without chromic sun-induced damage (non-CSD), while RAS mutations were more likely observed in acral melanomas. NF1 and Triple-WT subgroups were unbiased between melanomas arising in non-CSD and acral skin. BRAF, RAS, and NF1 mutations were significantly associated with lymph node metastasis or presence of ulceration, implying that these cancer driver genes were independent prognostic factors. In summary, our results suggest that mutational profiles of malignant melanomas in China are significantly different from Western countries, and both gene mutation and amplification play an important role in the development and progression of melanomas.

MiR-9 promotes synovial sarcoma cell migration and invasion by directly targeting CDH1.

BACKGROUND: Invasion and metastasis of synovial sarcoma is the leading cause of death in patients. Epithelial mesenchymal transition (EMT) accelerates tumor cell invasion and metastasis. MiR-9 promotes tumor metastasis by inducing EMT. However, the role of miR-9 in synovial sarcoma is still not clear. METHODS: Overexpression or knockdown of miR-9 in human synovial sarcoma (HSS) cell lines was carried out by miR-9 mimics or miR-9 inhibitors transfection. Cell proliferation, apoptosis, migration and invasion were detected using MTS and colony formation assays, flow cytometry, wound healing and transwell assays, respectively. Luciferase reporter assay was applied to study the interaction between miR-9 and CDH1. Nude mice xenograft model was established, and immunohistochemistry staining assessed Ki-67 level. The related mRNA and protein expression levels were evaluated by qRT-PCR and Western blotting. RESULTS: The bioinformatics analyses and luciferase reporter assay showed that miR-9 can target CDH1 3'-UTR. Moreover, miR-9 could induce EMT of HSS cells via targeting CDH1. The negative regulation of miR-9 on CDH1 expression was also confirmed in a mouse xenograft model of synovial sarcoma. Furthermore, miR-9 was observed to induce HSS cell proliferation, migration and invasion and inhibit apoptosis. MAPK/ERK and Wnt/ß-catenin signal pathways were activated by the miR-9 overexpression in HSS cells, and then further enhancing tumorigenesis of HSS, which was further confirmed in the mouse model. CONCLUSION: MiR-9 induces EMT by targeting CDH1, and activates MAPK/ERK and Wnt/ß-catenin signal pathways, thus promoting HSS tumorigenesis.

Propranolol induced G0/G1/S phase arrest and apoptosis in melanoma cells via AKT/MAPK pathway.

Both preclinical and epidemiology studies associate ß-adrenoceptors-blockers (ß-blockers) with activity against melanoma. However, the underlying mechanism is still unclear, especially in acral melanoma. In this study, we explored the effect of propranolol, a non-selective ß-blocker, on the A375 melanoma cell line, two primary acral melanoma cell lines (P-3, P-6) and mice xenografts. Cell viability assay demonstrated that 50µM-400µM of propranolol inhibited viability in a concentration and time dependent manner with an IC50 ranging from 65.33µM to 148.60µM for 24h -72h treatment, but propranolol (less than 200µM) had no effect on HaCaT cell line. Western blots showed 100µM propranolol significantly reduced the expression of Bcl-2 while increasing the expressions of Bax, cytochrome c, cleaved capase-9 and cleaved caspase-3, and down-regulated the levels of p-AKT, p-BRAF, p-MEK1/2 and p-ERK1/2 in melanoma cells, after a 24h incubation. The in vivo data confirmed the isolation results. Mice received daily ip. administration of propranolol at the dose of 2 mg/kg for 3 weeks and the control group was treated with the same volume of saline. The mean tumor volume at day 21 in A375 xenografts was 82.33 ± 3.75mm3vs. 2044.67 ± 54.57mm3 for the propranolol-treated mice and the control group, respectively, and 31.66 ± 4.67 mm3vs. 1074.67 ± 32.17 mm3 for the P-3 xenografts. Propranolol also reduced Ki67, inhibited phosphorylation of AKT, BRAF, MEK1/2 and ERK1/2 in xenografts. These are the first data to demonstrate that propranolol might inhibit melanoma by activating the intrinsic apoptosis pathway and inactivating the MAPK and AKT pathways.

Distraction osteogenesis and arthrodesis as a new surgical option for chondrosarcoma in the distal tibia.

Recent advances in the management of bone tumors have led to a significant increase in the survival rates of patients with malignant bone tumors. Thus, limb salvage surgery has gained importance for preserving limb function in the management of bone tumors. However, surgery presents unique difficulties in terms of the biomechanics and obtaining a soft-tissue cover, such as when the ankle is involved in the primary malignant bone tumor. We report a case of chondrosarcoma of the distal tibia treated with wide en bloc resection arthrodesis and reconstruction of the defect using distraction osteogenesis, which offers an effective alternative protocol for limb salvage. The patient has remained disease free for 3 years since the initial surgery and can maintain normal limb athletic function.