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Xian Chang,1,* Yang-Fan Lv,2,* Jing He,3,* Ya Cao,2 Chang-Qing Li,1 Qiao-Nan Guo2 1Department of Orthopedics, Xinqiao Hospital, Third Military Medical University (Army Medical University), Chongqing, 400037, People’s Republic of China; 2Department of Pathology, Xinqiao Hospital, Third Military Medical University (Army Medical University), Chongqing, 400037, People’s Republic of China; 3Department of Pediatric Surgery, Guangzhou Institute of Pediatrics, Guangdong Provincial Key Laboratory of Research in Structural Birth Defect Disease, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, 510623, Guangdong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qiao-Nan Guo; Chang-Qing Li Email qiaonan85@263.net; changqli1970@126.comBackground: N6-methyladenosine (m6A) RNA methylation is the most prevalent modification of mammalian RNA, and it is associated with tumorigenesis and cancer progression. Its regulation is mediated via m6A-related regulators, including “erasers,” “readers,” and “writers”. The present study evaluated the expression profile, risk signature and prognostic value of 13 m6A regulators in hepatocellular carcinoma (HCC) using different datasets, including The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and clinical samples.Methods: We used 374 HCC samples derived from the TCGA database, 569 HCC samples from 2 GEO datasets, and clinical tumour and nontumour tissues derived from 60 patients with HCC who underwent surgery in Xinqiao Hospital Chongqing to assess the gene expression profiles and prognostic values of m6A-related regulators in HCC.Results: Eight of 13 core m6A-related regulators were overexpressed in all databases, including TCGA, GSE, clinical tumour and nontumour tissues of HCC. Two clusters (Cluster 1 and Cluster 2) were identified via consensus clustering. Cluster 2 was associated with poorer prognosis, higher tumour grade, higher AFP levels, and worse outcome compared to Cluster 1, which indicates that these m6A-related regulators are highly correlated with HCC malignancy. We performed survival analyses using the Log rank tests and a Cox regression model. Gene enrichment analysis was used to detect the related KEGG and GO pathways. We derived a prognostic risk signature using five selected m6A-related regulators.Conclusion: Our work suggested that m6A-related regulators might be key participants in the tumour progression of HCC and potential biomarkers with prognostic value.Keywords: RNA modification, N6-methyladenosine, m6A, hepatocellular carcinoma, prognosis, RNA methylation regulators

Dioscin has been used as a treatment for Hashimoto’s thyroiditis (HT) in China. However, the molecular mechanisms governing the modes of action of dioscin have not been elucidated. In this study, f...

Ying Zeng,1,2 Xiangfeng Zhu,2 Yali Wang,1 Bo Liu,1 Xin Yang,2 Qiushi Wang,2 Juan Du,2 Yu Ma,2 Li Lin,2 Ping Fu,2 Hualiang Xiao,2 Qiao-Nan Guo1 1Department of Pathology, Second Affiliated Hospital, Amy Medical University (Third Military Medical University), Chongqing 400037, People’s Republic of China; 2Department of Pathology, Daping Hospital, Amy Medical University (Third Military Medical University), Chongqing 400042, People’s Republic of ChinaCorrespondence: Qiao-Nan Guo; Hualiang Xiao Email qiaonan85@263.net; dpbl_xhl@126.comPurpose: To observe the clinicopathological, immunohistochemical, and molecular genetic features of epithelioid glioblastoma (E-GBM), and identify tumor-associated prognostic factors.Patients and Methods: The clinical and radiological data of fifteen cases of E-GBM were collected, and their pathological, immunohistochemical, and molecular features were examined. A 1p/19q analysis via FISH, MGMT promoter methylation by MS-PCR, and IDH1 and BRAF V600E mutation analysis by HRM-PCR were performed. The level of EZH2 expression was valuated by immunohistochemistry in 15 E-GBM cases, and the prognostic factors were analyzed in E-GBM patients. Fifteen non-E-GBM cases were used as a control.Results: The fifteen cases of E-GBM included twelve males and three females, with fourteen cases supratentorially located. Headache was the main symptom. Microscopy revealed that the tumors were composed of epithelioid cells and some rhabdoid cells. The epithelioid and rhabdoid cells displayed focal discohesion, scant intervening neuropil, a distinct cell membrane, eosinophilic cytoplasm, and a laterally positioned nucleus. Most tumors showed high mitosis, zonal necrosis, and microvascular hyperplasia. Immunohistochemical findings included epithelioid cells positive for GFAP, vimentin, nestin, S-100, and INI-1. The molecular findings included no deletions of 1p/19q, EGFR amplifications, or IDH1 mutations in any case, a methylated MGMT promoter in 46.7% (7/15) cases, and a BRAFV600E mutation in 46.7% (7/15) cases. EZH2 overexpression occurred in 60.0% (9/15) of E-GBM cases. E-GBM patients with OS (≤ 12 months) exhibited extensive necrosis (6/6), EZH2 overexpression (6/6), MGMT promoter unmethylation (5/6), BRAFV600E mutation (3/6), and treatment (surgery4/6). E-GBM patients with OS (> 12 months) exhibited focal or limited necrosis, low or negative EZH2 expression, MGMT promoter methylation (2/3), BRAFV600E mutation (3/3), and treatment (surgery+radiotherapy/chemo-radiotherapy, 2/3).Conclusion: E-GBM was a rare variant of glioblastoma, with histological epithelioid features and poor prognosis. Extensive necrosis, MGMT promoter unmethylation, EZH2 overexpression, and lack of adjuvant chemo-radiotherapy may indicate a poor prognosis.Keywords: central nervous system tumor, epithelioid glioblastoma, immunohistochemistry, clinicopathological features, molecular genetics, differential diagnosis

Xue-Feng Tang,1 Ya Cao,1 Dong-Bin Peng,1 Guo-Sheng Zhao,2 Ying Zeng,1,3 Zi-Ran Gao,1 Yang-Fan Lv,1 Qiao-Nan Guo1 1Department of Pathology, Xinqiao Hospital, Army Medical University, Chongqing 400037, China; 2Department of Orthopedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; 3Department of Pathology, Daping Hospital, Army Medical University, Chongqing 400037, China Background: Notch signaling abnormalities are associated with the development of various tumors, including hematopoietic and epithelium-derived tumors. However, the role of Notch signaling in tumors originating from mesenchymal cells is unclear. The effect of Notch3 expression on the prognosis of osteosarcoma and its role and mechanism in osteosarcoma cells have never been reported. Materials and methods: In this study, we performed a clinicopathological analysis of 70 cases of osteosarcoma, with primary focus on survival. Osteosarcoma cell lines MTH and U2OS were used. After knockdown of Notch3 by lentiviral transfection and siRNA, the cell cycle, cell viability, and wound healing capacity were assessed. Subsequently, the Transwell assay was performed, and the expression levels of hairy and enhancer of split-1 (Hes1) and matrix metalloproteinase 7 (MMP7) were detected by RT-PCR and Western blot assay. The expression of MMP7 was also detected after knockdown of Hes1. Animal experiments were performed by injecting the cell lines MTH of Notch3 knockdown into mice tail veins and comparing the development of lung metastasis with the control group. Results: Comparison of survival curves showed that Notch3 expression significantly impacts patient survival. Additionally, multivariate analysis revealed that Notch3 is an independent prognostic factor for osteosarcoma. In in vivo experiments, osteosarcoma-associated pulmonary metastasis in nude mice was reduced after Notch3 silencing. The expression of downstream effector molecule, Hes1, and that of the invasion and metastasis-associated proteolytic enzyme, MMP7, were reduced, and MMP7 was further decreased by Hes1 knockdown in in vitro experiments. Conclusion: Notch3 is a prognostic factor for osteosarcoma and might regulate its invasion and metastasis through the downstream target gene Hes1 and effector MMP7. Keywords: osteosarcoma, prognosis, metastasis, Notch3, Hes1, MMP7

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY‐box 30 (Sox30) is an age‐related and essential regulator of meiosis in the postnatal testis. Sox30‐null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ‐cell differentiation and irregular Leydig cell proliferation. In aged Sox30‐null mice, the observed testicular impairments were more severe. Furthermore, the germ‐cell arrest occurred at the stage of meiotic zygotene spermatocytes, which is strongly associated with critical regulators of meiosis (such as Cyp26b1, Stra8 and Rec8) and sex differentiation (such as Rspo1, Foxl2, Sox9, Wnt4 and Ctnnb1). Mechanistically, Sox30 can activate Stra8 and Rec8, and inhibit Cyp26b1 and Ctnnb1 by direct binding to their promoters. A different Sox30 domain required for regulating the activity of these gene promoters, providing a “fail‐safe” mechanism for Sox30 to facilitate germ‐cell differentiation. Indeed, retinoic acid levels were reduced owing to increased degradation following the elevation of Cyp26b1 in Sox30‐null testes. Re‐expression of Sox30 in Sox30‐null mice successfully restored germ‐cell meiosis, differentiation and Leydig cell proliferation. Moreover, the restoration of actual fertility appeared to improve over time. Consistently, Rec8 and Stra8 were reactivated, and Cyp26b1 and Ctnnb1 were reinhibited in the restored testes. In summary, Sox30 is necessary, sufficient and age‐associated for germ‐cell meiosis and differentiation in testes by direct regulating critical regulators. This study advances our understanding of the regulation of germ‐cell meiosis and differentiation in the postnatal testis., We reported that Sox30‐null mice exhibit uniquely impaired testis with irregular Leydig cell proliferation and abnormal germ‐cell arrest at zygotene, and the observed testicular impairments were more severe as mice aged. Sox30 controls male germ‐cell meiosis by a “fail‐safe” regulation of retinoic acid signalling, and re‐expression of Sox30 in Sox30‐null mice can successfully restore germ‐cell meiosis and differentiation. Moreover, the restoration of actual fertility appeared to improve over time.

Background Over the last two or three decades, the pace of development of treatments for osteosarcoma tends has been slow. Novel effective therapies for osteosarcoma are still lacking. Previously, we reported that tumor-suppressing STF cDNA 3 (TSSC3) functions as an imprinted tumor suppressor gene in osteosarcoma; however, the underlying mechanism by which TSSC3 suppresses the tumorigenesis and metastasis remain unclear. Methods We investigated the dynamic expression patterns of TSSC3 and autophagy-related proteins (autophagy related 5 (ATG5) and P62) in 33 human benign bone tumors and 58 osteosarcoma tissues using immunohistochemistry. We further investigated the correlations between TSSC3 and autophagy in osteosarcoma using western blotting and transmission electronic microscopy. CCK-8, Edu, and clone formation assays; wound healing and Transwell assays; PCR; immunohistochemistry; immunofluorescence; and western blotting were used to investigated the responses in TSSC3-overexpressing osteosarcoma cell lines, and in xenografts and metastasis in vivo models, with or without autophagy deficiency caused by chloroquine or ATG5 silencing. Results We found that ATG5 expression correlated positively with TSSC3 expression in human osteosarcoma tissues. We demonstrated that TSSC3 was an independent prognostic marker for overall survival in osteosarcoma, and positive ATG5 expression associated with positive TSSC3 expression suggested a favorable prognosis for patients. Then, we showed that TSSC3 overexpression enhanced autophagy via inactivating the Src-mediated PI3K/Akt/mTOR pathway in osteosarcoma. Further results suggested autophagy contributed to TSSC3-induced suppression of tumorigenesis and metastasis in osteosarcoma in vitro and in vivo models. Conclusions Our findings highlighted, for the first time, the importance of autophagy as an underlying mechanism in TSSC3-induced antitumor effects in osteosarcoma. We also revealed that TSSC3-associated positive ATG5 expression might be a potential predictor of favorable prognosis in patients with osteosarcoma. Electronic supplementary material The online version of this article (10.1186/s13046-018-0856-6) contains supplementary material, which is available to authorized users.

Microenvironmental regulation of cancer stem cells (CSCs) strongly influences the onset and spread of cancer. The way in which glioma cells interact with their microenvironment and acquire the phenotypes of CSCs remains elusive. We investigated how communication between vascular endothelial cells and glioma cells promoted the properties of glioma stem cells (GSCs). We observed that CD133+ GSCs were located closely to Shh+ endothelial cells in specimens of human glioblastoma multiforme (GBM). In both in vitro and in vivo studies, we found that endothelial cells promoted the appearance of CSC-like glioma cells, as demonstrated by increases in tumourigenicity and expression of stemness genes such as Sox2, Olig2, Bmi1 and CD133 in glioma cells that were co-cultured with endothelial cells. Knockdown of Smo in glioma cells led to a significant reduction of their CSC-like phenotype formation in vitro and in vivo. Endothelial cells with Shh knockdown failed to promote Hedgehog (HH) pathway activation and CSC-like phenotype formation in co-cultured glioma cells. By examination of glioma tissue specimens from 65 patients, we found that the survival of glioma patients was closely correlated with the expression of both Shh by endothelial cells and Gli1 by perivascular glioma cells. Taken together, our study demonstrates that endothelial cells in the tumour microenvironment provide Shh to activate the HH signalling pathway in glioma cells, thereby promoting GSC properties and glioma propagation. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Promoter hypermethylation can lead to a loss of genetic imprinting in carcinogenesis. The mechanism for the loss of expression of the imprinted gene TSSC3 has not been investigated in cases of osteosarcoma. In this study, we treated osteosarcoma cell lines with 5-Aza-CdR, which is a widely-used DNA methyltransferase inhibitor, and found dose-dependent reduction in cell growth, conversion of cell morphology to a non-motile phenotype, and obvious increase in apoptosis. In addition, we also found that 5-Aza-CdR reactivated TSSC3 expression through demethylation of the promoter regions. These findings indicate that the TSSC3 gene is silenced through hypermethylation of the promoter regions, a mechanism commonly associated with gene silencing in cancer. Finally, we examined the role of TSSC3 in human osteosarcoma SaOS2 cells. We showed that TSSC3 overexpression suppressed SaOS2 cell growth and increased apoptosis through caspase-3 upregulation, thereby, suggesting that TSSC3 may play a pro-apoptosis role to maintain the normal balance of growth. Taken together, these observations suggest that the epigenetic regulation of TSSC3, a pro-apoptosis gene, provides valuable insights into possible osteosarcoma therapies.

To explore the polymorphisms and mutations of mitochondrial ATPase6 gene in Chinese patients with osteosarcoma and their possible association with carcinogenesis, direct DNA sequencing method was used to detect the variants of the mitochondrial ATPase6 gene in 39 patients with osteosarcoma. We found mutations of the mitochondrial ATPase6 gene in 24/39 (61.5%) of the tested osteosarcoma samples, and identified 27 variant sites in ATPase6 coding regions. We did not detect any new polymorphisms in osteosarcoma, nor was there any association between variants and the three histopathological subtypes. These data demonstrated that mtDNA mutations within the ATPase6 gene are a frequent event in Chinese patients with osteosarcoma.

Objective: Immune cells are key mediators of secondary damage following spinal cord injury (SCI), and dendritic cell (DC)-based vaccines have received considerable interest for treatment of SCI. We previously showed that vaccination with DCs pulsed with homogenate proteins of the spinal cord (hpDCs) promotes functional recovery from SCI in mice. However, the underlying molecular mechanisms remain unclear. Here, changes of neurotrophins, cytokines and T cells at the site of SCI in mice after vaccination with hpDCs were investigated and correlated with recovery from SCI. Methods: hpDCs, DCs (control) or PBS (control) were injected intraperitoneally into injured mouse spinal cords. Functional recovery of the spinal cord was measured weekly using the Basso Mouse Scale (BMS) and confirmed by histological and immunohistochemical analysis. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), interleukin-4 (IL-4) and interferon-γ (IFN-γ) levels in T cell culture supernatants and spinal cord tissues were determined by ELISA. Results: Eighty-four days after immunization, the BMS score of the hpDCs group (6.92 ± 0.20) was significantly higher than those of the DCs and PBS groups (p < 0.01). Meanwhile, the injury area and number of cysts in the hpDCs group decreased significantly compared with control groups. BDNF, NT-3, IL-4 and IFN-γ levels at the injured site as well as BDNF and NT-3 levels in the supernatant of cultured T cells from the hpDCs group were significantly higher than in control groups (p < 0.05). Conclusion: These results reveal that vaccination with hpDCs can promote SCI repair potentially by upregulating BDNF, NT-3, IL-4 and IFN-γ at the injury site.

This study aimed to determine whether formyl peptide receptors (FPRs) regulated the differentiation of neural stem cells (NSCs). FPRs promote the migration of NSCs both in vitro and in vivo. However, the role of FPRs during differentiation of NSCs is unknown. Analysis by Western blot showed significantly increased expression of FPR1 and FPR2 during differentiation of NSCs. The activation of FPRs promotes NSCs to differentiate into neurons with more primary neurites and branch points and longer neurites per cell. Meanwhile, this activation also inhibits the differentiation of NSC into astrocytes. This bidirectional effect can be inhibited by the FPRs-specific inhibitor. Moreover, it was found that the activation of FPRs increased the generation of reactive oxygen species (ROS) and phosphorylation of AKT in the NSCs, while N-acetylcysteine and LY294002 inhibited the FPRs-stimulated increase in ROS generation and AKT phosphorylation, and blocked the FPRs-stimulated neural differentiation into neurons. Therefore, FPRs-stimulated neural differentiation was mediated via ROS and PI3K-AKT signaling pathways. Collectively, the present findings provided a novel insight into the functional role of FPRs in neurogenesis, with important implications for its potential use as a candidate for treating brain or spinal cord injury.

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents, typically presenting with poor prognosis. Recent studies suggested that tumor initiating cells (T-ICs) drive tumor formation and relapse or metastasis and are relatively resistant to cell death induced by conventional chemo- and radiotherapies. Therefore, the poor prognosis of OS appears to be associated with T-ICs. Here, we enriched T-ICs in OS cell lines and evaluated whether the imprinted gene TSSC3 (tumor-suppressing STF cDNA 3) associated with apoptosis could affect T-ICs in OS. Sarcosphere selection and serial clone-forming unit assays were successfully used to enrich T-ICs from OS cell lines. Enrichment of T-ICs from a malignantly transformed hFOB1.19 osteoblast cell line (MThFOB1.19) indicated that OS T-ICs could originate from differentiated cells, and most of these MThFOB1.19 cells showed stem-like features. TSSC3 was expressed at a low level in T-ICs, while overexpression of TSSC3 could efficiently downregulate the expression of stem cell markers Nanog, Oct4 and Sox2 in T-ICs and decrease the clone formation rate, as well as downregulate tumorigenesis in MThFOB1.19 cells, supporting a suppressive role for TSSC3 in OS T-ICs. Furthermore, overexpression of TSSC3 was found to induce apoptosis of OS T-ICs through increasing cleaved caspase-3 (active form), increasing the release of Cyt c and decreasing pro-caspase-9 (pro-enzyme form), as well as disruption of the mitochondrial membrane potential (ΔΨ). Taken together, our findings provide preliminary evidence that TSSC3 inhibits OS tumorigenicity through reducing stemness and promoting apoptosis of T-ICs. Thus, targeting TSSC3 may be a promising approach to suppressing tumorigenicity in OS.

Loss of expression of TSSC3, an apoptosis-related imprinted gene, has been reported in several cases of malignant tumors. However, the roles and mechanisms of TSSC3 in human osteosarcoma remain to be defined. In this study, we found TSSC3 to be downregulated during osteosarcoma transformation and progression in osteosarcoma cell lines and tissues. The SaOS2 cell line was used to further evaluate the precise role of TSSC3 in osteosarcoma development. Overexpression of TSSC3 markedly reduced cell vitality and growth, colony formation, Ki67 expression as well as cell cycle arrest in the G(0)/G(1) phase. Consistently, TSSC3 overexpression was associated with increased apoptosis assayed by annexin V/propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. Subcutaneous injection of TSSC3 overexpressing SaOS2 cells into athymic nude mice showed that TSSC3 also inhibited tumorigenesis through growth inhibition and apoptosis induction in vivo. Further mechanistic studies revealed that the mitochondrial apoptosis pathway was required for TSSC3-mediated cell apoptosis. These findings support a suppressor role for TSSC3 in osteosarcoma development by regulating apoptosis. In addition, constitutive TSSC3 expression greatly enhanced the sensitivity of human osteosarcoma cells to the chemotherapeutic drugs cisplatin and epirubicin. Conversely, TSSC3 knockdown increased SaOS2 cell growth and decreased apoptosis in vitro and in vivo and reduced sensitivity of the cells to chemotherapy. This is the first study to demonstrate that TSSC3 has a potent tumor suppressor role in osteosarcoma, probably by inhibition of growth and induction of apoptosis via the mitochondrial apoptosis pathway.

Genomic imprinting, a heritable form of epigenetic information, is thought to play an important role in tumor progression. DNA methylation is a common mechanism of genomic imprinting. To evaluate the genome-wide effects of malignant transformation on osteosarcoma progression, we examined multiple biological properties, including DNA methylation, in human osteoblast hFOB1.19 cells (ATCC Catalog No. CRL-11372) transformed by treatment with carcinogenic agent N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.0 microg/ml) and carcinogenic promoting agent 12-O-tetradecanoyl phorbol-13-acetate (TPA, 200 ng/ml). We also examined global changes in expression of imprinted genes during transformation using microarray analysis. Ten imprinted genes, including H19, MKRN3, NDN, CDKN1C, PHLDA2, MEST, CD81, GRB10, SLC22A18, and SLC22A3 were aberrantly regulated in transformed cells, suggesting roles in tumorigenesis. Moreover, we analyzed the methylation state of the promoter regions of H19, PHLDA2, and SLC22A18 genes by bisulfite sequencing array and observed a correlation between upregulated expression of H19 and PHLDA2 genes and hypomethylation of their promoter regions, although this was not observed for SLC22A18. Our results suggest that changes in expression of imprinted genes caused by changes in methylation are involved, and are among the earliest events, in neoplastic progression.

Epithelial to mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining the invasive and metastatic behavior of cells during cancer progression. Our previous study showed that loss of expression of TSSC3 is positively associated with osteosarcoma malignancy and progression. However, whether TSSC3 mediates EMT in osteosarcoma is poorly understood. In the present study, we determined that TSSC3 downregulation induced cell migration and invasion ability and promoted mesenchymal transition of osteosarcoma cells by upregulating mesenchymal markers and inhibiting the epithelial markers. Furthermore, TSSC3 downregulation elicited a signaling cascade that included increased levels of Wnt3a and LRP5, inactivation of GSK-3β, accumulation of nuclear β-catenin and Snail, the augmented binding of β-catenin to TCF-4, and accordingly increased the expression of Wnt target genes (CD44, MMP7). The gene knockdown of these signaling proteins could inhibit TSSC3 downregulation-promoted EMT, migration, and invasion in osteosarcoma. Finally, TSSC3 overexpression obviously inhibited cell migration, invasion, and repressed mesenchymal phenotypes, reducing lung metastasis through GSK-3β activation. Collectively, TSSC3 downregulation promotes the EMT of osteosarcoma cells by regulating EMT markers via a signal transduction pathway that involves Snail, Wnt-β-catenin/TCF, and GSK-3β.

The enhancer of zeste homolog 2 (EZH2) methyltransferase is the catalytic subunit of polycomb repressive complex 2 (PRC2), which acts as a transcription repressor via the trimethylation of lysine 27 of histone 3 (H3K27me3). EZH2 has been recognised as an oncogene in several types of tumors; however, its role in osteosarcoma has not been fully elucidated. Herein, we show that EZH2 silencing inhibits tumor growth and lung metastasis in osteosarcoma by facilitating re-expression of the imprinting gene tumor-suppressing STF cDNA 3 (TSSC3). Our previous study showed that TSSC3 acts as a tumor suppressor in osteosarcoma. In this study, we found that EZH2 was abnormally elevated in osteosarcoma and its overexpression was associated with poor prognosis in osteosarcoma. Silencing of EZH2 resulted in tumor growth inhibition, apoptosis and chemosensitivity enhancement. Moreover, suppression of EZH2 markedly inhibited tumor growth and lung metastasis in vivo. Furthermore, EZH2 knockdown facilitated the re-expression of TSSC3 by reducing H3K27me3 in the promoter region. Cotransfection with siEZH2 and siTSSC3 could partially reverse the ability of siEZH2 alone. We have demonstrated that EZH2 plays a crucial role in tumor growth and distant metastasis in osteosarcoma; its oncogenic role is related to its regulation of the expression of TSSC3.

Methyl-CpG-binding protein 2 (MeCP2) is a DNA methylation-related gene of the methyl-CpG-binding protein family. Here, we investigated the epigenetic function of the MeCP2 in SaOS2 and U2OS cell lines, and explored the antitumor effects of the gene silencing for osteosarcoma. In this study, chromatin immunoprecipitation assay was used to detect MeCP2 binding activity with TSSC3 gene. RT-PCR and western blot assay were used to analyze the MeCP2 expression in osteosarcoma cell lines after transfection with LV-MECP2-RNAi. Transwell invasion and migration assays were used to detect the cell invasion and migration. The cell apoptosis was examined by using the flow cytometry assay. The tumor size was also assessed to determine the therapeutic effects of gene silencing. The results indicated that MeCP2 indicated the highest combining power with TSSC3 gene. LV-MECP2-RNAi could decrease MeCP2 level in tumor cells compared with the untreated cells (P

Suppression of anoikis is a prerequisite for tumor cell metastasis, which is correlated with chemoresistance and poor prognosis. We characterized a novel interaction between RanBP9 SPRY domain and TSSC3 PH domain by which RanBP9/TSSC3 complex exerts transcription and post-translation regulation in osteosarcoma. RanBP9/TSSC3 complex was inversely correlated with a highly anoikis-resistant phenotype in osteosarcoma cells and metastasis in human osteosarcoma. RanBP9 cooperated with TSSC3 to inhibit anchorage-independent growth and to promote anoikis in vitro and suppress lung metastasis in vivo. Moreover, RanBP9 SPRY domain was required for RanBP9/TSSC3 complex-mediated anoikis resistance. Mechanistically, RanBP9 formed a ternary complex with TSSC3 and Src to scaffold this interaction, which suppressed both Src and Src-dependent Akt pathway activations and facilitated mitochondrial-associated anoikis. Collectively, the newly identified RanBP9/TSSC3 complex cooperatively suppress metastasis via downregulation of Src-dependent Akt pathway to expedite mitochondrial-associated anoikis. This study provides a biological basis for exploring the therapeutic significance of dual targeting of RanBP9 and TSSC3 in osteosarcoma.

Gene hypomethylation and hypermethylation can lead to a loss of genetic imprinting in malignancies. The mechanism responsible for overexpression of the imprinted insulin-like growth factor II (IGF2) gene has not been investigated in osteosarcoma. In this study, the expression levels, imprinting status, and the extent of cytosine methylation of the IGF2 gene was evaluated in 20 of 24 cases of osteosarcoma using immunohistochemistry, reverse transcriptase polymerase chain reaction, restriction fragment length polymorphism, and bisulfite genomic sequencing. Promoter use analysis indicated that P3- and P4-derived messenger RNAs were more highly expressed than P1 transcripts in the osteosarcoma samples. Loss of imprinting of IGF2 was observed in 3 of 20 of the osteosarcoma samples, but this was not associated with IGF2-specific transcripts. Methylation analysis revealed that the methylation patterns of the differentially methylated region of IGF2 were not uniform, regardless of IGF2-P3 expression. However, the average degree of methylation of the 14 CpG sites in the IGF2-P3 promoter was significantly lower in osteosarcoma samples with P3 transcripts than in osteosarcoma samples without P3 expression (P < .05). This observation was also observed in nontumor samples. These data suggest that hypomethylation of the IGF2-P3 promoter correlates with expression of P3 transcripts in osteosarcoma. Furthermore, elevated IGF2-P3 expression in osteosarcoma tissues is due to P3 promoter hypomethylation, which may represent an early event in progression of osteosarcoma.

Calcineurin (CN) is a heterodimer of a catalytic subunit, calcineurin A (CNA), and a regulatory subunit, calcineurin B (CNB), and is unique in being regulated by Ca2+, together with calmodulin (CaM). Previously, RII peptide undp- nitrophenyl phosphate were often applied as substrates to explore how CNA activity was regulated by CNB and CaM. Here, by using tau, a physiological substrate of CN, and its truncation mutant tau50 as substrates, the specific effects of CNB and CaM were compared. Activity assays and kinetic analyses revealed that alone CNB failed to regulate CNA and its truncation mutant CNAabc, and CaM not only regular CNA, but also stimulate the regulation of CNB on CNA. It was found that CNB and CaM were all not required by CNA to associate with tau, and the efficiencies of CN to dephosphorylate tau keep constant regardless of whether CNB and/or CaM are present or not. The findings may be valuable for revealing how CN take part in controlling phosphorylation of tau in vivo and the pathogenesis of tauopathies such as Alzheimer's Disease.

To explore the polymorphisms and mutations of mitochondrial D-Loop region in osteosarcoma and their possible association with carcinogenesis, direct DNA sequencing method was used to detect the variants of the mitochondrial D-Loop in 20 patients with osteosarcoma. We found mutations of the mitochondrial D-Loop in 14/20 (70%) of the tested osteosarcoma samples, including 34 novel mutations. We did not detect any new polymorphisms in osteosarcomas, nor was there any association between variants and the three histopathological subtypes. We identified 89 variant sites, most of which were within the hypervariable I and II (HV I and HV II) regions. These data suggest that mtDNA mutations within the D-Loop region, particularly the HV I and HV II segments, are a frequent event in osteosarcomas.

Background Numerous epidemiological studies have evaluated the association between TGFBR1 polymorphisms and the risk of cancer, however, the results remain inconclusive. To derive a more precise estimation of the relation, we conducted a comprehensive meta-analysis of all available case-control studies relating the TGFBR1*6A and IVS7+24G>A polymorphisms of the TGFBR1 gene to the risk of cancer. Methods Eligible studies were identified by search of electronic databases. Overall and subgroup analyses were performed. Odds ratio (OR) and 95% confidence interval (CI) were applied to assess the associations between TGFBR1*6A and IVS7+24G>A polymorphisms and cancer risk. Results A total of 35 studies were identified, 32 with 19,767 cases and 18,516 controls for TGFBR1*6A polymorphism and 12 with 4,195 cases and 4,383 controls for IVS7+24G>A polymorphism. For TGFBR1*6A, significantly elevated cancer risk was found in all genetic models (dominant OR = 1.11, 95% CI = 1.04∼1.18; recessive: OR = 1.36, 95% CI = 1.11∼1.66; additive: OR = 1.13, 95% CI = 1.05∼1.20). In subgroup analysis based on cancer type, increased cancer risk was found in ovarian and breast cancer. For IVS7+24G>A, significant correlation with overall cancer risk (dominant: OR = 1.39, 95% CI = 1.15∼1.67; recessive: OR = 2.23, 95% CI = 1.26∼3.92; additive: OR = 1.43, 95% CI = 1.14∼1.80) was found, especially in Asian population. In the subgroup analysis stratified by cancer type, significant association was found in breast and colorectal cancer. Conclusions Our investigations demonstrate that TGFBR1*6A and IVS7+24G>A polymorphisms of TGFBR1 are associated with the susceptibility of cancer, and further functional research should be performed to explain the inconsistent results in different ethnicities and cancer types.