Osteosarcoma Cell Proliferation Research Articles

Identification of ATG7 as a Regulator of Proferroptosis and Oxidative Stress in Osteosarcoma.

Background Ferroptosis has gained significant attention from oncologists as a vital outcome of oxidative stress. The aim of this study was to develop a prognostic signature that was based on the ferroptosis-related genes (FRGs) for osteosarcoma patients and explore their specific role in osteosarcoma. Methods The training cohort dataset was extracted from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Different techniques like the univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, multivariate Cox regression analyses, and the Kaplan-Meier (KM) survival analyses were utilized to develop a prognostic signature. Then, the intrinsic relationship between the developed gene signature and the infiltration levels of the immune cells was further investigated. An external validation dataset from the Gene Expression Omnibus (GEO) database was employed to assess the predictive ability of the developed gene signature. Subsequently, the specific function of potential FRG in affecting the oxidative stress reaction and ferroptosis of osteosarcoma cells was identified. Results A prognostic signature based on 5 FRGs (CBS, MUC1, ATG7, SOCS1, and PEBP1) was developed, and the patients were classified into the low- and high-risk groups (categories). High-risk patients displayed poor overall survival outcomes. The risk level was seen to be an independent risk factor for determining the prognosis of osteosarcoma patients (p < 0.001, hazard ratio: 7.457, 95% CI: 3.302-16.837). Additionally, the risk level was associated with immune function, which might affect the survival status of osteosarcoma patients. Moreover, the findings of the study indicated that the expression of ATG7 was related to the regulation of oxidative stress in osteosarcoma. Silencing the ATG7 gene promoted the proliferation and migration in osteosarcoma cells, suppressing the oxidative stress and ferroptosis process. Conclusions A novel FRG signature was developed in this study to predict the prognosis of osteosarcoma patients. The results indicated that ATG7 might regulate the process of oxidative stress and ferroptosis in osteosarcoma cells and could be used as a potential target to develop therapeutic strategies for treating osteosarcoma.

Lnc-SELPLG-2:1 enhanced osteosarcoma oncogenesis via hsa-miR-10a-5p and the BTRC cascade

BackgroundTo investigate the potential role of Long Non-coding RNAs (lncRNAs) in the progression of osteosarcoma.MethodsThe candidate lncRNAs were screened with RNA-seq and confirmed with quantitative real-time PCR. Using MTS, transwell assay, and flow cytometric analysis, the effects of overexpressed lnc-SELPLG-2:1 on cell functions were determined. Immunohistochemical staining, fluorescence in situ hybridization, and luciferase reporter assay were used to evaluate the potential mechanism of lnc-SELPLG-2:1 in vivo and in vitro using a tumor model. Moreover, the effects of overexpression of hsa-miR-10a-5p on the functions of SaOS2 cells were determined using functional cell analysis. A response test was used to confirm the mechanism by which lnc-SELPLG-2:1 sponge hsa-miR-10a-5p promotes the expression of BTRC to regulate osteosarcoma.ResultsLnc-SELPLG-2:1 was highly expressed in osteosarcoma compared to normal cells and bone and marrow samples. Inhibition of lnc-SELPLG-2:1 accelerated cell apoptosis and suppressed cell proliferation, migration, and invasion, whereas lnc-SELPLG-2:1 overexpression had the opposite effect. Moreover, inhibiting lnc-SELPLG-2:1 in an in vivo model decreased tumor size and suppressed the expression of cell migration-related proteins. The prediction, dual luciferase assay, and response test results indicated that hsa-miR-10-5p and BTRC were involved in the lnc-SELPLG-2:1 cascade. Unlike lnc-SELPLG-2:1, hsa-hsa-miR-10a-5p had opposite expression and function. Competitive binding of lnc-SELPLG-2:1 to hsa-hsa-miR-10a-5p prevented BTRC from miRNA-mediated degradation, thereby activating the expression of VIM, MMP9, and MMP2, promoting osteosarcoma cell proliferation, migration, and invasion, and inhibiting apoptosis.ConclusionLnc-SELPLG-2:1 is an oncogenesis activator in osteosarcoma, and its functions are performed via hsa-miR-10a-5p /BTRC cascade.

Curculigoside Represses the Proliferation and Metastasis of Osteosarcoma via the JAK/STAT and NF-κB Signaling Pathways.

Curculigoside (Cur) is a natural component from Curculigo orchioides Gaertn, with various bioactivities. The function of Cur in the nervous system and osteoarthritis has been reported. However, its role in osteosarcoma (OS) needs to be investigated. Hence, we focus on probing the impact of Cur on OS. In vitro, cell counting kit 8 (CCK-8), flow cytometry and Transwell assay were used to investigate the effects of Cur on OS cell proliferation, apoptosis, migration and invasion. In vivo, we developed a xenograft model to figure out the effect of Cur on tumor growth in nude mice. Western blotting (WB) was conducted to compare the levels of Cur on apoptosis-related proteins (C-caspase-3, Bax, and Bcl-2), epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, Snail, and E-cadherin) and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and nuclear factor-κB (NF-κB) pathways in vitro and in vivo. In-vitro data testified that Cur treatment markedly hampered OS cells' growth, migration and invasion and intensified their apoptosis compared to that of the control group. In vivo, Cur treatment notably hampered the growth of OS tumors in mice. In addition, both in vitro and in vivo experiments demonstrated that the phosphorylation of JAK2, STAT3, and NF-κB were inhibited through Cur treatment. Furthermore, the inhibition of Cur in OS cells was demonstrated by up-regulating the expression of JAK/STAT and NF-κB pathways protein levels. In summary, the data suggest that Cur curbs OS growth by down-regulating the JAK/STAT and NF-κB pathways, which is an underlying therapeutic option for OS treatment.

LRIG2 regulates cell proliferation, migration and apoptosis of osteosarcoma

BackgroundOsteosarcoma (OS) is one of the malignant bone tumors with strong aggressiveness and poor prognosis. Leucine-rich repeats and immunoglobulin-like domains2 (LRIG2) is closely associated with the poor prognosis of a variety of tumors, but the role of LRIG2 in osteosarcoma and the underlying molecular mechanism remains unclear.ObjectiveThe aim of this study was to determine the function of LRIG2 in OS and the related molecular mechanism on cell proliferation, apoptosis and migration of OS.MethodsThe mRNA and protein expression of LRIG2 in OS tissues and cells was detected by qRT-PCR, western blot (WB) assay and immunohistochemistry (IHC). The cell counting Kit-8 (CCK-8), clone formation, transwell, TdT-mediated dUTP Nick-End Labeling (TUNEL) and WB assay were applied to determine the proliferation, migration and apoptosis abilities of OS cells and its molecular mechanisms. Spontaneous metastasis xenografts were established to confirm the role of LRIG2 in vivo.ResultsLRIG2 exhibited high expression in OS tissues and OS cell lines and the expression of which was significantly correlated with Enneking stage of patients, knockdown LRIG2 expression significantly inhibited OS cell proliferation, migration and enhanced apoptosis. Silencing LRIG2 also suppressed the growth of subcutaneous transplanted tumor in nude mice. Further, the mechanism investigation revealed that the protein level of cell proapoptotic proteins (Bax, caspase9 and caspase3) all increased attributed to LRIG2 deficiency, whereas expression of anti-apoptotic protein BCL2 decreased. LRIG2 silencing led to the decrease phosphorylation of AKT signaling, a decrease expression of vimentin and N-cadherin. Additionally, silencing LRIG2 significantly decreased the rate of tumor growth and tumor size.ConclusionsLRIG2 acts as an oncogene in osteosarcoma, and it might become a novel target in the treatment of human OS.

Effect of Long Noncoding RNA HULC on Proliferation, Migration, and Invasion of Osteosarcoma Cells.

Background Previous studies had shown that lncRNA HULC exhibited different effects in human cancers. However, the role of HULC was not reported in osteosarcoma. Hence, we designed this research to explore the function of HULC in osteosarcoma. Methods Firstly, HULC expression was measured in osteosarcoma tissues and cells via the RT-qPCR assay. The protein expression was detected through western blot. Then, CCK-8 and Transwell assays were conducted to measure cell proliferation, migration, and invasion. Results The expression of HULC was obviously higher in osteosarcoma tissues and cells compared with normal control. Moreover, cell proliferation, migration, and invasion were inhibited by HULC knockdown in osteosarcoma cells. HULC overexpression markedly increased osteosarcoma cell proliferation and tumor size in vivo. Furthermore, HULC increased the activity of AKT-PI3K-mTOR pathway by blocking PTEN in osteosarcoma cells. LY294002 inhibited the phosphorylation of AKT, mTOR, and PI3K. Overexpressing HULC enhanced cell migration and invasion of SAOS-2 cells and MG63 cells, while LY294002 reversed the effects. Conclusion HULC enhanced the progression of osteosarcoma through targeting PTEN.

Activation of PGRN/MAPK axis stimulated by the hypoxia-conditioned mesenchymal stem cell-derived HIF-1α facilitates osteosarcoma progression

BackgroundProgranulin (PGRN) is an important survival factor in the progression of multiple cancers. PurposeTo explore the effects and mechanisms of PGRN on malignant biological behavior of osteosarcoma (OS) cells and the effects of mesenchymal stem cells (MSCs) and the hypoxic microenvironment on PGRN alteration. Material and methodsThe expression pattern of PGRN in OS were evaluated in OS tissues and cell lines. Next, a loss-of-function assay investigated the function of PGRN on the proliferation, migration and cell death of OS cells. The activation of MAPK signaling in the process was examined by western blot and functional experiments accompanied by skatole. Additionally, we internally silenced hypoxia-inducible factor-1α (HIF-1α) in MSCs along with exogenously added HIF-1α (exo-HIF-1α) to explore how MSCs affect PGRN alteration and the malignant behavior of OS cells. ResultsAn aberrantly high expression of PGRN was observed in OS and associated with the poor prognosis of OS patients. PGRN knockdown repressed the proliferation, migration and induced cell death of OS cells, and activating MAPK pathway reversed these effects. Further evidence showed that MSCs regulated PGRN to mediate the malignant biological behavior of OS cells. Hypoxia enhanced HIF-1α expression in MSCs. HIF-1α silencing in MSCs under hypoxia suppressed the oncogenic effects of MSCs and reduced PGRN expression in OS cells, while the treatment of exo-HIF-1α reversed the depressive effects of HIF1α silencing on OS progression. ConclusionOverall, we concluded that PGRN, which was activated by the increase of hypoxic-MSCs-derived HIF-1α, promoted OS progression through the activation of MAPK signaling.

N6-methyladenosine reader YTHDF3 contributes to the aerobic glycolysis of osteosarcoma through stabilizing PGK1 stability.

N6-methyladenosine (m6A) modification is a pivotal transcript chemical modification of eukaryotics, which has been identified to play critical roles on tumor metabolic reprogramming. However, the functions of m6A-reading protein YTH N6-methyladenosine RNA-binding protein 3 (YTHDF3) in osteosarcoma is still unclear. This research planned to investigate the bio-functions and mechanism in osteosarcoma tumorigenesis. The aerobic glycolysis of osteosarcomacells were calculated byglucose uptake,lactate production analysis, ATP analysis andmetabolic flux analysis for extracellular acidification rate (ECAR).Molecular binding was identified byRIP-qPCR,RNA decay analysis. Results indicated that YTHDF3 is upregulated in the osteosarcoma tissue samples and cells, and closely correlated to the poor prognosis of osteosarcoma patients. Functionally, gain and loss-of-functional assays illustrated that YTHDF3 promoted the proliferation and aerobic glycolysis of osteosarcoma cells in vitro, and accelerated the tumor growth in vivo. Mechanistically, a m6A-modified PGK1 mRNA functioned as the target of YTHDF3, and YTHDF3 enhanced the PGK1 mRNA stability via m6A-dependent manner. In conclusion, these findings indicated that YTHDF3 functioned as an oncogene in osteosarcoma tumorigenesis through m6A/PGK1 manner, providing a therapeutic strategy for human osteosarcoma.

Silencing of circCYP51A1 represses cell progression and glycolysis by regulating miR-490-3p/KLF12 axis in osteosarcoma under hypoxia

BackgroundHypoxia is a key characteristic of osteosarcoma (OS). Increasing data suggested that circular RNA (circRNAs) were involve in the progression of cancers and the regulation of hypoxia, including OS. This study aims to examine the biological mechanism of circRNA cytochrome P450 family 51 subfamily A member 1 (circCYP51A1) in OS under hypoxia. MethodsThe expression levels of circCYP51A1, microRNA-490-3p (miR-490-3p) and kruppel-like factor 12 (KLF12) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in OS tissues and cells. Cell proliferation, migration and invasion were determined by colony formation assay and transwell assay. Lactate production and glucose consumption in OS cells were measured by using lactate assay kit and glucose assay kit, respectively. Western blot assay and immunohistochemistry assay were used to test protein levels. The associated relationship between miR-490-3p and circCYP51A1 or KLF12 was predicted using Starbase or DIANA online database and verified by dual-luciferase reporter assay. The xenograft model was used to explore the role of circCYP51A1 in vivo. ResultsCircCYP51A1 and KLF12 expression were dramatically increased, whereas miR-490-3p was decreased in OS cells under hypoxia condition. Deficiency of circCYP51A1 hindered hypoxia-induced cell proliferation, migration, invasion and glycolysis in OS cells. CircCYP51A1 enhanced KLF12 expression by sponging miR-490-3p. MiR-490-3p inhibitor weakened the inhibition effect of circCYP51A1 knockdown on the progression of OS under hypoxia. Besides, overexpression of miR-490-3p inhibited cell progression of OS under hypoxia condition, while the effects were attenuated by KLF12 overexpression. Importantly, knockdown of circCYP51A1 inhibited tumor growth in vivo. ConclusionCircCYP51A1 mediated cell proliferation, migration, invasion and glycolysis by regulating miR-490-3p/KLF12 axis in OS cells under hypoxia condition.

Cyy260 suppresses the proliferation, migration and tumor growth of osteosarcoma by targeting PDGFR-β signaling pathway

Osteosarcoma (OS) is a group of malignant tumors with high rates of malignancy and metastasis. OS most commonly affects adolescents and young individuals. However, owing to the lack of effective targeted treatments, the 5-year survival rate for OS is still around 20%. Thus, it is essential to develop effective drugs with low toxicity for OS treatment. In the present study, we investigated the antitumor effect and underlying mechanism of cyy260 in OS via suppressing PDGFR-β and its downstream pathway. We demonstrated that cyy260 inhibits OS cell proliferation and promotes apoptosis via inducing DNA damage and causing cell cycle arrest. More importantly, cyy260 also significantly inhibits tumor migration. Further analysis of molecular mechanisms confirmed that PDGFR-β and its downstream AKT, STAT3, and ERK were involved in the cyy260-mediated antitumor effect. Analysis of subcutaneously transplanted tumors in mice showed that cyy260 suppressed tumor cell growth and exhibited low toxicity in vivo. Collectively, these findings proved that cyy260 could serve as a promising PDGFR-β inhibitor for the treatment of OS.

Expression of Sex Hormone Receptors in Canine Osteosarcoma.

Simple SummaryOsteosarcoma is a bone tumor that arises from normal bone cells that become malignant. This is a disease that is relatively common in dogs and also affects humans. Dogs are a useful model for human osteosarcoma as they share many similarities including genetic changes, manner of spread and how they respond to treatment. In dogs, the tumors are frequently seen in large breeds and may be caused by environmental and genetic factors. Sex steroids have been shown to regulate bone metabolism directly and indirectly through receptors on bone. The aim of this study was to investigate the expression levels of sex hormone receptors in canine osteosarcoma tissue and cell lines. Our results demonstrate varying levels of receptors in tissue and a high expression of progesterone receptors in the canine osteosarcoma cell lines. Low oxygen levels further increased progesterone receptor expression. Lastly, estradiol decreased the expression of progesterone receptor in one of the cell lines, and progesterone decreased cell proliferation in the same cell line. Further investigation is recommended to determine whether or not sex steroid receptor levels and sensitivity to hormones are related.Sex steroids regulate bone metabolism directly and indirectly through receptors on bone. Estrogen receptors (ER-∝, ER-β), progesterone receptor (PR), and androgen receptor (AR), have been previously identified on human osteosarcoma (OSA) cells, and are considered to influence tumor growth, but their expression and role in canine OSA is unknown. The aim of this study was to characterize sex hormone receptor expression levels in naturally occurring OSA tissue and in three canine OSA cell lines. The expression of ER-α, ER-β, PR, and AR was investigated using RT-PCR. PR expression levels were also quantified in OSA cells cultured under hypoxic conditions or in the presence of estradiol. The effects of progesterone on cell proliferation were quantified. Results demonstrated varying expression levels of these receptors in five OSA subtypes. OSA cell lines demonstrated high gene expression levels of PR and low gene expression levels of ER-α and ER-β and no gene expression of AR. PR expression was increased in OSA cells cultured under hypoxic conditions in a HIF-∝ independent manner. Interestingly, one cell line expressed very high levels of PR, expression of which decreased in response to estradiol. In addition, progesterone decreased OSA cell proliferation in this particular cell line. Further investigation of the role of sex steroids, particularly PR and its ligands, in regulation of canine OSA is recommended.

Circ_0051079 functions as an oncogenic regulator in osteosarcoma by leading to MAFB expression upregulation by competitively interacting with miR-1286

BackgroundCircular RNAs are involved in various cellular processes of bone diseases by acting as miRNA sponges to regulate gene expression levels, including osteosarcoma (OS). This research concentrated on the molecular mechanism of circ_0051079 in OS progression.MethodsReverse transcription-quantitative polymerase chain reaction assay was used for expression detection of circ_0051079, microRNA-1286 (miR-1286), and musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB). Cell Counting Kit-8 assay and Edu assay were used for cell proliferation analysis. Cell apoptosis was evaluated using flow cytometry. Western blot was performed to measure protein levels. Migration and invasion were assessed via transwell assay. Interaction of circ_0051079/miR-1286 or miR-1286/MAFB was explored through a dual-luciferase reporter assay. In vivo research was carried out via tumor xenograft assay and immunohistochemistry staining.ResultsCirc_0051079 expression was upregulated in OS. Downregulation of circ_0051079 reduced OS cell proliferation, migration, invasion, and accelerated apoptosis. Circ_0051079 interacted with miR-1286, and the tumor-inhibitory function of si-circ_0051079 was abolished by miR-1286 inhibition in OS cells. MAFB served as a target for miR-1286. OS cell progression was suppressed by miR-1286 overexpression via downregulating MAFB. Circ_0051079/miR-1286 resulted in expression change of MAFB in OS cells. Silencing circ_0051079 inhibited tumor growth in vivo via regulating the miR-1286/MAFB axis.ConclusionThe collective results elucidated that circ_0051079 contributed to OS progression via miR-1286-mediated upregulation of MAFB, confirming the interaction of circ_0051079/miR-1286/MAFB axis in OS.

KMT5A Knockdown Suppresses Osteosarcoma Cell Proliferation and Metastasis Through Ꞵ-Catenin Signalling.

Osteosarcoma (OS) is the most common malignant solid bone tumor in children and young adults. We aimed to investigate the effects and cellular mechanisms of KMT5A on OS cell activity. The protein expression was evaluated in the clinical normal, adjacent and OS osteogenic tissues. Knockdown of KMT5A was achieved by KMT5A siRNAs in a human OS cell line, MG63, to detect cell proliferation and metastasis. KMT5A expression was upregulated in clinical OS tissues. Knockdown of KMT5A inhibited cell proliferation but enhanced cell death, with significantly reduced cyclinD1 and Bcl2 and increased cleaved-caspase9 levels. KMT5A knockdown also suppressed OS cell migration and invasion capacity and deceased MMP3 and vimentin expression. β-catenin levels were upregulated in OS tissues and blocking KMT5A resulted in a significant decline in β-catenin expression in the OS cells. Further administration of β-catenin activator remarkably increased protein levels of KMT5A, cyclinD1, Bcl2, MMP3, and vimentin, which showed reversed effects of KMT5A knockdown on OS cell activity. KMT5A knockdown plays an inhibitory role in OS cell proliferation and metastasis through β-catenin signalling, which provides basic evidence and suggests potential targets for OS therapeutic research.

ZCCHC12 promotes the progression of osteosarcoma via PI3K/AKT pathway.

Researchers have reported that zinc finger CCHC domain containing 12 gene (ZCCHC12) plays a role in the progression and tumorigenesis of papillary thyroid cancer. However, the biological role of ZCCHC12 in osteosarcoma (OS) remains unknown. ZCCHC12 was highly upregulated in OS cell lines according to our present study. Also, our subsequent assays demonstrated that ZCCHC12 enhanced the proliferation, tumor growth and migration of OS cells. Moreover, the epithelial-mesenchymal transition (EMT) of OS cells was also promoted by ZCCHC12. In addition, downregulation of ZCCHC12 induced apoptosis and S-phase arrest in OS cells. Then, our study indicated that ZCCHC12 exerts its oncogenic function in OS cells by activating the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway greatly limits the oncogenic function of ZCCHC12 in OS cells. Also, overexpression of ZCCHC12 promotes tumor growth in vivo. Altogether, our study suggests ZCCHC12 promotes OS cells progression by activating the PI3K/AKT pathway. The ZCCHC12 gene may be a novel diagnostic and therapeutic target for OS.

Long non-coding RNA MALAT1 promotes cell proliferation, migration and invasion by targeting miR-590-3p in osteosarcoma.

Osteosarcoma (OS) is a common malignant bone cancer and commonly occurs in adolescents and children. Long non-coding RNAs (lncRNAs) play major roles in cancer cell proliferation and metastasis. The present study aimed to investigate the potential molecular mechanism of lncRNA MALAT1 in OS. The levels of lncRNA MALAT1 and microRNA-590-3p were detected by reverse transcription-quantitative PCR in OS tissues and cells. Cell Counting Kit-8 and flow cytometry assays were conducted to assess cell proliferation and apoptosis. Cell migration and invasion were examined by Transwell assay. The levels of E-cadherin, N-Cadherin, Vimentin and Snail were measured by western blotting. The target of MALAT1 was predicted using online software and confirmed by luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. The results indicated that MALAT1 was highly expressed in OS tissues and cell lines. MALAT1 knockdown promoted apoptosis and suppressed proliferation, migration, invasion and epithelial- mesenchymal transition (EMT) of OS cells. Overexpression of miR-590-3p increased cell apoptosis and hampered cell proliferation, migration, invasion and EMT in OS cells. In addition, MALAT1 knockdown upregulated the expression of miR-590-3p in OS cells. In conclusion, MALAT1 was demonstrated to suppress cell apoptosis and induce cell proliferation, migration, invasion and EMT by inhibiting miR-590-3p in OS, which indicated that MALAT1 has potential value in the diagnosis and treatment of OS.

LncRNA JPX modulates malignant progress of osteosarcoma through targeting miR-33a-5p and PNMA1 regulatory loop

Osteosarcoma (OS) is a common type of bone tumor, present worldwide, that has distal metastasis ability. Although continuous development in cancer therapy has taken place, there are still no effective metastasis-curbing strategies for OS available. Hence, a better understanding of the biological characteristics and molecular mechanisms of OS carcinogenesis is urgently needed. Long noncoding RNAs (lncRNAs) have captured great interest among cancer scientists with considerable potential implications for cancer treatment. In this study, we found that lncRNA JPX was up-regulated in OS tissues and cells. We subsequently examined the functional role of JPX in OS cells through knocked-down JPX by using siRNA. JPX down-regulation was observed to suppress OS cell proliferation, migration and invasion. Furthermore, it was verified that JPX acts as a sponge for miR-33a-5p, and that JPX regulated OS cell proliferation, migration and invasion through miR-33a-5p. Moreover, down-regulation of miR-33a-5p in OS contributed to PNMA1 upregulation, and PNMA1 depletion inhibited OS cell proliferation, migration and invasion in vitro. Taken together, our data support an important role of JPX in regulating OS cell proliferation, invasion and migration that highlights JPX may be a potential therapeutic target for OS.

Rhizoma Paridis saponins suppresses vasculogenic mimicry formation and metastasis in osteosarcoma through regulating miR-520d-3p/MIG-7 axis

Osteosarcoma (OS) is a highly metastatic bone cancer that usually affects children. Rhizoma Paridis saponins (RPS) have been identified to show a broad-spectrum anti-tumor activity. Our previous study has identified vasculogenic mimicry (VM) as an indicator of poor prognosis for OS. Rhizoma Paridis ethanol extract exhibits potent anti-OS property. However, the anti-metastatic effect of RPS on OS and the detailed mechanisms remain unknown. RPS was characterized by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) analysis. The anti-OS, anti-metastasis and anti-VM activities of RPS were investigated using in vitro biological assays and a xenograft mouse model. Western blot, qRT-PCR, ELISA, Phalloidin staining and immunohistochemistry assays were conducted to investigate the molecular mechanism of RPS. A total of 34 phytochemicals from RPS were identified by LC/Q-TOF/MS. RPS dose-dependently suppressed the OS cell proliferation, metastasis and VM formation in vitro and in vivo. Mechanically, we found that RPS downregulated migration-inducing gene 7 (MIG-7) expression, resulting in inhibition of the PI3K/MMPs/Ln-5γ2 pathway and cell protrusion formation. Additionally, we confirmed that RPS downregulated MIG-7 by upregulating miR-520d-3p expression. Our results suggests that RPS inhibits the VM formation and metastasis of OS by modulating the miR-520d-3p/MIG-7 signaling axis.

Long non-coding RNA DUXAP10 exerts oncogenic properties in osteosarcoma by recruiting HuR to enhance SOX18 mRNA stability

Recent studies have demonstrated that several long non-coding RNAs (lncRNAs) play an important role in the occurrence and development of osteosarcoma (OS). However, more lncRNAs and their mechanisms in regulating growth and migration of OS cells remain to be investigated. In this study, we identified an lncRNA called DUXAP10 by analysis of GEO data, which was significantly up-regulated in OS tissues and cell lines. Experiments in vitro revealed that lncRNA DUXAP10 promoted proliferation, migration, and invasion of OS cells and inhibited their apoptosis. We also demonstrated that DUXAP10 promoted the formation and growth of OS by tumor formation assay. Furthermore, SOX18 was identified as a critical downstream target of DUXAP10 by transcriptome RNA-seq. Mechanistically, DUXAP10 mainly localized in cytoplasm and could specifically bind to HuR to increase the stability of SOX18 mRNA. Meanwhile, SOX18 knockdown largely reversed increased proliferation of OS cells induced by DUXAP10 overexpression. Findings from this study indicate that lncRNA DUXAP10 can act as an oncogene in osteosarcoma by binding HuR to up-regulate the expression of SOX18 at a post-transcriptional level, which may provide a new target for OS clinical diagnosis and treatment.

Down-Regulation of Ubiquitin-Specific Peptidase 9X Inhibited Proliferation, Migration and Invasion of Osteosarcoma via ERK1/2 and PI3K/Akt Signaling Pathways.

Ubiquitin-specific peptidase 9X (USP9X) has been reported to be closely associated with the formation and progression of a variety of malignant tumors. However, the mechanism by which USP9X is involved in osteosarcoma and development has not been clearly studied. This work aimed to probe the influence of USP9X on osteosarcoma cell proliferation, migration and invasion. This study recruited sixty-seven patients with histologically definited osteosarcoma. Osteosarcoma samples and cell-line were used to reflect the expression level of USP9X. Analysis of cell proliferation by thiazolium blue (MTT) assays. Transwell experiments and wound healing were used to verify cell migration and invasion capabilities. The effect of USP9X was investigated through in vivo experiments. USP9X-related pathway proteins were detected by Western blot and quantitative real-time PCR (qRT-PCR). The expression of USP9X in osteosarcoma was higher than that in adjacent tissues. The overall survival of patients with USP9X-negative patients was better than that of patients with USP9X-positive. The growth of osteosarcoma cells in vivo and in vitro was inhibited by USP9X inhibitor. Cell migration and invasion were significantly inhibited by down-regulation of USP9X. USP9X was involved in extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3-kinases/protein-serine-threonine kinase (PI3K/Akt) pathway in osteosarcoma cells. Proliferation, migration and invasion of osteosarcoma cells were inhibited by down-regulation of USP9X, and were related to the ERK1/2 and PI3K/Akt signaling pathways, therefore, it might probably become a new target for the prevention and treatment of osteosarcoma.

Cisplatin and Exogenous Hydrogen Sulfide Inhibit the Progression of Osteosarcoma via EGFR/P13K/Akt Signaling Pathway

We attempted to analyze the effects of cisplatin and exogenous hydrogen on the invasion and proliferation of human osteosarcoma cells through EGFR/P13K/Akt signaling pathway. The effects of exogenous hydrogen sulfide and cisplatin on the invasion, apoptosis and proliferation of MG-63 cells were detected by transwell chamber invasion experiment, flow cytometry and CCK-8 method. After 24 and 48 hours of drug action, the inhibition rate of cell proliferation was significantly higher than in the combined treatment. The number of cells decreased significantly, compared with the control group. Western blotting and qRT-PCR showed that the expression of Akt mRNA in MG-63 cells treated with cisplatin+hydrogen sulfide at different concentrations for 48 hours decreased. The expression of Akt, P13K and EGFR were decreased Flow cytometry analysis showed that after the drug treatment of MG-63 cells, the S phase of cisplatin and cisplatin+exogenous hydrogen concentrations with different prolonged concentrations was significantly shortened phase and the G0/G1 was significantly, and the apoptosis rate of each group was significantly increased. Cisplatin and exogenous hydrogen sulfide can synergistically inhibit the proliferation and invasion of human osteosarcoma MG-63 cells, and regulate the apoptosis mechanism of MG-63 cells through EGFR/P13K/Akt signaling pathway.

LncRNA FGD5-AS1 potentiates autophagy-associated doxorubicin resistance by regulating the miR-154-5p/WNT5A axis in osteosarcoma.

Osteosarcoma is prevalent in children and adolescent. The oncogenic function of long-chain noncoding RNA (lncRNA) FGD5 antisense RNA 1 (FGD5-AS1) has been reported. However, the function of FGD5-AS1 in doxorubicin-resistance in osteosarcoma remains to be illucidated. Quantitative real-time PCR (qRT-PCR) and western blot analysis (WB) were used to measure the expression of FGD5-AS1, miR-154-5p, WNT5A and autophagy proteins. MTT assay was used to assess cell viability and transwell assay was performed to evaluate migration. A nude mouse xenograft model was developed to verify the function of FGD5-AS1 in vivo. FGD5-AS1was upregulated in doxorubicin-resistant (DXR) osteosarcoma cells. Knockdown of FGD5-AS1 suppressed osteosarcoma cell proliferation, migration, and autophagy. FGD5-AS1 upregulated WNT5A expression via sponging miR-154-5p. Furthermore, FGD5-AS1 enhanced osteosarcoma cell chemotherapy resistance through upregulation of WNT5A by inhibiting miR-154-5p. Suppression of FGD5-AS1 significantly suppressed tumor growth in nude mice. FGD5-AS1 may promote chemoresistance through WNT5A-induced autophagy by sponging miR-154-5p in osteosarcoma cells.