Suppressed Osteosarcoma Cell Proliferation Research Articles

Long noncoding RNA DHRS4 antisense RNA 1 suppresses osteosarcoma cell proliferation and promotes apoptosis through a competitive endogenous RNA mechanism

Osteosarcoma (OS) is the most common primary malignant bone tumor. Recent evidence suggests that the novel long noncoding RNA DHRS4 antisense RNA 1 (DHRS4-AS1) serves an important role in cancer progression and metastasis. However, its function and molecular mechanism in OS remain largely unknown. In the present study, DHRS4-AS1 expression was detected in OS cells by quantitative PCR. Gain- and loss-of-function experiments were conducted to study the effects of DHRS4-AS1 on the proliferation and apoptosis of OS cells. The potential mechanism of DHRS4-AS1 was examined through bioinformatics analysis and rescue experiments. DHRS4-AS1 was downregulated in OS cell lines. DHRS4-AS1 depletion promoted proliferation and inhibited apoptosis in OS cells, whereas DHRS4-AS1 overexpression had the opposite effects. Further research suggested that DHRS4-AS1 inhibited OS progression by regulating the microRNA-362-5p/aminopeptidase puromycin sensitive axis. The present findings suggested that DHRS4-AS1 may serve as a potential therapeutic target for OS.

Design, synthesis, and biological evaluation of Flavokavain B derivatives as potent TRF2 inhibitors for the treatment of Osteosarcoma.

Telomere repeat-binding factor 2 (TRF2) is a crucial component of the shelterin complex, commonly overexpressed in osteosarcoma (OS) and positively correlated with its progression. To date, effective TRF2 inhibitors for in vivo applications remain limited. In this study, a series of Flavokavain B derivatives were designed and synthesized, and their TRF2 inhibition and antitumor activity were evaluated. Among the tested compounds, the active compound F2 showed remarkable inhibition of TRF2 expression, along with potent antiproliferative activity in U2OS and MG63cells, with IC50 values of 5.28μM and 1.52μM, respectively. Moreover, F2 significantly suppressed OS cell proliferation and induced apoptosis by accelerating telomere shortening and loss due to TRF2 inhibition. Mechanically, F2 selectively inhibited TRF2 protein expression and telomeric localization by directly binding to the TRF2TRFH domain. Furthermore, F2 demonstrated strong antitumor efficacy with minimal toxicity in an MG63-derived xenograft mouse model. These findings demonstrate that F2 is a promising drug candidate for the treatment of osteosarcoma.

Scutellarein Inhibits Osteosarcoma Growth by Targeting the TLR4/TRAF6/NF-κB Pathway.

Osteosarcoma (OS) is the most common malignant tumor associated with poor patient outcomes and a limited availability of therapeutic agents. Scutellarein (SCU) is a monomeric flavone bioactive compound with potent anti-cancer activity. However, the effects and mechanisms of SCU on the growth of OS remain unknown. The Cell Counting Kit-8, colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays were used to analyze cell proliferation ability in vitro. TLR4/TRAF6/NF-κB signaling transduction was investigated by RNA sequencing analysis, quantitative real-time polymerase chain reaction, Western blotting, NF-κB luciferase reporter assay, immunofluorescent staining, and immunoprecipitation. Molecular docking and cellular thermal shift assay were employed to confirm the binding interaction between SCU and TLR4. The effects of SCU and TLR4 overexpression on OS growth were analyzed using a xenograft tumor model and immunohistochemical staining. SCU was found to significantly inhibit OS cell proliferation, and RNA sequencing analysis suggested that the NF-κB pathway is closely associated with this process. Further studies revealed that SCU inhibited the canonical NF-κB pathway through its binding with TLR4, which disrupted the interaction of TLR4 and TRAF6. Moreover, SCU also repressed NF-κB signal transduction by inhibiting TLR4 expression. Furthermore, SCU was revealed to suppress OS cell proliferation by targeting TLR4 in vitro and in vivo. SCU exhibited a dual impact by inhibiting TLR4 expression and disrupting TLR4-TRAF6 interaction, resulting in NF-κB inactivation, thereby blocking OS growth.

SQLE promotes osteosarcoma progression via activating TGFβ/SMAD signaling pathway

BackgroundThe prognosis of advanced osteosarcoma (OS) has remained stagnant in last decades, requiring the identification of novel therapeutic targets. Recently, much attention was paid to the role of squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol metabolism, in the field of oncology, while the specific role of SQLE in OS has not been sufficiently elucidated. The present study aims to investigate the role of SQLE in the progression of OS and explore the potential mechanisms. MethodsThe expression levels of SQLE in OS tissues and adjacent normal tissues were compared using bioinformatic methods and experiments. Kaplan-Meier survival analysis and univariate and multivariate Cox analysis were performed to detect the association of SQLE expression and patient’ prognosis. Stably cell lines with SQLE knockdown or overexpression were constructed by lentivirus infection. CCK-8, colony formation, scratch healing, and Transwell invasion assays were carried out to explore the effect of SQLE knockdown or overexpression on the proliferation, migration, and invasion of OS cells. Gene set enrichment analysis was conducted to reveal signaling pathways associated with SQLE expression. The effect of SQLE on TGFβ/SMAD signaling pathway were explored by Western blot assay. ResultsHere, we found a notable rise of SQLE expression in OS tissues and cell lines. Survival analysis showed that individuals with high SQLE expression had a lower median overall survival time compared to those with low SQLE expression. Univariate and multivariate Cox regression analyses showed that SQLE might have the potency to serve as an independently prognostic biomarker in OS. Loss- and gain-of-function experiments indicated that silence of SQLE suppressed OS cell proliferation, migration, and invasion, while overexpression of SQLE exerted the opposite effects. Mechanistically, TGF-β signaling pathway was identified as the downstream pathway of SQLE through bioinformatic methods, and the results of Western blot assay showed that SQLE positively regulated the activity of TGFβ1/SMAD2/3 signaling in OS. Resue experiments demonstrated that SB431542, a small molecule that inhibits TGFβ/SMAD signaling, could partly reverse the promoting effects of SQLE on OS cell proliferation, migration, and invasion. ConclusionOur results provided preliminary evidences that SQLE was a tumor-promoting factor and prognosis predictor in OS. SQLE promoted OS cell proliferation, migration, and invasion via activating TGFβ/SMAD signaling and targeting SQLE might be a potential strategy for the treatment of OS.

MicroRNA-605-3p Inhibited the Growth and Chemoresistance of Osteosarcoma Cells via Negatively Modulating RAF1.

Osteosarcoma (OS) is the leading cancer-associated mortality in childhood and adolescence. Increasing evidence has demonstrated the key function of microRNAs (miRNAs) in OS development and chemoresistance. Among them, miRNA-605-3p acted as an important tumor suppressor and was frequently down-regulated in multiple cancers. However, the function of miR-650-3p in OS has not been reported. The aim of this work is to explore the novel role of miR-605-3p in osteosarcoma and its possible involvement in OS chemotherapy resistance. The expression levels of miR-605-3p in OS tissues and cells were assessed by reverse transcription quantitative PCR (RT-qPCR). The relevance of miR-605-3p with the prognosis of OS patients was determined by the Kaplan-Meier analysis. Additionally, the influence of miR-605-3p on OS cell growth was analyzed using the cell counting kit-8, colony formation assay, and flow cytometry. The mRNA and protein expression of RAF1 were detected by RT-qPCR and western blot. The binding of miR-605-3p with the 3'-UTR of RAF1 was confirmed by dual-luciferase reporter assay. Our results showed that miR-605-3p was markedly decreased in OS tissues and cells. A lower level of miR-605-3p was strongly correlated with lymph node metastasis and poor 5-year overall survival rate of OS patients. In vitro assay found that miR-605-3p suppressed OS cell proliferation and promoted cell apoptosis. Mechanistically, the proto-oncogene RAF1 was seen as a target of miR-605-3p and strongly suppressed by miR-605-3p in OS cells. Restoration of RAF1 markedly eliminated the inhibitory effect of miR-605-3p on OS progression, suggesting RAF1 as a key mediator of miR-605-3p. Consistent with the decreased level of RAF1, miR-605-3p suppressed the activation of both MEK and ERK in OS cells, which are the targets of RAF1. Moreover, lower levels of miR-605-3p were found in chemoresistant OS patients, and downregulated miR-605-3p increased the resistance of OS cells to therapeutic agents. Our data revealed that miR-605-3p serves as a tumor suppressor gene by regulating RAF1 and increasing the chemosensitivity of OS cells, which provided the novel working mechanism of miR-605-3p in OS. Engineering stable nanovesicles that could efficiently deliver miR-605-3p with therapeutic activity into tumors could be a promising therapeutic approach for the treatment of OS.

Inhibition of the galactosyltransferase C1GALT1 reduces osteosarcoma cell proliferation by interfering with ERK signaling and cell cycle progression

Novel therapeutic strategies are urgently required for osteosarcoma, given the early age at onset and persistently high mortality rate. Modern transcriptomics techniques can identify differentially expressed genes (DEGs) that may serve as biomarkers and therapeutic targets, so we screened for DEGs in osteosarcoma. We found that osteosarcoma cases could be divided into fair and poor survival groups based on gene expression profiles. Among the genes upregulated in the poor survival group, siRNA-mediated knockdown of the glycosylation-related gene C1GALT1 suppressed osteosarcoma cell proliferation in culture. Gene expression, phosphorylation, and glycome array analyses also demonstrated that C1GALT1 is required to maintain ERK signaling and cell cycle progression. Moreover, the C1GALT1 inhibitor itraconazole suppressed osteosarcoma cell proliferation in culture, while doxycycline-induced shRNA-mediated knockdown reduced xenograft osteosarcoma growth in mice. Elevated C1GALT1 expression is a potential early predictor of poor prognosis, while pharmacological inhibition may be a feasible treatment strategy for osteosarcoma.

Combined SET7/9 and CDK4 inhibition act synergistically against osteosarcoma

Osteosarcoma is the most common malignant bone tumor. It has a poor prognosis because of a lack of therapeutic targets and strategies. The SET domain-containing lysine-specific methyltransferase, SET7/9, has various functions in different cancer types in tissue-type and signaling context-dependent manners. The role of SET7/9 in osteosarcoma cells is currently controversial and its potential as a therapeutic candidate in osteosarcoma is unknown. In the present study, SET7/9 inhibition or ablation suppressed osteosarcoma cell proliferation by causing G1 arrest. Mechanistically, SET7/9 inhibition disrupted the interaction between cyclin-dependent kinase 4 (CDK4) and cyclin D1, which affected CDK4–cyclin D1 complex function, leading to decreased phosphorylation of retinoblastoma protein. CDK4 was overexpressed in osteosarcoma tissues and was closely related to a poor prognosis in patients with osteosarcoma. We therefore hypothesized that SET7/9 inhibition might increase the sensitivity of osteosarcoma cells to CDK4 inhibitors, potentially decreasing the risk of adverse effects of CDK4 inhibitors. The combination of SET7/9 and CDK4 inhibition enabled dose reductions of both inhibitors and had a synergistic effect against osteosarcoma growth in vivo. Collectively, these findings indicate that SET7/9 plays an oncogenic role in osteosarcoma by regulating CDK4–cyclin D1 complex interaction and function. The combination of SET7/9 and CDK4 inhibition may thus provide a novel effective therapeutic strategy for osteosarcoma with no significant toxicity.

MTORC1 accelerates osteosarcoma progression via m6A-dependent stabilization of USP7 mRNA

Osteosarcoma (OS) is considered a sex steroid hormone-dependent bone tumor. The development and progression of OS are regulated by 17β-estradiol (E2). However, the detailed mechanisms of E2-modulated OS progression remained to be elucidated. Here, we found that E2-activated mammalian target of rapamycin (mTOR) signaling promoted N6-methyladenosine (m6A) modification through regulating WTAP. Inhibition of mTOR complex 1 (mTORC1) reversed E2-activated WTAP expression. Meanwhile, inhibition of mTORC1 suppressed OS cell proliferation and migration. Deficiency of TSC2 activated mTORC1 signaling and enhanced OS cell proliferation and migration, while abrogated by Rapamycin. Interestingly, mTOMC1 promoted mRNA stability of ubiquitin-specific protease 7 (USP7) through m6A modification. Loss of USP7 suppressed the proliferation, migration, and ASC specks, while promoted apoptosis of OS cells. USP7 interacted with NLRP3 and deubiquitinated NLRP3 through K48-ubiquitination. USP7 was upregulated and positive correlation with NLRP3 in OS patients with high level of E2. Loss of USP7 suppressed the progression of OS via inhibiting NLRP3 inflammasome signaling pathway. Our results demonstrated that E2-activtated mTORC1 promoted USP7 stability, which promoted OS cell proliferation and migration via upregulating NLRP3 expression and enhancing NLRP3 inflammasome signaling pathway. These results discover a novel mechanism of E2 regulating OS progression and provide a promising therapeutic target for OS progression.

Shikonin suppresses proliferation of osteosarcoma cells by inducing ferroptosis through promoting Nrf2 ubiquitination and inhibiting the xCT/GPX4 regulatory axis.

Osteosarcoma (OS) is a prevalent primary malignant bone tumor which lacks effective therapeutic interventions. Ferroptosis is a new form of programmed cell death characterized by iron-dependent accumulation of lethal lipid oxidation, which provides a potential alternative intervene for the OS treatment. Shikonin is the major bioactive component extracted from the roots of lithospermum erythrorhizon which is also known as "Zicao" in traditional Chinese medicine, has been proved to have exhibits remarkable anti-tumor properties in several cancers. However, whether ferroptosis participated in the shikonin mediated anti-OS activity still remains to be clarified. Herein, we provide evidence that shikonin possesses the capability to induce the ferroptosis, and elucidate the underlying mechanisms in the treatment of OS. In the present study, it was found that shikonin significantly suppressed OS cells proliferation and blocked the cell cycle progression in vitro. Subsequent results revealed that shikonin could trigger ferroptosis in OS cells by promoting the Fe2+ accumulation, reactive oxygen species and lipid peroxidation formation, malondialdehyde production and mitochondrial damage. Further study showed that the effects of OS cell proliferation and death caused by shikonin could be successfully reversed by ferroptosis inhibitor ferrostatin-1, indicating that ferroptosis participated in the shikonin mediated anti-OS activity. Mechanistically, shikonin physically interacted with Nrf2, a critical regulator of ferroptosis, and influenced Nrf2 stability via inducing ubiquitin degradation, which suppressed the expression of Nrf2 downstream targets xCT and GPX4, and led to stimulating ferroptosis. Collectively, our findings indicated that shikonin induced OS cells ferroptosis through Nrf2/xCT/GPX4 regulatory axis, which might shed light on exploiting shikonin as a promising candidate for the future OS therapy.

Corilagin Induces ROS-mediated Apoptosis and Triggers Cell-Cycle Arrest at G0/G1 Stage in Osteosarcoma Cells

Background Osteosarcoma (OS) is an extremely aggressive primary bone cancer (BC) malignancy. A variety of malignancies can develop in the bones, including BC. Primary BCs are tumors that start in the bone. Bones can also become affected by tumors that start in the body’s organs or other tissues. Objectives Several reports suggested that corilagin (CL) exerts anticancer properties on various kinds of tumor cells; however, its molecular action on OS cells remains undefined. Materials and Methods The CL activity of OS cells’ cytotoxicity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, and cell-cycle distribution was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA), Rh-123, acridine orange and ethidium bromide (AO/EB), 4′,6-diamidino-2-phenylindole (DAPI), and flow cytometry analysis. Results Results revealed that CL could suppress OS cell proliferation via enhanced intracellular ROS, and MMP loss, and triggered apoptosis in a dose-dependent has an inferior manner. Our findings demonstrated that CL alleviates U2OS and MG-63 cell proliferation by the ROS-mediated apoptosis, which triggers G0/G1 cell-cycle arrest. Conclusion Thus, CL might be a protective therapeutic agent against BC.

Co-delivery of curcumin and si-STAT3 with a bioinspired tumor homing for polydopamine nanoparticles for synergistic osteosarcoma therapy

PurposeOwing to the complexity of cancer, a synergistic combination of chemotherapy and gene therapy can be a promising therapeutic strategy. This study aimed to use stem cell membrane (SCM)-camouflaged polydopamine nanoparticles for simultaneous delivery of curcumin (CUR) and siRNA-targeting STAT3 (CPDA/siSTAT3@SCM NPs) for osteosarcoma (OS).MethodsTransmission electron microscopy, UV–Vis absorbance spectra, zeta potential, cell co-localization, and Coomassie bright blue staining were used to characterize CPDA/siSTAT3@SCM NPs constructed by the self-assembly method. Drug release, cellular uptake, cell proliferation, apoptosis, wound healing, and transwell assays were evaluated in vitro. The expression levels of epithelial–mesenchymal transition (EMT)- and apoptosis-related proteins were measured by western blotting. Furthermore, the biodistribution, antitumor efficacy, and biosafety of CPDA/siSTAT3@SCM NPs in an MG63 xenograft mouse model were evaluated.ResultsCPDA/siSTAT3@SCM NPs were successfully synthesized to deliver CUR and siRNA simultaneously, and they showed osteosarcoma-targeting ability. Furthermore, it showed high cellular uptake and excellent synergistic antitumor effects in vitro. CPDA/siSTAT3@SCM NPs suppressed OS cell proliferation, migration, invasion, and EMT progression, and promoted the apoptotic process. In tumor-bearing mice, the treatment with CPDA/siSTAT3@SCM NPs showed an excellent antitumor effect with no side effects in major organs.ConclusionThis study revealed that CPDA/siSTAT3@SCM NPs can target drug delivery by biomimetic multifunctional nanoparticles to treat OS through chemo-gene combined therapy.

Retraction: MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7.

Retraction: MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7.

Retraction: SRCIN1 Suppressed Osteosarcoma Cell Proliferation and Invasion.

Retraction: SRCIN1 Suppressed Osteosarcoma Cell Proliferation and Invasion.

PITX1 suppresses osteosarcoma metastasis through exosomal LINC00662-mediated M2 macrophage polarization

Paired-like homeodomain transcription factor 1 (PITX1) is frequently downregulated in cancers, including osteosarcoma (OS). However, its role in OS remains unknown. Therefore, we aimed to explore the functions and potential mechanisms of PITX1 in OS malignant progression. Elevated PITX1 suppressed OS cell proliferation and migration, based on transwell, proliferation, and colony formation assays. Pathway enrichment analysis of differentially-expressed genes between PITX1-overexpressing and control OS cells indicated that PITX1 expression was associated with the FAK/Src and PI3k/Akt signaling pathways. Mechanistically, ubiquitination assays and rescue experiments showed that PITX1 interacted with transcription factor STAT3, leading to decreased STAT3 transcriptional activity, which repressed the expression of LINC00662. Specific knockdown of LINC00662 reduced the tumor growth and invasion of OS cells induced by downregulated PITX1. Moreover, exosomal LINC00662, derived from PITX1 knockdown OS cell lines activated M2 macrophages in cell co-culture assays. M2 macrophage secreted several cytokines, among which CCL22 was found to cause OS cell EMT. Collectively, our data indicate that PITX1 suppresses OS cell proliferation and metastasis by downregulating LINC00662. Moreover, LINC00662 can be packaged into OS cell-derived exosomes to mediate M2 macrophage polarization to promote OS metastasis via CCL22.

LncRNA PCED1B-AS1 knockdown inhibits osteosarcoma via methylation-mediated miR-10a downregulation

BackgroundLncRNA PCED1B-AS1 (PCED1B-AS1) promotes glioma. This study aimed to investigate its role in osteosarcoma (OS).MethodsThe study included 60 OS patients. Accumulation of miR-10a and PCED1B-AS1 in tissues from OS patients and cell lines was determined by RT-qPCR. Cell transfections were performed for interaction analysis. Participation of PCED1B-AS1 siRNA silencing and miR-10a overexpression in proliferation, invasion, and migration of U2OS and MG-63 cells was analyzed by cell proliferation assay and Transwell assay.ResultsPCED1B-AS1 level was increased in OS and positively correlated with miR-10a level. In OS cells, PCED1B-AS1 siRNA silencing downregulated miR-10a. Methylation-specific PCR analysis showed that PCED1B-AS1 siRNA silencing decreased the methylation of miR-10a gene promoter. Moreover, PCED1B-AS1 siRNA silencing suppressed OS cell proliferation, invasion, and migration. In addition, miR-10a overexpression attenuated the effects of PCED1B-AS1 siRNA silencing.ConclusionPCED1B-AS1 knockdown may inhibit OS cell proliferation and movement by regulating miR-10 gene methylation.

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.

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.

α-Mangostin suppresses proliferation and invasion in osteosarcoma cells via inhibiting fatty acid synthase

• α-mangostin was able to induce human osteosarcoma cells apoptosis via downregulating fatty acid synthase (FASN) expression in dose-dependent manner. • α-mangostin, as a FASN inhibitor, plays a great role in regulated ER stress-related protein and signal protein expression. • α-mangostin induced FASN inhibition, which might influence osteosarcoma progression, invasion and migration. Fatty acid synthase (FASN) is highly expressed in multiple types of human cancers and is recognized as one of the therapeutic targets for treating cancer metastasis. α-Mangostin is a natural xanthone extracted from mangosteen fruit, and possesses various biological activities. In our previous studies, α-mangostin was found to inhibit FASN activity. The present study was designed to reveal the effects of α-mangostin on osteosarcoma and to reveal whether the mechanism of α-mangostin in anticancer activity is related to FASN inhibition. Cytotoxicity was assessed in osteosarcoma MG63, 143B and U2OS cells. Cell viability was detected by an MTT assay. The protein expression levels were detected by western blotting. Flow cytometry, Annexin V/propidium iodide dual staining and Hoechst 33258 staining were performed to assess the apoptotic effects. Wound healing and Transwell assays were used to detect the inhibitory effect of α-mangostin on osteosarcoma cells invasion and migration. We found that α-mangostin blocked FASN expression, inhibited osteosarcoma cell proliferation, invasion and migration in a dose-dependent manner. In addition, α-mangostin regulated endoplasmic reticulum transmembrane receptors and signal protein expression in osteosarcoma cells. These findings suggested that α-mangostin suppresses osteosarcoma cell proliferation and metastasis by inhibiting FASN expression, which provides a basis as a potential target for osteosarcoma treatment.

Downregulation of hsa_circ_0000885 suppressed osteosarcoma metastasis and progression via regulating E2F3 expression and sponging miR-16-5p.

IntroductionAccumulating evidence has shown that circular RNAs (circRNAs) have indispensable functions during tumor progression by regulating gene expression. A previous study found that upregulation of hsa_circ_0000885 indicated a poor clinical outcome of osteosarcoma (OS). However, the regulatory mechanism of this process is unclear.MethodsThis investigation aimed to elucidate how hsa_circ_0000885 regulated OSs. The study used RT-qPCR to investigate hsa_circ_0000885 expression in OS cells. We conducted luciferase reporter assays and analyses to confirm the hsa_circ_0000885 downstream target. We transfected OS cells using different vectors and used Transwell migration, colony formation, western blotting, Matrigel invasion, proliferation, in vivo tumorigenesis, and metastasis assays to identify the role of hsa_circ_0000885 in OS.ResultsThe results showed that hsa_circ_0000885 expression altered OS cell lines, and that hsa_circ_0000885 downregulation suppressed OS cell proliferation and invasion using in vivo and in vitro experiments. Luciferase reporter assays verified that miR-16-5p and E2F3 were downstream targets of hsa_circ_0000885. E2F3 overexpression or miR-16-5p inhibition reversed OS cell invasion and proliferation after silencing hsa_circ_0000885. Furthermore, hsa_circ_0000885 affected cancer stem cell differentiation by regulating miR-16-5p/E2F3.ConclusionsOverall, the results showed that hsa_circ_0000885 downregulation suppressed OS progression and metastasis via regulating E2F3 expression and sponging miR-16-5p.

Convallatoxin suppresses osteosarcoma cell proliferation, migration, invasion, and enhances osteogenic differentiation by downregulating parathyroid hormone receptor 1 (PTHR1) expression and inactivating Wnt/β-catenin pathway

ABSTRACT Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Convallatoxin, a natural cardiac glycoside, exhibits potent anti-tumor activities. Literature has confirmed that PTHR1 is highly expressed in OS tissues and cells and downregulation of PTHR1 could decrease the invasion and growth of OS cells and increase tumor differentiation. In addition, PTHR1 could activate Wnt signaling pathway to promote the malignant functions of OS. In the present study, MG63 and U2OS cells were treated with 0, 12.5, 25, and 50 nM convallatoxin in order to elucidate the precise function of convallatox on the malignant behaviors of OS cells. Moreover, MG63 and U2OS cells treated with convallatoxin were transfected with Ov-PTHR1 or sh-DKK1, aiming to explore whether convallatoxin impeded the malignant progression of OS by modulating PTHR1 and Wnt/β-catenin pathway. CCK-8, wound healing and transwell assays were employed to assess the proliferation, migration, and invasion of OS cells. Differentiation markers (collagen 1, osteopontin, RANKL, Runx2, osteocalcin) were measured to evaluate OS cell differentiation. Results illuminated that convallatoxin suppressed proliferation, migration, and invasion as well as promoted osteogenic differentiation of OS cells. Besides, convallatoxin inhibited PTHR1 expression and inactivated Wnt/β-catenin pathway and PTHR1 overexpression activated Wnt/β-catenin pathway. Furthermore, PTHR1 overexpression or DKK1 knockdown reversed the suppressing effects of convallatoxin on OS cell proliferation, migration, and invasion, as well as the enhancing effect of convallatoxin on OS cell osteogenic differentiation. Collectively, convallatoxin may repress the malignant progression of OS by blocking PTHR1 and Wnt/β-catenin pathway.