Proliferation Of Human Osteosarcoma Cells Research Articles

Echinatin inhibits the growth and metastasis of human osteosarcoma cells through Wnt/β-catenin and p38 signaling pathways

Osteosarcoma (OS) is a highly aggressive malignant bone tumor that mainly occurs in adolescents. At present, chemotherapy is the most commonly used method in clinical practice to treat OS. However, due to drug resistance, toxicity and long-term side effects, chemotherapy can’t always provide sufficient benefits for OS patients, especially those with metastasis and recurrence. Natural products have long been an excellent source of anti-tumor drug development. In the current study, we evaluated the anti-OS activity of Echinatin (Ecn), a natural active component from the roots and rhizomes of licorice, and explored the possible mechanism. We found that Ecn inhibited the proliferation of human OS cells and blocked cell cycle at S phase. In addition, Ecn suppressed the migration and invasion, while induced the apoptosis of human OS cells. However, Ecn had less cytotoxicity against normal cells. Moreover, Ecn inhibited the xenograft tumor growth of OS cells in vivo. Mechanistically, Ecn inactivated Wnt/β-catenin signaling pathway while activated p38 signaling pathway. β-catenin over-expression and the p38 inhibitor SB203580 both attenuated the inhibitory effect of Ecn on OS cells. Notably, we demonstrated that Ecn exhibited synergistic inhibitory effect with cisplatin (DDP) on OS cells in vitro and in vivo. Therefore, our results suggest that Ecn may exert anti-OS effects at least partly through regulating Wnt/β-catenin and p38 signaling pathways. Most meaningfully, the results obtained suggest a potential strategy to improve the DDP-induced tumor-killing effect on OS cells by combining with Ecn.

Cinobufacini injection suppresses the proliferation of human osteosarcoma cells by inhibiting PIN1-YAP/TAZ signaling pathway.

Cinobufacini injection (CI), an aqueous extract of Cutis Bufonis, is clinically used for cancer therapy in China, but its molecular mechanism for the treatment of osteosarcoma (OS) remains unclear. We constructed U2OS ectopic subcutaneous tumor model to verify the anti-OS effect of CI in vivo. Meanwhile, cell proliferation of U2OS and MG63 cells was monitored in vitro using the CCK-8 assay, colony formation and morphological changes. Cell cycle arrest and apoptosis were detected by flow cytometry and western blot, which showed that CI significantly inhibited proliferation, induced cell cycle arrest and apoptosis in human OS cells. The further RNA-seq results identified that the Hippo signaling pathway was involved in the anti-OS effect of CI. YAP/TAZ are two major components of the Hippo pathway in breast cancer and are positively regulated by prolyl isomerase PIN1, we assessed their role in OS using both clinicopathological sections and western blots. CI also inhibited PIN1 enzyme activity in a dose-dependent manner, which resulted in impaired PIN1, YAP, and TAZ expression in vitro and in vivo. Additionally, 15 potential compounds of CI were found to occupy the PIN1 kinase domain and inhibit its activity. In summary, CI plays an anti-OS role by down-regulating the PIN1-YAP/TAZ pathway.

PSKH1 affects proliferation and invasion of osteosarcoma cells via the p38/MAPK signaling pathway.

Malignant osteosarcoma (OS) is a tumor of bone and soft tissue that metastasizes early and has a high mortality rate. Protein serine kinase H1 (PSKH1), an autophosphorylating human protein serine kinase, controls the trafficking of serine/arginine-rich domain, with downstream effects on mRNA processing. It is also associated with tumor progression. However, how this protein contributes to OS progression and metastasis is unknown. The present study evaluated the potential effect of PSKH1 on proliferation of human OS cells. OS cell lines were used in Cell Counting Kit-8, colony formation, wound-healing and Transwell assays, to investigate cellular processes such as proliferation, migration and invasion and underlying molecular mechanisms. Expression of PSKH1 in OS tissue was significantly greater than in adjacent non-malignant tissue. PSKH1 knockdown inhibited the proliferation, migration and invasion of OS cells. Conversely, PSKH1 overexpression promoted proliferation of OS cells. PSKH1 upregulated phosphorylated-p38 in OS cells. Moreover, the p38 MAPK inhibitor SB203580 effectively blocked the tumor-promoting action of PSKH1. Furthermore, PSKH1 knockdown inhibited tumor growth and metastasis in vivo. In conclusion, these findings suggested that PSKH1 promoted OS proliferation, migration and invasion. Thus, PSKH1 may serve an oncogenic role in the development of human OS.

ML792 inhibits growth and TGF-β1-induced EMT of osteosarcoma cells via TGF-β1/Smad and PI3K/AKT pathways

Osteosarcoma (OS), a common bone malignancy, is highly metastatic and featured by poor prognosis. SUMOylation is deeply involved in tumorigenesis and cancer progression. SUMO-activating enzyme (SAE) inhibitor has been found to play an anti-proliferative role in multiple cancers. However, their functions in osteosarcoma remain unknown. Based on this, it was hypothesized that ML792, an SAE inhibitor, might be an anti-tumor candidate against osteosarcoma in this paper. The role of ML792 in the treatment of osteosarcoma was investigated in vitro and in vivo. The results showed ML792 significantly arrested human osteosarcoma cells (HOCs) at the G2/M phase, inhibited the proliferation of HOCs, and elicited apoptosis. To better assess the inhibitory role of ML792 in human osteosarcoma, we induced HOCs with TGF-β1. Our study showed that ML792 significantly suppressed the migration, invasion, and EMT of HOCs induced by TGF-β1. Moreover, ML792 inhibited TGF-β1-induced phosphatidylinositol- 3-kinase (PI3K) and AKT activation, as well as Smad2/3 phosphorylation in HOCs. In vivo, ML792 inhibits the growth of osteosarcoma. Thus, ML792 is a promising new antitumor agent to suppress the progress of OS cells undergoing proliferation and EMT by targeting the PI3K/Akt and TGF-β1/Smad pathway and may prevent or reduce the growth and metastasis 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.

Andrographolide inhibits the growth of human osteosarcoma cells by suppressing Wnt/β-catenin, PI3K/AKT and NF-κB signaling pathways

Osteosarcoma (OS) is an aggressive malignant skeletal tumor characterized by an extremely poor prognosis and a high tendency to recur. The frequently used anti-OS chemotherapy regents are often limited by drug resistance and severe adverse events. It is urgent to develop more effective, tolerable and safe drugs for the treatment of OS. Andrographolide (AG), a diterpenoid lactone isolated from Andrographis paniculata, has been proved to possess anti-tumor activity against several human cancer types. In this current study, we evaluated the inhibitory effect of AG on human OS cells and probed the possible mechanism. We found that AG inhibited the proliferation of human OS cells and blocked cell cycle at G2/M phase. Furthermore, AG impeded the migration and invasion, while promoted the apoptosis of human OS cells. Moreover, we found that AG inhibited OS growth and lung metastasis in orthotopic transplantation model. Mechanistically, we demonstrated that AG suppressed the activity of Wnt/β-catenin, PI3K/AKT and NF-κB signaling pathways. Notably, we validated that AG synergized with the inhibitors of Wnt/β-catenin, PI3K/AKT and NF-κB to suppress the proliferation, migration and invasion of human OS cells. Collectively, our study conclusively demonstrates that AG inhibits the growth of human OS cells, thus, may be a promising candidate for the treatment of OS.

CTGF/CCN2 promotes the proliferation of human osteosarcoma cells via cross-talking with the stromal CXCL12/CXCR4-AKT-αvβ3 signaling axis in tumor microenvironment

CTGF/CCN2 promotes the proliferation of human osteosarcoma cells via cross-talking with the stromal CXCL12/CXCR4-AKT-αvβ3 signaling axis in tumor microenvironment

Effects of butein on human osteosarcoma cell proliferation, apoptosis, and autophagy through oxidative stress.

Osteosarcoma (OS) is a primary malignant bone tumor, and the cure rate has stagnated in the past three decades. Butein, a plant polyphenol extracted from many herbs, has been proved to possess anti-tumor activity. However, the effect of butein on human OS and the underlying mechanisms remain to be elucidated. The OS cell line 143B was used. The effects of butein were evaluated through the cell proliferation assay, flow cytometry, florescence and transmission electron microscopy, and western blotting. All statistical analyses were performed using GraphPad Prism 7.0. Butein was found to inhibit cell proliferation by causing G2/ M phase arrest in the 143B cells. In addition, butein suppressed the invasion of 143B cells upon IL-6 treatment. Additionally, we found that butein inhibited the invasion of 143B cells stimulated with IL-6 via the p-STAT3-MMP9 signaling pathway. Remarkably, butein triggered extrinsic and intrinsic apoptosis and autophagy of 143B cells. The process of autophagy may have tumor-supporting effects. Furthermore, butein induced oxidative stress as evidenced by ROS generation, increase in malondialdehyde (MDA) level, and decrease in GSH/GSSH ratio and GPX4 expression. N-acetylcysteine can reverse the change of ROS. Further experiments indicated apoptosis and autophagy could be attenuated by the N-acetyl-L-cysteine and c-Jun N-terminal kinase (JNK) inhibitor SP600125. Additionally, butein inhibited the Akt/mammalian target of rapamycin (mTOR) signaling pathway, and suppressed the Akt kinase activity increased apoptosis and autophagy. Our results revealed butein induced apoptosis and autophagy by regulating oxidative stress, activating the JNK signaling pathway and blocking the Akt/mTOR signaling pathway in OS cells. Additionally, butein inhibited the invasion of 143B cells stimulated with IL-6 through the pSTAT3- MMP9 signaling pathway. In view of these results, butein may be a potential anti-tumor drug targeting osteosarcoma.

Circle RNA circCSPP1 promotes human osteosarcoma cell proliferation and increases glucose metabolism by suppressing miR-200c maturation.

MiR-200c plays a central role in glucose metabolism in cancer cells. However, its upstream regulators in this process are unknown. CircRNA CSPP1 (circCSPP1) was predicted to bind to premature miR-200c, an oncogenic miRNA. Therefore, we explored their interaction in osteosarcoma (OS). Differential circCSPP1 and miR-200c expression in OS was analyzed using RT-qPCR. Glucose metabolism was analyzed by glucose uptake assay. Subcellular circCSPP1 location in OS cells was detected using cellular fractionation assay. The direct interaction between circCSPP1 and miR-200c was explored using RNA-RNA pull-down assay. The role of circCSPP1 in miR-200c maturation was investigated by analyzing both mature and premature miR-200c levels in OS cells with circCSPP1 overexpression. CircCSPP1 and premature miR-200c levels were increased while mature miR-200c level was decreased in OS. CircCSPP1 was detected in both the nuclear and cytoplasm fractions of OS cells. CircCSPP1 directly interacted with premature miR-200c. CircCSPP1 overexpression increased premature miR-200c level, glucose uptake, and cell proliferation, but decreased mature miR-200c level. MiR-200c overexpression suppressed the role of circCSPP1 in OS cells. CircCSPP1 promotes OS cell proliferation and increases glucose metabolism by suppressing miR-200c maturation.

SRPK1 promotes cell proliferation and tumor growth of osteosarcoma through activation of the NF-κB signaling pathway.

To explore the expression and the functions of SRPK1 in osteosarcoma, we retrieved transcription profiling dataset by array of human bone specimens from patients with osteosarcoma from ArrayExpress (accession E-MEXP-3628) and from Gene Expression Omnibus (accession GSE16102) and analyzed expression level of SRPK1 and prognostic value in human osteosarcoma. Then we examined the effect of differential SRPK1 expression levels on the progression of osteosarcoma, including cell proliferation, cell cycle, apoptosis, and investigated its underlying molecular mechanism using in vitro osteosarcoma cell lines and in vivo nude mouse xenograft models. High expression level of SRPK1 was found in human osteosarcoma tissues and cell lines as compared to the normal bone tissues and osteoblast cells, and predicted poor prognosis of human osteosarcoma. Overexpression of SRPK1 in osteosarcoma U2OS cells led to cell proliferation but inhibition of apoptosis. In contrast, knockdown of SRPK1 in HOS cells impeded cell viability and induction of apoptosis. Moreover, silencing SRPK1 inhibited osteosarcoma tumor growth in nude mice. Mechanistic studies revealed that SRPK1 promoted cell cycle transition in osteosarcoma cells and activation of NF-κB is required for SRPK1 expression and its pro-survival signaling. SRPK1 promoted human osteosarcoma cell proliferation and tumor growth by regulating NF-κB signaling pathway.

A c-Met Inhibitor Suppresses Osteosarcoma Progression via the ERK1/2 Pathway in Human Osteosarcoma Cells.

IntroductionOsteosarcoma is the most common primary malignancy of the bone among adolescents and children. Despite intensive chemotherapy and aggressive surgery, the 5-year survival rate of osteosarcoma still falls under 70%, mainly due to its tendency to metastasize and to develop drug resistance. Therefore, new treatments for osteosarcoma are urgently needed. HGF/c-Met signaling pathway, when dysregulated, is involved in the onset, progression and metastasis of various cancers, making the HGF/c-Met axis a promising therapeutic target.MethodsIn this study, we found Met to be a cancer-promoting gene in osteosarcoma as well, and aimed to investigate the role of a c-met inhibitor (PHA-665752) in osteosarcoma. For this purpose, two human osteosarcoma cell lines (143B and U2OS) were introduced in this study and treated with PHA-665752. CCK8 cell proliferation assay was performed to obtain the IC50 value of PHA-665752 for 143B and U2OS. After that, colony formation assay, transwell migration and invasion assay and wound-healing assay were performed. Furthermore, a tumor-transplanted mouse model was used for in vivo experiments.ResultsOur results showed that PHA-665752 could suppress osteosarcoma progression, promote apoptosis and inhibit proliferation of human osteosarcoma cells. Moreover, we found ERK1/2 pathway to be an important mediator underlying the osteosarcoma-suppressing function of PHA-665752. LY3214996, a highly selective inhibitor of the ERK1/2 pathway, was able to antagonize the effects of PHA-665752 in osteosarcoma. Finally, in vivo experiments indicated that PHA-665752 suppressed tumor growth in a tumor-transplanted mouse model.ConclusionTaken together, Met provided a druggable target for osteosarcoma and PHA-665752 is a promising candidate for anti-osteosarcoma treatments.

Ginsenoside Rg5 Inhibits Human Osteosarcoma Cell Proliferation and Induces Cell Apoptosis through PI3K/Akt/mTORC1-Related LC3 Autophagy Pathway.

The function and mechanism underlying the suppression of human osteosarcoma cells by ginsenoside-Rg5 (Rg5) was investigated in the present study. MG-63, HOS, and U2OS cell proliferation was determined by MTT assay after Rg5 treatment for 24 h. Rg5 inhibited human osteosarcoma cell proliferation effectively in a dose-dependent manner. The range of effective inhibitory concentrations was 160-1280 nM. Annexin V-FITC and PI double-staining assay revealed that Rg5 induced human osteosarcoma cell apoptosis. Western blotting, qRT-PCR, and FACS experiments revealed that Rg5 inhibited human osteosarcoma cells via caspase-3 activity which was related to the LC3-mediated autophagy pathway. Rg5 decreased the phosphorylation of PI3K, Akt, and mTORC1 activation. In contrast, LC3-mediated autophagy and caspase-3 activity increased significantly. A PI3K/AKT stimulator, IGF-1, reversed Rg5-induced cell autophagy and apoptosis in MG-63 cells. Collectively, the current study demonstrated that Rg5 induced human osteosarcoma cell apoptosis through the LC3-mediated autophagy pathway. Under physiological conditions, activation of PI3K/AKT/mTORC1 inhibits LC3 activity and caspase-3-related cell apoptosis. However, Rg5 activated LC3 activity by inhibiting the activation of PI3K/AKT/mTORC1. The present study indicated that Rg5 could be a promising candidate as a chemotherapeutic agent against human osteosarcoma.

Phosphotyrosine picked threonine kinase stimulates proliferation of human osteosarcoma cells in vitro and in vivo.

IntroductionOsteosarcoma (OS) is the most common primary bone tumor, and the main affected population is adolescents. The survival of OS patients was 10–20% when surgery was used as a single treatment. There is less basic research on OS than other tumors, and we need more ways to improve the survival rate. Phosphotyrosine picked threonine kinase (TTK) has been widely reported as an oncogene in multiple types of cancers, and it is also known as a clinical therapeutic target. This study aims to assess TTK expression levels in human OS tissues and its link with the clinical characteristics of OS patients, and to evaluate the potential role in OS development.Material and methodsImmunohistochemical (IHC) assays were conducted to detect the expression levels of TTK in a total of 74 OS tissues and the corresponding adjacent tissues. Furthermore, according to the staining intensity of TTK in tumor tissues, patients were divided into TTK high and low expression groups. The possible correlation between TTK expression levels and clinical features were analyzed, and the effects of TTK on OS cell proliferation were detected through colony formation and cell counting kit-8 (CCK8) assays. The effects of TTK on tumor growth were detected using an animal model.ResultsPhosphotyrosine picked threonine kinase was abnormally highly expressed in human OS tissues. Meanwhile, TTK was significantly correlated with the clinical characteristics such as tumor size (p = 0.004*) and clinical stage (p = 0.014*) of OS patients. Our results also revealed that the inhibition of TTK dramatically suppressed the proliferation of OS cells in vitro and blocked tumor growth in mice.ConclusionsWe demonstrated the involvement of TTK in the development of OS, and therefore we suggest that TTK should be considered as a promising therapy target for OS.

Asiatic Acid Induces Mitochondrial Apoptosis via Inhibition of JAK2/STAT3 Signalling Pathway in Human Osteosarcoma

Osteosarcoma (OS), a severe malignant bone tumour, usually occurs in adolescents and children and has a poor prognosis. Asiatic acid (AA), an active component isolated from Centella asiatica (L.) Urb., exhibits appreciable anti-oxidant and anti-tumour activities. So far, the effects and underlying mechanisms of AA against OS have not been clarified. Here, we explored the anti-tumour effects of AA against human OS and the involved mechanism mediating its actions. To evaluate effects of AA on the cell proliferation of human OS cells, cell viability and colony formation assays were performed. Flow cytometry was used to evaluate apoptosis in OS cells exposed to AA and mitochondrial membrane potential. Western blotting and RT-PCR were applied to determine expression of the relevant proteins and their mRNA levels. Our explorations showed that AA inhibits proliferation of human OS cells in a concentration- and time-dependent manner, and induces apoptosis of OS cells by the intrinsic (mitochondrial) pathway. Importantly, we found that inhibition of the AA-induced phosphorylation of JAK2/STAT3 signalling molecules and the decrease in MCL-1 contributed to the anti-tumour efficacy of AA. Collectively, our results suggest that AA could evoke mitochondrial- induced apoptosis in human OS cells by suppression of the JAK2/STAT3 pathway and MCL-1 expression. These results strongly demonstrate that AA could be a potential anti-tumour agent for OS treatment.

LncRNA FOXD2-AS1 knockdown inhibits the resistance of human osteosarcoma cells to cisplatin by inhibiting miR-143 expression.

The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) FOXD2-adjacent opposite strand RNA 1 (FOXD2-AS1) on the sensitivity of osteosarcoma cells to cisplatin and its possible underlying mechanism. Our findings might help to provide a certain reference for clinically preventing the drug resistance of osteosarcoma cells. Cisplatin with a certain concentration gradient was used to induce the stable acquired resistance of human osteosarcoma U2-OS cell line. Subsequently, the expression level of lncRNA FOXD2-AS1 was determined in osteosarcoma cells in non-resistance group (Control group) and Cisplatin-resistance group (Cisplatin-RES group), respectively. Next, the cell line with stable lncRNA FOXD2-AS1 knockdown was constructed in Cisplatin-RES group using small interfering RNA (siRNA). The effects of stable knockdown of lncRNA FOXD2-AS1 on the proliferation of human osteosarcoma cells and the half-maximal inhibitory concentration (IC50) of cisplatin were detected by Cell Counting Kit-8 (CCK-8) assay. 5-ethynyl-2'-deoxyuridine (EdU) staining was performed to measure deoxyribonucleic acid (DNA) replication level in each group of cells. The protein expression levels of apoptosis-associated genes B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) in each group of cells were measured via Western blotting. The migration and invasion abilities of cells in each group were determined using wound-healing assay and transwell assay. In addition, the expression of micro RNA (miR)-143 in each group of cells was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Compared with Control group, the expression level of lncRNA FOXD2-AS1 rose significantly in cells in Cisplatin-RES group (p<0.05). Knockdown of FOXD2-AS1 evidently decreased the IC50 of cisplatin in human osteosarcoma cells (p<0.05). According to EdU staining results, the knockdown of FOXD2-AS1 distinctly inhibited the proliferation of osteosarcoma cells (p<0.05). Western blotting results demonstrated that the knockdown of FOXD2-AS1 remarkably upregulated the expression of pro-apoptotic protein Bax and repressed that of anti-apoptotic protein Bcl-2 in drug-resistant human osteosarcoma cells (p<0.05). Moreover, the knockdown of FOXD2-AS1 significantly weakened the migration and invasion abilities of drug-resistant human osteosarcoma cells (p<0.05). Finally, it was found that the expression level of miR-143 was distinctly elevated in drug-resistant human osteosarcoma cells after knockdown of FOXD2-AS1 (p<0.05). LncRNA FOXD2-AS1 knockdown inhibits the resistance of human osteosarcoma cells to cisplatin, promotes their apoptosis and weakens their invasion and migration abilities. The possible underlying mechanism may be related to the inhibition of miR-143 expression by lncRNA FOXD2-AS1 in drug-resistant cell lines.

MiR-128-3p serves as an oncogenic microRNA in osteosarcoma cells by downregulating ZC3H12D.

Osteosarcoma is the second leading cause of cancer-associated mortality worldwide in children and adolescents. ZC3H12D has been shown to negatively regulate Toll-like receptor signaling and serves as a possible tumor suppressor gene. MicroRNAs (miRNAs/miRs) are known to play an important role in the proliferation of human osteosarcoma cells. However, whether miRNAs can affect tumor development by regulating the expression of ZC3H12D has not yet been investigated. The aim of the present study was to investigate the role of miR128-3p in regulating ZC3H12D expression, as well as its function in tumor cell proliferation, apoptosis, and metastasis. Reverse transcription-quantitative PCR, western blotting and dual luciferase reporter assays were performed to analyze the regulation of ZC3H12D expression by miR-128-3p. MTT, colony formation and flow cytometry assays were also used to analyze the effect of miR-128-3p on cell proliferation and apoptosis. A wound healing assay was performed to investigate the cell migration ability. The results demonstrated that miR-128-3p directly targeted ZC3H12D and downregulated its expression, thereby promoting cell proliferation and migration. miR-128-3p overexpression also improved resistance to cisplatin in MG-63 and 143B cell lines, supporting the hypothesis that miR-128-3p may function as an oncogene in osteosarcoma cells. The potential clinical significance of miR-128-3p as a biomarker and therapeutic target provides rationale for further investigation into the miR-128-3p-mediated molecular pathway and how it is associated with osteosarcoma development.

3D printed poly(lactic acid) scaffolds modified with chitosan and hydroxyapatite for bone repair applications

3D printed poly(lactic acid) (PLA) scaffolds surface modified with chitosan (CS) and hydroxyapatite (HA) to produce a novel bioactive composite scaffold is reported. Excellent mechanical properties of PLA, the bioactivity of CS, and osteogenic characteristics of HA are combined to fabricate composite scaffolds using a simple desktop 3D printer. Scaffolds were characterized through attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD) and water contact angle measurements before and after modification. Formic acid was used as a solvent to prepare stable CS/HA dispersions and was found to be a suitable solvent for producing PLA/CS/HA composites. Surface properties of modified scaffolds were superior in terms of hydrophilicity and bioactivity, which resulted in enhanced attachment and proliferation of human osteosarcoma cells in vitro compared to the unmodified PLA scaffolds.

MicroRNA-744 promotes proliferation of osteosarcoma cells by targeting PTEN

MicroRNAs (miRNAs/miRs) are non-coding RNAs that regulate protein synthesis by targeting mRNAs for translational repression or degradation. Previous studies have reported that aberrant expression of miR‑744 may be involved in human osteosarcoma; however, the underlying mechanisms remain elusive. In the present study, the expression levels of miR‑744 and its downstream signals were determined by reverse transcription‑quantitative PCR and western blotting. Cell proliferation was assessed using the bromodeoxyuridine assay, and the target of miR‑744 was investigated using a dual‑luciferase activity assay. The present study identified a significant upregulation of miR‑744 in osteosarcoma tissues compared with adjacent non‑tumor tissues. Furthermore, it was demonstrated that ectopic overexpression of miR‑744 induced by a miR‑744 precursor significantly enhanced proliferation of the osteosarcoma cell line MG63, whereas opposite results were observed following suppression of miR‑744 with its inhibitor. Moreover, as a unique anti‑oncogene, PTEN was identified as a direct target of miR‑744. It was confirmed that miR‑744 downregulated PTEN expression in MG63 cells by targeting the PTEN 3'untranslated region, and that the downstream AKT signal was also regulated by miR‑744. Collectively, the present results suggested that miR‑744 promoted proliferation of human osteosarcoma cells by directly regulating the PTEN/AKT signaling pathway.

Research of cryptochrome gene 1 in human osteosarcoma

Objective Aim at investigating the function and mechanism of cryptochrome gene 1 in human osteosarcoma. Methods The lentivirus vector with short hairpin RNA (shRNA) targeting CRY1 was transfected into human osteosarcoma cells (HOS). The knockdown of CRY1 in HOS was confirmed by Western blotting; the proliferation of HOS cells was detected by cell counting kit-8 (CCK-8) assay; the downstream molecules was confirmed by Western blotting; the mRNA expression level of clock gene cryptochrome gene (CRY1, CRY2), period gene (PER1, PER2), BMAL1, CLOCK was tested by quantitative real-time polymerase chain reaction (RT-qPCR). Results After the knockdown of CRY1, the proliferation of HOS was enhanced (0.839±0.020 vs. 0.553±0.019, P<0.01) and the canonical Wnt signaling was found activated (0.729±0.067 vs. 1.119±0.018, P<0.01), as well as the mRNA levels of all clock network key genes but CRY1 were increased. What’s more, proliferation of HOS cell was inhibited in rescue experiment (1.012±0.151 vs. 1.483±0.095 vs. 0.541±0.129, P<0.01). Conclusion CRY1 controls proliferation of human osteosarcoma cells via Wnt signaling pathway and regulates the expression levels of other clock network key genes, including CRY2, PER1, PER2, BMAL1 and CLOCK, which indicates that CRY1 plays an important role in human osteosarcoma progression. Key words: Cryptochrome gene 1; Wnt signaling pathway; Clock gene networks; Osteosarcoma

Effect of Hydroxyapatite Formation on Titanium Surface with Bone Morphogenetic Protein-2 Loading through Electrochemical Deposition on MG-63 Cells.

Calcium phosphate ceramics used in dentistry and orthopedics are some of the most valuable biomaterials, owing to their excellent osteoconduction, osteoinduction, and osseointegration. Osteoconduction and osteoinduction are critical targets for bone regeneration, and osseointegration is essential for any dental implantations. In this study, a hydroxyapatite (HAp) hybrid coating layer with the sequential release of bone morphogenetic protein 2 (BMP-2) was deposited onto an etched titanium substrate by electrochemical deposition. The resulting release of BMP-2 from Ti–HAp was assessed by immersing samples in a simulated buffer fluid solution. Through coculture, human osteosarcoma cell proliferation and alkaline phosphatase activity were assessed. The characteristics and effect on cell proliferation of the hybrid coatings were investigated for their functionality through X-ray diffraction (XRD) and cell proliferation assays. Findings revealed that −0.8 V vs. Ag/AgCl (3 M KCl) exhibited the optimal HAp properties and a successfully coated HAp layer. XRD confirmed the crystallinity of the deposited HAp on the titanium surface. Ti-0.8 V Ti–HAp co-coating BMP sample exhibited the highest cell proliferation efficiency and was more favorable for cell growth. A successful biocompatible hybrid coating with optimized redox voltage enhanced the osseointegration process. The findings suggest that this technique could have promising clinical applications to enhance the healing times and success rates of dental implantation.