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Objective:To investigate the effect and mechanism of RNaseH-1 on the radiosensitivity of the osteosarcoma cells via the alternative lengthening of telomeres (ALT) mechanism to maintain the telomere length.Methods:ALT osteosarcoma cell U2OS and telomerase-positive osteosarcoma cell 143B over-expressing RNaseH-1 were constructed by lentiviral transfection. After cell transfection, cell proliferation and cell cycle were determined using CCK-8 assay and flow cytometry. The effect of RNaseH-1 on the radiosensitivity of osteosarcoma cells was examined by colony formation assay. DNA injury (γ-H 2AX foci) was assessed by immunofluorescent assay. The expression levels of related proteins were detected by Western blot. Results:The proliferation abilities of U2OS cells were significantly declined following the over-expression of RNaseH-1, and G 1 cell cycle arrest was noted (all P<0.05). Over-expression of RNaseH-1 in U2OS cells increased the phosphorylated levels of ATM and Chk 2, down-regulated the expression of homologous recombination (HR)-related proteins RAD51 and BRCA1significantly aggravated DNA damage and remarkably enhanced the radiosensitivity (all P<0.05). Over-expression of RNaseH-1 exerted no inhibitory effect upon the telomerase-positive 143B cells ( P>0.05). Conclusion:RNaseH-1 over-expression suppresses telomerase-negative osteosarcoma cells and enhances the radiosensitivity probably via the role of RNaseH-1 in inhibiting the homologous recombination repair and activating the ATM signaling pathway.
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Objective To investigate the effect of microRNA361-5p on radiosensitivity of osteosarcoma cells and its downstream regulatory mechanisms.Methods The radioresistant osteosarcoma cell line HOS-R was constructed and the expression of microRNA-361-Sp in HOS and HOS-R cells was detected by RT-qPCR.The survival rate and apoptosis rate were detected by clone formation assay and flow cytometry in HOS-R cells treated with up-regulated or down-regulated of miR-361-Sp,or FOXM1 depletion.Dual fluorescent luciferase reporter and western blot assays were used to measure the relationship between miR-361-5p and FOXM1.he effects of miR-361-5p on radiosensitivity of osteosarcoma cells were determined by cloning formation assay and flow cytometry.Results RT-qPCR results showed that miR-361-5p was low expressed in HOS-R cells.Colony formation and flow cytometry assays demonstrated that overexpression of miR-361-5p significantly decreased the survival rate and increased the apoptosis rate of HOS-R cells.In contrast,FOXM1 downregulation inhibited cell survival rate and induced apoptosis.Moreover,miR-361-5p negatively regulated FOXM1 expression.Besides,FOXM1 overexpression attenuated the miR-361-5p upregulation-mediated promotion of radiosensitivity in HOS-R cells.Conclusion miR-361-5p was involved in the radiosensitivity of osteosarcoma cells by inhibiting FOXM1.
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Objective To investigate the effects of inositol hexaphosphate (IP6) on proliferation of human osteosarcoma cell line MG-63 and primary cultured osteoblasts so as to explore the optimal concentration for achieving anti-cancer effects.Methods We primary cultured and identified human osteoblasts.Then we made recovery and normal culture of human osteosarcoma cell line MG-63.We tested the proliferation of two kinds of cell lines under different concentrations of IP6 by MTT to determine the optimal concentration and then detected MG-63 cell cycle and apoptosis by flow cytometry.Results When IP6 concentration was more than 1 mmol/L,IP6 began to inhibit the proliferation of MG-63 cell line in the time-dose dependent manner.When the concentration reached 4 mmol/L,this inhibitory effect was the maximum.When IP6 concentration was 0.5 mmol/L or 1 mmol/L,the proliferation of osteoblasts was not obviously inhibited.When it was 2 mmol/L,the proliferation was slightly inhibited.A concentration of 4 mmol/L caused the apoptosis of osteoblasts.Conclusion IP6 can inhibit the proliferation of osteosarcoma cell line MG-63 and lead to its apoptosis.The optimal concentration is 2 mmol/L for achieving anti-cancer effects.
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Objective To investigate the inhibition of Hedyotic diffusa injection on the proliferation of hu-man osteosarcoma cell line MG -63 in vitro.Methods The human osteosarcoma cell line MG -63 was subcultivated aseptically in vitro.Different concentration of Hedyotic diffusa injection (50μL/mL,100μL/mL,200μL/mL,400μL/mL) successively acted on such cell .A total three time points were selected to determine the activity and numbers of cells by MTT assay including 12h,24h and 48h.Results The cellular proliferation inhibition rates of human osteosarcoma cell line MG-63 in drug groups of 50μL/mL concentration holes were (2.87 ±2.22)%,(13.42 ±2.14)% and (30.80 ±3.67)%after 12 h,24 h and 48 h.The rates of 100μL/mL concentration holes were (22.25 ±1.58)%, (43.34 ±2.84)%and (66.46 ±2.64)%,after 12 h,24 h and 48 h.The rates gradually increased and had statis-tical meaning,t12h =12.319,t24h =14.573,t48h =12.319,P<0.05;the cell in drug groups of 200μL/ml and 400μL/ml concentration holes was totally dead , and pathological findings showed that there were circular and floating cells in which nucleoplasmic ratio decreased and small fragments of cells increased .Conclusion Hedyotic diffusa extract has a certain inhibition in vitro on the proliferation of human osteosarcoma cell line MG -63.Moreover,the strength of its inhibition is relevant probably with drug concentration ,which deserves a further research .
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Objective To study the inhibitory effect of fluorouracil combined with DDP on the growth of human osteosarcoma cell line MG-63,and to explore its influence on the expressions of transient receptor potential vanilloid 5 (TRPV5 )and transient receptor potential vanilloid 6 (TRPV6 )proteins.Methods The MG-63 cells were cultured by the density of 5 × 104 mL-1 .Fluorouracil group,DDP group,fluorouracil+ DDP group and control group containing 10% FBS were set up.The inhibitory rates of growth of MG-63 cells at different time were detected by CCK-8 assay.The apoptosis of MG-63 cells after treated with different drugs was determined by Hoechst staining Kit.The immunocytochemical staining was used to treatent to detect the expressions of TRPV5 and TRPV6 before and after treatment.Results Fluorouracil and DDP both inhibited the growth of MG-63 cells in a time-and dose- dependent manner.There were a lot of black particles in the MG-63 cells and the cells were smaller,aging or death when they were exposed to fluorouracil or DDP.Compared with 24 h group,the inhibitory rates of proliferation of MG-63 cells after treated with the sigle drug of fluorouracil or DDP for 48 and 72 h were increased significantly (P <0.05).Compared with control group,the apoptotic rates of MG-63 cells in fluorouracil group and DDP group 24,48,and 72 h after treatment were increased (P < 0.01)in a time-dependent manner. The expression levels of TRPV5 and TRPV6 in MG-63 cells 72 h after treatment of fluorouracil and DDP were decreased significantly compared with before treatment (P < 0.05 ). Conclusion Fluorouracil, DDP and fluorouracil combined with DDP could significantly inhibit the proliferation of MG-63 cells,induce the apoptosis, and decrease the expression levels of TRPV5 and TRPV6.
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AIM:To investigate the effect of Notch-1 knockdown on the growth of dihydroartemisinin-inhibited human osteosarcoma cell line U-2OS.METHODS:U-2OS cells treated with different concentrations of dihydroartemisinin (5, 10, 15 and 20μmol/L) were collected.The expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively.U-2OS cells were transfected with Notch-1 siRNA for 24 h and incubated with dihydroartemisinin for another 24 h.The cell apoptotic rate , protein expression of MMP-2, MMP-9 and Hes-1, and the migration ability were measured by MTT assay , Western blotting and Transwell experiment , respectively.RESULTS:Dihydroartemisinin (5, 10, 15 and 20 μmol/L) decreased the expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels in a dose-dependent manner .Down-regulation of Notch-1 significantly en-hanced the effect of dihydroartemisinin on the cell apoptosis , the protein expression of MMP-2, MMP-9 and Hes-1, and mi-gration ability ( P<0.05 ) .CONCLUSION: Notch-1 pathway is involved in the process of dihydroartemisinin-inhibited U-2OS cell growth.Knockdown of Notch-1 augments the inhibitory effect of dihydroartemisinin on U-2OS cell viability.
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Objective To investigate the effect of miR-30a on human osteosarcoma cell 143B in migration,invasion andcellviability.Methods 143BcellswereinfectedortransfectedwithrecombinantadenovirusmiR-30a(Ad-miR30a) and miR-30a inhibitor respectively .Wound healing assay was performed to detect the cell healing ability ( P<0.05 ) .Cell migration and invasion ability were determined by Transwell assay ( P<0.05 ) .The cell viability was analyzed by MTT assay ( P<0.01 ) .Real-time quantitative PCR was performed to analyze the expression of RUNX2 mRNA level and confirmed the adenovirus miR-30a expressed in 143B cells.The expression of RUNX2 was analyzed by Western blot .miR-30a target to RUNX2 was verified by luciferase reported gene assay .Results The ability of migration and invasion was suppressed in osteosarcoma cell 143B by overexpression miR-30a,and the cell viability also decreased .After the endogenous miR-30 a being inhibited , the cell motility and invasion enhanced and the cell viability was promoted .The RUNX2 protein decreased after overexpression miR-30 a as compared with controlgroup.TheluciferaseactivityofRUNX2decreasedbyaddingmiR-30a.Conclusions 143Bcellmigration, invasion and viability were suppressed by miR-30a,and this process is potentially achieved via suppressing RUNX 2 protein expression .
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AIM:To investigate the effect of Ginsenoside Rh2 (Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS:The cell viability was determined by MTT assay .MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining.The expression of Bcl-2, Bax, cytochrome C ( Cyt C) and cleaved caspase-3 were measured by Western blot .RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner.Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased , while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group ( P<0.05 ) .The protein level of cleaved caspase-3 was also increased (P<0.05).CONCLUSION:Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway , suggesting that Rh2 is a novel approach for the treatment of osteosarcoma .
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The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca2+]o) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca2+]i) was increased by an increment of [Ca2+]o. This [Ca2+]i increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca2+]o- induced [Ca2+]i increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR- related disorders in the body.
Subject(s)
Humans , Bone Neoplasms , Calcium/metabolism , Caveolins/metabolism , Cell Fractionation , Cell Line, Tumor , Cell Membrane/metabolism , Microscopy, Confocal , Oligoribonucleotides, Antisense/pharmacology , Osteosarcoma , Receptors, Calcium-Sensing/antagonists & inhibitors , Up-RegulationABSTRACT
Objective To study the effects of 50 Hz magnetic fields on the in vitro proliferation of human osteosareoma cell line MG-63 with different cell densities.Methods Four different magnetic intensities(1 mT, 2 mT,3 roT,4 mT)were used to stimulate the cells,and the experiment was repeated with different cell densities. The method of MTT was employed to evaluate the level of proliferation.Results Fifty Hz magnetic fields signifi- cantly affected the level of proliferation of human osteosareoma cell line MG-63,and the 2 mT intensity exerted the greatest influence on it.The effects of the magnetic field differed with different cell densities.Conclusion The effect of 50 Hz magnetic fields on the in vitro proliferation of human osteosarcoma cell line MG-63 was not only relat- ed to the magnetic intensity,but also the cell density,
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HCMV infection can evoke the broad spectrum of symptoms, which may be caused by the infection of responsible cell types. It is important to identify the cell types to be infected and replicated with HCMV infection for characterizing the property of HCMV infection and symptoms. Bone marrow stroma consists of heterogeneous cells, which have many cellular functions. This study was performed to verify the infectivity of HCMV to osteoblasts using the osteogenic sarcoma cell line, Saos-2, and the effect of HCMV infection to them on the cellular function. Immediate-early antigens, IE1 and IE2, were detected from 1 day postinfection (d.p.i.), and early (ppUL44) and late (gB) antigen were detected from 2 d.p.i. by the immunoperoxidase staining. All the antigens were expressed as far as observed (9 days). It was found that the virus titer in the culture supernatant and the cell pellet were 150 to 2,200 pfu/ml and 50 to 800 pfu/ml, respectively, after 4 days when the cells were infected with 2 m.o.i. Alkaline phosphatase production in Saos-2 cells infected with the different amount of HCMV was decreased to 8 to 15%, 31 to 47%, and 11 to 52% on 4, 6, and 11 d.p.i., respectively, as compared with mock-infected cells. This result suggested that HCMV could replicate in some bone marrow stromal cells and disturb the cellular function such as production of alkaline phosphatase.
Subject(s)
Humans , Alkaline Phosphatase , Bone Marrow , Cell Line , Cytomegalovirus , Mesenchymal Stem Cells , Osteoblasts , Osteosarcoma , Permissiveness , Viral LoadABSTRACT
Objective To determine the expression of insulin receptor substract(IRS) family in human osteosarcoma cell line MG-63 and cultured normal human osteoblast-like cells (HOB). Methods The mRNA and protein expression of IRS family was measured using semi-quantitative RT-PCR and western blot analysis, respectively. Results MG-63 cells and HOB had the mRNA and protein expressions of IRS-1,-2,-3, and -4. Among IRS family, the expressions levels of IRS-1 mRNA and protein were the highest, and those of the IRS-4 mRNA and protein were the lowest in MG-63 cell line and HOB. Conclusion MG-63 and HOB can express the mRNA and protein of IRS family members, and the expressional levels of IRS family members were different.