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Objective To investigate the effect of fluoride exposure on expression of miRNA (miR)-200c and its target in human osteoblast Saos-2 cells.Methods Saos-2 cells were cultured in DMEM/F-12 medium and treated with fluoride (sodium fluoride,NaF).There were two groups including:control group (0 mg/L) and fluoride group (4 mg/L).Cells were harvested after 48 hours of culture with fluoride.The expression of miR-200c,the mRNA of alkaline phosphatase (ALP),osteocalcin (BGP),the target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and dual-specific phosphatase 1 (DUSP1) of miR-200c was detected by qRT-PCR.The protein expression of PTEN and DUSP1 was detected by Western blotting.Results The expressions of ALP,BGP mRNA and miR-200c in Saos-2 cells in the fluoride group (23.60 ± 1.87,9.41 ± 0.94,8.61 ± 0.26) were higher than those in the control group (1.00 ± 0.11,1.00 ± 0.07,1.00 ± 0.12).The differences were statistically significant (t =-24.084,-18.388,-8.687,P ＜ 0.05).The mRNA expressions of PTEN and DUSP1 in the fluoride group (0.63 ± 0.02,0.38 ± 0.02) were lower than those in the control group (1.02 ± 0.24,1.02 ± 0.24).The differences were statistically significant (t =3.327,5.454,P ＜ 0.05).The protein expressions of PTEN and DUSP1 in Saos-2 cells in the fluoride group (1.19 ± 0.10,0.83 ± 0.07) were lower than those in the control group (1.81 ± 0.14,1.44 ± 0.25).The differences were statistically significant (t =6.250,4.171,P ＜ 0.05).Conclusion Exposure to fluorine may increase the expression of miR-200c in Saos-2 cells,and fluorine may act on PTEN and DUSP1 through miR-200c,downregulates the mRNA and protein expression levels of PTEN and DUSP1.
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Objective Microenvironment plays important roles in the proliferation , viability, and apoptosis of tumor cells. This study was to investigate the effects of different functional groups on the biological characteristics of the osteosarcoma Saos -2 cell line in vitro. Methods Using self-assembled monolayers of alkanethiols on gold , we prepared different terminal chemical groups , including methyl (-CH3 ) , amino (-NH2 ) , hydroxyl (-OH) , and carboxyl (-COOH ) .We determined the similar density of different functional groups by contact angle measurement and x-ray photoelectron spectroscopy , and observed the effects of different functional groups on the adhesion , proliferation, viability, and apoptosis of the osteosarcoma Saos-2 cells by fluorescence microscopy , CCK-8 as-say, flow cytometry, and scan electron microscopy (SEM). Results The surface of -COOH and -NH2 promoted the adhesion and proliferation of the of the Saos-2 cells, with a good compatibility , while that of -CH3 was unfavorable for their adhesion and proliferation and even increased their apoptosis . The promoting effects of the functional groups on the adhesion and proliferation of the cells were listed in the following order: -COOH ≥ -NH2 >-OH -CH3 , while their toxicity and apoptosis-increasing effect ranked as -CH3 -OH >-NH2 >-COOH. Conclusion The-CH3 group inhibits the adhesion and proliferation and promotes the apoptosis of Saos-2 cells, which has provided some evidence for the surface design of biomaterials.
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Objective To investigate the effects of c?myc promoter binding protein 1(MBP?1)gene on the proliferation of human Saos?2 osteo?sarcoma cells in vitro. Methods Saos?2 cells were divided into three groups:blank control group(untransfected cells),negative group(cells transfected with missense sequence)and experimental group(cells transfected with MBP?1 shRNA). Two MBP?1 shRNA sequences and one neg?ative control shRNA sequence were designed ,synthesized and cloned into pSIREN?retroQ plasma. Then the recombinant plasmids were construct?ed and transfected into human Saos?2 osteosarcoma cells by Lipofectamine 2000. The expressions of MBP?1 mRNA and protein in Saos?2 cells were detected by real?time PCR and Western blot ,respectively. The effects of altered expression of MBP?1 on cell proliferation were measured by CCK?8 cell proliferation assay. The expressions of cyclin D1 and cyclin E in Saos?2 were determined by Western blot. Results PCR and sequenc?ing results indicated that the recombinant plasmids pSIREN?retroQ was constructed. The relative expression level of MBP?1 mRNA in the MBP?1 siRNA transfection group was significantly decreased than that in blank control group(P<0.05). Compared with the blank control group,the ex?pression levels of MBP?1 protein in the experimental group also significantly decreased. The proliferation abilities of Saos?2 cells at 48,72,and 96 hours after MBP?1 siRNA transfection were significantly increased than those in the blank control group(P<0.05). Compared with the blank con?trol group,the expression levels of cyclin D1 and cyclin E protein in the experimental group also significantly increased(P<0.05). Conclusion Knockdown of the expression of MBP?1 gene promotes the proliferation of human Saos?2 osteosarcoma cells. MBP?1 gene may become the new tar?get of gene therapy for osteosarcoma.
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AIM:ToexploretheeffectofcelastrolonapoptosisofSaos-2cellsanditsmechanism.METH-ODS:Saos-2 cells were treated with various concentrations of celastrol , and the cell viability was measured by MTT assay . Apoptosis and reactive oxygen species ( ROS) production were determined by flow cytometry .The protein levels of cleaved caspase-9, cleaved caspase-3 and phosphorylated JNK were evaluated by Western blot .RESULTS:The viability of Saos-2 cells was significantly inhibited by celastrol .Celastrol significantly induced apoptosis of Saos-2 cells.Celastrol signifi-cantly induced ROS production in the Saos-2 cells.Western blot analysis demonstrated that celastrol significantly increased the protein levels of cleaved caspase-9, cleaved caspase-3 and phosphorylated JNK in the Saos-2 cells.CONCLUSION:Celastrol induces caspase-dependent apoptosis through ROS/JNK pathway in Saos-2 cells.
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inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity of [14C]L-leucine uptake and the inhibition profiles of [14C]L-leucine uptake by various amino acids in the Saos2 cells were comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [14C]Lleucine uptake is, therefore, mediated by LAT1 in the Saos2 cells. These results suggest that the transports of neutral amino acids including several essential amino acids into Saos2 human osteogenic sarcoma cells are for the most part mediated by LAT1. Therefore, the Saos2 human osteogenic sarcoma cells are excellent tools for examine the properties of LAT1. Moreover, the specific inhibition of LAT1 in tumor cells might be a new rationale for anti-tumor therapy.