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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 To study the effect of T-2 toxin on proliferation and cell cycle of rat chondrocytes,in order to provide a new idea in molecular mechanism of T-2 toxin-induced chondrocyte damage.Methods Primary chondrocytes of neonatal Wistar rats were isolated and stained by toluidine blue staining and type Ⅱ collagen immunofluorescence staining.The effects of different concentrations of T-2 toxin [0 (control),1,5,10,20,50,100 μg/L)] on proliferation of chondrocytes for 24 h were detected by cell counting kit-8 (CCK-8) method,and control,1 (low dose),5 (medium dose),and 10 μg/L (high dose) T-2 toxin were selected for subsequent experiment;cell cycle changes were detected by flow cytometry;Real-time PCR and Western blotting were used to detect the effects of T-2 toxin on mRNA and protein expressions of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in chondrocytes.Results With increase of T-2 toxin concentration (control,1,5,10,20,50,100 μg/L),the cell survival rates [(100.00 ± 0.00)％,(93.12 ± 1.66)％,(77.12 ± 1.11)％,(59.44 ± 4.09)％,(46.64 ± 3.86)％,(38.15 ± 3.37)％,(33.79 ± 0.99)％] were decreased,and the differences were statistically significant (F =139.21,P ＜0.05).The percentages of quiescent phase/pre-DNA synthesis phase (G0/G1 phase) ceils in 1,5,10 μg/L T-2 toxin groups [(22.03 ± 0.42)％,(30.54 ± 2.61)％,(36.01 ± 1.51)％] were significantly higher than that in control group [(13.79 ± 1.65)％,P ＜ 0.05];the percentages of DNA synthesis phase (S phase) cells [(60.27 ± 3.53)％,(53.88 ±4.38)％,(49.55 ± 2.49)％] were significantly lower than that in control group [(76.72 ± 4.24)％,P ＜ 0.05].The differences of mRNA levels of PCNA and Cyclin D1 between groups were statistically significant (F =46.80,17.97,P ＜ 0.05),and 5,10 μg/L T-2 toxin groups (0.77 ± 0.13,0.79 ± 0.08,0.60 ± 0.07,0.56 ± 0.05) were lower than the control group (0.99 ± 0.02,1.01 ± 0.01,P ＜ 0.05).The expressions of PCNA protein in 5,10 μg/L T-2 toxin groups (0.69 ± 0.03,0.49 ± 0.03) were lower than that in control group (0.92 ± 0.05,P ＜ 0.05);the expressions of Cyclin D1 protein in 1,5,10 μg/L T-2 toxin groups (0.80 ± 0.06,0.60 ± 0.07,0.33 ± 0.13) were lower than that in control group (0.95 ± 0.07,P ＜ 0.05).Conclusion T-2 toxin can inhibit the proliferation of chondrocytes,which may be worked through influencing the expression of cell cycle protein,causing cell cycle arrest,thereby inhibiting DNA synthesis.