氟暴露对人成骨细胞系Saos-2细胞微小RNA-200c表达及其靶点的影响

Effects of fluoride exposure on microRNA-200c expression and its target in human osteoblast Saos-2 cells

目的 探讨氟暴露对于人成骨细胞系Saos-2细胞（简称Saos-2细胞）微小RNA（miR）-200c表达及其靶点的影响。方法 采用DMEM／F-12培养液，体外培养Saos-2细胞。按培养液中氟化钠（NaF）剂量将Saos-2细胞分为对照组（0 mg／L）、染氟组（4 mg／L），染氟培养48 h后收集细胞。采用实时荧光定量PCR（qRT-PCR）检测Saos-2细胞中miR-200c表达，碱性磷酸酶（ALP）、骨钙素（BGP）及miR-200c作用靶点人第10号染色体缺失磷酸酶（PTEN）、双特异性磷酸酶（DUSP1）mRNA的表达；采用蛋白免疫印迹法（Western blot）检测PTEN、DUSP1蛋白表达。结果 染氟组Saos-2细胞中ALP、BGP mRNA和miR-200c表达（23.60 ± 1.87、9.41 ± 0.94、8.61 ± 0.26）高于对照组（1.00 ± 0.11、1.00 ± 0.07、1.00 ± 0.12），组间比较差异有统计学意义（t = - 24.084、- 18.388、- 8.687，P均 〈 0.05）；PTEN和DUSP1 mRNA表达（0.63 ± 0.02、0.38 ± 0.02 ）低于对照组（1.02 ± 0.24、1.02 ± 0.24），组间比较差异有统计学意义（t = 3.327、5.454，P均 〈 0.05）。染氟组Saos-2细胞PTEN、DUSP1蛋白表达（1.19 ± 0.10、0.83 ± 0.07）低于对照组（1.81 ± 0.14、1.44 ± 0.25），组间比较差异有统计学意义（t = 6.250、4.171，P均 〈 0.05）。结论 氟暴露能够使Saos-2细胞miR-200c表达水平升高，通过miR-200c作用于PTEN和DUSP1，下调PTEN、DUSP1 mRNA与蛋白表达水平。

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.