17β-雌二醇对人成骨肉瘤细胞CLCF1基因表达的影响

Effect of 17β-estradiol on CLCF1 Gene Expression in Human Osteosarcoma Cells

目的探讨17β-雌二醇对人成骨肉瘤细胞CLCF1基因表达的影响。方法将骨肉瘤细胞MG-63分为对照组、β-雌二醇低、中、高剂量组,对照组加入10-3mol/L DMSO,将17β-雌二醇用DMSO稀释后,分别加入β-雌二醇低、中、高剂量组,终浓度分别为10-10、10-9、10-8 mol/L,干预MG-63细胞24、48、72 h后,实时荧光定量PCR检测各组MG-63细胞中CLCF1 mRNA的表达情况;构建CLCF1基因启动子荧光素酶报告基因载体,采用10-8 mol/L 17β-雌二醇及雌激素受体抑制剂氟维司群(ICI)干预MG63细胞24 h后,双荧光素酶报告基因系统检测双荧光素酶活性。结果①干预24 h,β-雌二醇低、中、高剂量组CLCF1 mRNA的表达均高于对照组,差异有统计学意义(P<0.05);干预48 h,仅β-雌二醇高剂量组CLCF1 mRNA的表达高于对照组,差异有统计学意义(P<0.05);干预72 h,β-雌二醇低组CLCF1 mRNA的表达低于对照组,差异有统计学意义(P<0.05),β-雌二醇中、高剂量组CLCF1 mRNA的表达与对照组比较,差异无统计学意义(P>0.05);②与对照组相比,17β-雌二醇高剂量组荧光素酶活性提高,差异有统计学意义(P<0.05);加入雌激素受体抑制剂ICI后,荧光素酶活性较对照组降低,差异有统计学意义(P<0.05)。结论17β-雌二醇可通过提高CLCF1启动子活性促进MG63细胞株CLCF1基因的表达,这可能是绝经后骨质疏松症肾阴虚证的分子机制之一。

Objective To explore the effect of 17β-estradiol on CLCF1 gene expression in human osteosarcoma cells.Methods The osteosarcoma cells MG-63 were divided into control group,β-estradiol low,medium and high dose groups.In the control group,10-3mol/L DMSO was added,and 17β-estradiol was diluted with DMSO,and then added to the low,medium,and high-doseβ-estradiol groups.The final concentrations were 10-10,10-9,and 10-8 mol/L.After 24,48,and 72 h after intervention with MG-63 cells,real-time fluorescence quantitative PCR was used to detect the expression of CLCF1 mRNA in each group of MG-63 cells;Construction of CLCF1 gene promoter luciferase reporter gene vector,using 10-8 mol/L 17β-estradiol and estrogen receptor inhibitor Fulvestrant(ICI)to intervene in MG63 cells for 24 h,double luciferase reporter gene The system detects dual luciferase activity.Results①After 24 h of intervention,the expression of CLCF1 mRNA inβ-estradiol low,medium and high dose groups was higher than that of the control group,the difference was statistically significant(P<0.05);After 48 h of intervention,only the expression of CLCF1 mRNA in theβ-estradiol high-dose group was higher than that of the control group,the difference was statistically significant(P<0.05);After 72 h of intervention,the expression of CLCF1 mRNA in the lowβ-estradiol group was lower than that of the control group,the difference was statistically significant(P<0.05).There was no statistically significant difference in the expression of CLCF1 mRNA between the middle and high doses ofβ-estradiol group and the control group(P>0.05);②Compared with the control group,the luciferase activity of the 17β-estradiol high-dose group increased,the difference was statistically significant(P<0.05);After adding the estrogen receptor inhibitor ICI,the luciferase activity was lower than the control group,the difference was statistically significant(P<0.05).Conclusion 17β-estradiol can promote the expression of CLCF1 gene in MG63 cell line by increasing the activity of CLCF1 promoter,which may be one of the molecular mechanisms of kidney-yin deficiency syndrome in postmenopausal osteoporosis.