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目的研究莪术油通过hTert作为下游靶基因对乳腺癌MCF-7细胞端粒酶活性及DNA损伤反应的调控作用。方法以300μg/mL莪术油处理MCF-7细胞,采用MTT法测定细胞增殖活性,绘制生长曲线,筛选最佳药物处理时间。于加药后48 h,实时定量RT-PCR检测端粒酶催化亚单位hTert表达水平、TRAP Assay检测端粒酶活性变化;收集加药前以及加药后24,48,72 h细胞,采用免疫荧光及Western Blot检测DNA损伤反应相关53BP1蛋白水平。结果莪术油处理的MCF-7细胞24 h后增殖活力开始降低,至48 h生长明显减缓,实时定量RT-PCR结果表明,加药后48 h,hTert表达水平降低为对照细胞的2.43倍(2-△△Ct),TRAP Assay结果显示端粒酶活性降低为对照细胞的2.27倍,免疫荧光及Western Blot结果显示莪术油处理的细胞53BP1蛋白磷酸化水平自转染24 h后开始升高,至48 h左右达到高峰,至72 h时仍保持在较高水平。结论莪术油可以hTert作为下游靶分子,通过调控端粒酶活性及DNA损伤反应,抑制乳腺癌MCF-7细胞的增殖。
Objective To study the regulation of telomerase activity and DNA damage response of Curcuma oil by the use of hTert as a downstream target gene in breast cancer MCF-7 cells. Methods MCF-7 cells were treated with 300μg / mL zedoary turmeric oil, cell proliferation activity was measured by MTT assay, growth curve was drawn, and the best drug treatment time was screened. The expression level of telomerase catalytic subunit hTert was detected by real-time quantitative RT-PCR 48 h after the addition of dosing and the change of telomerase activity was detected by TRAP Assay. The cells were collected before and 24, 48 and 72 h after dosing, Fluorescence and Western Blot were used to detect the DNA damage response of 53BP1 protein. Results The proliferation of MCF-7 cells started to decrease after 48 h and the growth slowed down to 48 h. The real-time quantitative RT-PCR results showed that the expression level of hTert decreased 2.43 folds of the control cells at 48 h - △△ Ct). TRAP Assay results showed that the telomerase activity decreased 2.27 times that of control cells. The results of immunofluorescence and Western Blot showed that the level of 53BP1 phosphorylation increased from 24 h after transfection to Peaked at about 48 h and remained at a relatively high level at 72 h. Conclusion Ezhu oil can hTert as a downstream target molecule, inhibition of breast cancer MCF-7 cell proliferation by regulating telomerase activity and DNA damage response.