摘要
目的:研究20(S)-原人参二醇[20(S)-PPD]通过调控miRNA-19b的表达诱导乳腺癌MCF-7细胞凋亡以及相关作用的机制。方法:采用MTT比色法检测30、40、50、60、70、80μmol/L 20(S)-PPD处理48 h对MCF-7细胞存活率的影响;real-time PCR检测30、50、80μmol/L 20(S)-PPD处理MCF-7细胞48 h后miRNA-19b相对表达量的变化;DNA甲基化转移酶(DNMT)活性检测试剂盒检测30、50、80μmol/L 20(S)-PPD处理MCF-7细胞48 h对DNMT活性的影响。将MCF-7细胞分成瞬时转染miRNA阴性对照模拟物+空白培养液组、瞬时转染miRNA-19b模拟物+20(S)-PPD组、瞬时转染miRNA-19b模拟物+空白培养液组、瞬时转染miRNA阴性对照模拟物+20(S)-PPD组,real-time PCR检测4组细胞miRNA-19b表达量的变化,Western blot检测4组细胞中TNFα蛋白表达量的变化,流式细胞术检测4组细胞的凋亡率。结果:20(S)-PPD可剂量依赖性地降低MCF-7细胞的存活率。随着20(S)-PPD剂量的升高,miRNA-19b的表达量降低(P<0.05)。80μmol/L的20(S)-PPD可增强DNMT活性(P<0.05)。转染miRNA-19b模拟物后,miRNA-19b的表达量上升,TNFα蛋白的表达受到抑制,细胞凋亡率降低;而加入65μmol/L 20(S)-PPD后,miRNA-19b的表达下调,TNFα蛋白的表达上调,细胞凋亡率升高(P<0.05)。结论:20(S)-PPD可能通过甲基化作用抑制miRNA-19b的表达,进而促进TNFα的表达,诱导MCF-7细胞凋亡。
Aim： To investigate the possible mechanism of 20（S）-protopanaxadiol[20（S）-PPD] which could induce apoptosis of breast cancer MCF-7 cells via regulating miRNA-19b. Methods： MCF-7 cells were treated with 30,40,50,60,70,80 μmol/L 20（ S）-PPD for 48 h and the cell viability was detected by MTT method. MCF-7 cells were treated with 30,50,80 μmol/L 20（ S）-PPD for 48 h,the expression level of miRNA-19b was detected by real-time PCR,and DNMT activity assay kit was used to detect the DNMT activity. MCF-7 cells were divided into four groups： miRNA mimics plus blank medium group,miRNA-19b mimics plus 20（ S）-PPD group,miRNA-19b mimics plus blank medium group,and miRNA mimics plus 20（ S）-PPD group. The expression of miRNA-19b in the four groups was detected by real-time PCR. The protein expression level of TNFα in the four groups was detected by Western blot. The cell apoptosis in the four groups was tested by flow cytometry. Results： 20（ S）-PPD could suppress the viability of MCF-7 cells concentration-dependently.20（ S）-PPD could significantly decrease the expression of miRNA-19b with the increase of the concentration（ P〈0. 05）. 80μmol/L 20（ S）-PPD significantly enhanced the activity of DNMT（ P〈0. 05）. After transfecting miRNA-19b mimics,the expression of miRNA-19b upregulated,the expression of TNFα was repressed,and the apoptosis rate reduced（ P〈0. 05）.After joining 65 μmol/L 20（ S）-PPD,the expression of miRNA-19b decreased,the expression of TNFα increased,and the apoptosis rate increased（ P〈0. 05）. Conclusion： In MCF-7 cells,20（ S）-PPD may inhibit the expression of miRNA-19b,increase the expression of TNFα,and induce apoptosis through its methylation role.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2017年第6期706-710,共5页
Journal of Zhengzhou University(Medical Sciences)
基金
国家自然科学基金资助项目81101655
湖南省自然科学基金资助项目12JJ3099
