20(S)-原人参二醇通过调控miRNA-19b诱导乳腺癌MCF-7细胞凋亡的观察

摘要

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, 50,80μmol/L 20(S)-PPD for 48 h, the expression level of miRNA-19b was detected by real-time PCR, and DNMT activi-ty 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 pro-tein expression level of TNFαin 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 TNFαwas 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 TNFαincreased, 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 .%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.