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毛蕊异黄酮抑制肺腺癌细胞增殖和迁移的miR-21/PTEN信号通路机制研究
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| 篇名： | 毛蕊异黄酮抑制肺腺癌细胞增殖和迁移的miR-21/PTEN信号通路机制研究 |
| TITLE： | |
| 摘要： | 目的：探讨毛蕊异黄酮（CA）通过调控微RNA-21（miR-21）/人类第10号染色体缺失的磷酸酶及张力蛋白同源物（PTEN）信号通路对肺腺癌细胞增殖和迁移的抑制作用机制。方法：以人肺腺癌SPC-A1细胞为对象，采用MTT法检测不同剂量CA（5、15、25、50、75、100 μg/mL）作用12、24、48、72 h后的细胞增殖情况，并计算细胞存活率、30％细胞生长抑制浓度（IC30）和半数抑制浓度（IC50）；采用Transwell迁移试验检测低、中、高剂量CA（50、75、100 μg/mL）作用24 h后的细胞迁移情况，记录染色细胞数并计算细胞迁移抑制率；采用蛋白质印迹法和实时聚合酶链反应法检测低、中、高剂量CA（50、75、100 μg/mL）作用24 h后细胞miR-21以及PTEN、血管内皮生长因子（VEGF）、基质金属蛋白酶9（MMP-9）蛋白及其mRNA的表达情况；检测细胞在分别转染miR-21模拟物（mimic）和抑制物（inhibitor）后，CA（75 μg/mL）对其miR-21及PETN、VEGF、MMP-9蛋白表达的影响。结果：经50、75、100 μg/mL CA作用12、24、48 h，25、50、75、100 μg/mL CA作用72 h后，细胞存活率均显著降低（P＜0.05或P＜0.01）；12～72 h各时间点CA的IC30值分别为82.24、50.45、46.34、31.81 μg/mL，IC50值分别为108.06、73.35、70.08、49.89 μg/mL。与正常对照组比较，CA各剂量组染色细胞数，低剂量组细胞中VEGF蛋白以及中、高剂量组细胞中miR-21，VEGF、MMP-9蛋白及其mRNA的相对表达量均显著减少或降低，且中、高剂量组显著少于或低于低剂量组，高剂量组显著少于或低于中剂量组（P＜0.05或P＜0.01）；CA各剂量组细胞迁移率以及中、高剂量组细胞中PTEN蛋白及其mRNA的相对表达量均显著升高，且中、高剂量组显著高于低剂量组，高剂量组显著高于中剂量组（P＜0.05或P＜0.01）。转染miR-21 mimic后，miR-21 mimic组细胞miR-21及VEGF、MMP-9蛋白的相对表达量均较正常对照组显著升高，PTEN蛋白的相对表达量显著降低（P＜0.01）；加入CA干预后，细胞中miR-21及VEGF、MMP-9蛋白的相对表达量均较miR-21 mimic组显著降低，PTEN蛋白的相对表达量均显著升高（P＜0.05或P＜0.01）。转染miR-21 inhibitor后，miR-21 inhibitor组细胞中miR-21及VEGF、MMP-9蛋白的相对表达量均较正常对照组显著降低，PTEN蛋白的相对表达量显著升高（P＜0.05或P＜0.01）；加入CA干预后，细胞中miR-21及上述蛋白的表达较miR-21 inhibitor组均未见明显变化（P＞0.05）。结论：CA可剂量依赖性地抑制肺腺癌SPC-A1细胞的增殖和迁移，且这种作用可能与调控miR-21/PTEN信号通路有关。 |
| ABSTRACT： | OBJECTIVE： To investigate the mechanism of calycosin （CA） inhibiting the proliferation and migration of lung adenocarcinoma cells by regulating miR-21/PTEN signaling pathway. METHODS： Using lung adenocarcinoma SPC-A1 cells as objects， cell proliferation was detected by MTT method after treated with different doses of CA （5， 15， 25， 50， 75， 100 μg/mL） for 12， 24， 48， 72 h. Cell survival rate， 30％ cell growth inhibition concentration （IC30） and half inhibition concentration （IC50） were calculated. Transwell migration test was used to detect the migration of cells after treated with low-dose， medium-dose and high-dose of CA （50， 75， 100 μg/mL） for 24 h. The number of stained cells was recorded and inhibition rate of cell migration were calculated. Western blotting assay and real-time PCR were used to detect the expression of miR-21 as well as the proteins and their mRNAs expression of PTEN， VEGF， MMP-9 after treated with low-dose， medium-dose and high-dose of CA （50， 75， 100 μg/mL） for 24 h. After transfected with miR-21 mimics and miR-21 inhibitor， the effects of CA （75 μg/mL） on the expression of miR-21 and the protein expression of PETN， VEGF and MMP-9 were detected. RESULTS： After treated with 50， 75， 100 μg/mL CA for 12， 24， 48 h， 25， 50， 75， 100 μg/mL CA for 72 h， cell survival rate was decreased significantly （P＜0.05 or P＜0.01）. IC30 of CA were 82.24， 50.45， 46.34， 31.81 μg/mL ； IC50 of CA were 108.06， 73.35， 70.08， 49.89 μg/mL during 12-72 h. Compared with normal control group， the number of stained cells in CA groups， protein expression of VEGF in CA low-dose group， expression of miR-21 as well as proteins and their mRNAs expression of VEGF， MMP-9 in CA medium-dose and high-dose groups were decreased significantly； the medium-dose and high-dose groups were significantly less or lower than low-dose group； the high-dose group was significantly less or lower than medium-dose group （P＜0.05 or P＜0.01）. Cell migration rate of CA groups as well as protein and its mRNA expression of PTEN in CA medium-dose and high-dose groups were increased significantly； the medium-dose and high-dose groups were significantly higher than the low-dose group； the high-dose group was significantly higher than the medium-dose groups （P＜0.05 or P＜0.01）. After transfected with miR-21 mimics， expression of miR-21 as well as protein expression of VEGF and MMP-9 were increased significantly in miR-21 mimic group， compared with normal control group； protein expression of PTEN was decreased significantly （P＜0.01）. After intervened by CA， expression of miR-21 as well as protein expression of VEGF and MMP-9 in cells were decreased significantly， compared with miR-21 mimic group； protein expression of PTEN was increased significantly （P＜0.05 or P＜0.01）. After transfected with miR-21 inhibitor， expression of miR-21 as well as protein expression of VEGF and MMP-9 were decreased significantly in miR-21 inhibitor group， compared with normal control group； protein expression of PTEN was increased significantly （P＜0.05 or P＜0.01）. After intervened by CA， the expression of miR-21 and above protein had no significant change in cells， compared with miR-21 inhibitor group （P＞0.05）. CONCLUSIONS： CA can inhibit the proliferation and migration of lung adenocarcinoma SPC-A1 cells in a dose-dependent manner， which may be associated with the regulation of miR-21/PTEN signaling pathway. |
| 期刊： | 2019年第30卷第12期 |
| 作者： | 周立霞，关洪全，王淳，马贤德，王丹 |
| AUTHORS： | ZHOU Lixia，GUAN Hongquan，WANG Chun，MA Xiande，WANG Dan |
| 关键字： | 毛蕊异黄酮；微RNA-21/人类第10号染色体缺失的磷酸酶及张力蛋白同源物信号通路；肺腺癌；SPC-A1细胞；增殖；迁移；抑制作用；机制 |
| KEYWORDS： | Calycosin； miR-21/PTEN signaling pathway； Lung adenocarcinoma； SPC-A1 cells； Proliferation； Migration； Inhibitory effect； Mechanism |
| 阅读数： | 269 次 |
| 本月下载数： | 8 次 |
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