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、100μg/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、100μg/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、100μg/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(75μg/mL)对其miR-21及PETN、VEGF、MMP-9蛋白表达的影响。结果:经50、75、100μg/mLCA作用12、24、48 h,25、50、75、100μg/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、31.81μg/mL,IC50值分别为108.06、73.35、70.08、49.89μg/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-21mimic后,miR-21mimic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-21mimic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。
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 μ g/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 μ g/mL) for 24 h. After transfected with miR-21 mimics and miR-21 inhibitor, the effects of CA(75 μ g/m L) 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 μ g/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.
作者
周立霞
关洪全
王淳
马贤德
王丹
ZHOU Lixia;GUAN Hongquan;WANG Chun;MA Xiande;WANG Dan(College of Basic Medicine,Liaoning University of TCM,Shenyang 110032,China;Graduate School,Jinzhou Medical University,Liaoning Jinzhou 121001,China)
出处
《中国药房》
CAS
北大核心
2019年第12期1595-1602,共8页
China Pharmacy
基金
国家自然科学基金资助项目(No.81774184)
辽宁省自然科学基金资助项目(No.20170540376)
