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
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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
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