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
|
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(IC_(30))和半数抑制浓度(IC_(50));采用Transwell迁移试验检测低、中、高剂量CA(50、75、100μg/m L)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/mL CA作用12、24、48 h,25、50、75、100μg/m L 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IC_(30)值分别为82.24、50.45、46.34、31.81μg/mL,IC_(50)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-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。
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(IC_(30))and half inhibition concentration(IC_(50))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). IC_(30) of CA were 82.24,50.45,46.34,31.81 μg/mL;IC_(50) 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.
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(IC_(30))and half inhibition concentration(IC_(50))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). IC_(30) of CA were 82.24,50.45,46.34,31.81 μg/mL;IC_(50) 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.
引文
[1]Global Burden of Disease Cancer,FITZMAURICE C,AKINYEMIJU TF,et al.Global,regional,and national cancer incidence,mortality,years of life lost,years lived with disability,and disability-adjusted life-years for 29cancer groups,1990 to 2016:a systematic analysis for the global burden of disease study[J].JAMA Oncol,2018,4(11):1553-1568.
[2]BROWN NA,AISNER DL,OXNARD GR.Precision medicine in non-small cell lung cancer:current standards in pathology and biomarker interpretation[J].Am Soc Clin Oncol Educ Book,2018.DOI:10.1200/EDBK_209089.
[3]KUMARAKULASINGHE NB,VAN ZANWIJK N,SOORA.Molecular targeted therapy in the treatment of advanced stage non-small cell lung cancer:NSCLC[J].Respirology,2015,20(3):370-378.
[4]ZAMAY TN,ZAMAY GS,KOLOVSKAYA OS,et al.Current and prospective protein biomarkers of lung cancer[J].Cancers:Basel,2017.DOI:10.3390/cancers9110155.
[5]NAKAMURA H,NISHIMURA T.History,molecular features,and clinical importance of conventional serum biomarkers in lung cancer[J].Surg Today,2017,47(9):1037-1059.
[6]ZHENG Y,XIE J,JIANG F,et al.Inhibition of miR 21promotes cell apoptosis in oral squamous cell carcinoma by upregulating PTEN[J].Oncol Rep,2018,40(5):2798-2805.
[7]HUANG Y,HU Q,DENG Z,et al.MicroRNAs in body fluids as biomarkers for non-small cell lung cancer:a systematic review[J].Technol Cancer Res Treat,2014,13(3):277-287.
[8]LONG ZW,WU JH,CAI H,et al.miR-374b promotes proliferation and inhibits apoptosis of human GIST cells by inhibiting PTEN through activation of the PI3K/Akt pathway[J].Mol Cells,2018,41(6):532-544.
[9]YUAN Y,XU XY,ZHENG HG,et al.Elevated miR-21 is associated with poor prognosis in non-small cell lung cancer:a systematic review and meta-analysis[J].Eur Rev Med Pharmacol Sci,2018,22(13):4166-4180.
[10]LIU C,YANG Z,DENG Z,et al.Downregulated miR-144-3p contributes to progression of lung adenocarcinoma through elevating the expression of EZH2[J].Cancer Med,2018,7(11):5554-5566.
[11]TSAI CC,WU HH,CHANG CP,et al.Calycosin-7-O-beta-D-glucoside reduces myocardial injury in heat stroke rats[J].J Formos Med Assoc,2019,118(3):730-738.
[12]YANG J,JIA M,ZHANG X,et al.Calycosin attenuates MPTP-induced Parkinson’s disease by suppressing the activation of TLR/NF-κB and MAPK pathways[J].Phytother Res,2018,33(2):309-318.
[13]SUN H,YIN M,QIAN W,et al.Calycosin,a Phytoestrogen isoflavone,induces apoptosis of estrogen receptor-positive MG-63 osteosarcoma cells via the phosphatidylinositol 3-kinase(PI3K)/AKT/mammalian target of rapamycin(mTOR)pathway[J].Med Sci Monit,2018.DOI:10.12659/MSM.910201.
[14]DEY P,GHOSH RK.Fine-needle aspiration cytology of non-small cell lung carcinoma:a paradigm shift[J].Diagn Cytopathol,2019,47(4):351-358.
[15]HAJIASGHARZADEH K,SOMI MH,SHANEHBANDID,et al.Small interfering RNA-mediated gene suppression as a therapeutic intervention in hepatocellular carcinoma[J].J Cell Physiol,2019,234(4):3263-3276.
[16]LI M,AN W,XU L,et al.The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in human lung cancer cells[J].J Exp Clin Cancer Res,2019.DOI:10.1186/s13046-019-1064-8.
[17]LI YL,ZHANG XY,LENG Y,et al.Global protein expression analysis of molecular markers of DS-1-47,a component of implantation-promoting traditional chinese medicine[J].J Huazhong Univ Sci Technolog Med Sci,2016,36(6):910-915.
[18]ZHOU Y,LIU QH,LIU CL,et al.Calycosin induces apoptosis in human ovarian cancer SKOV3 cells by activating caspases and Bcl-2 family proteins[J].Tumor Biol,2015,36(7):5333-5339.
[19]CHEN J,HOU R,ZHANG X,et al.Calycosin suppresses breastcancer cell growth via ERβ-dependent regulation of IGF-1R,p38 MAPK and PI3K/Akt pathways[J].PLoSOne,2014.DOI:10.1371/journal.pone.0091245.
[20]CHEN J,ZHAO X,LI X,et al.Calycosin induces apoptosis by the regulation of ERβ/mi R-17 signaling pathway in human colorectal cancer cells[J].Food Funct,2015,6(9):3091-3097.
[21]QIU R,MA G,LI X,et al.Clinical case report of patients with osteosarcoma and anticancer benefit of calycosin against human osteosarcoma cells[J].J Cell Biochem,2018,120(6):10697-10706.
[22]CHENG XD,GU JF,YUAN JR,et al.Suppression of A549 cell proliferation and metastasis by calycosin via inhibition of the PKC-α/ERK1/2 pathway:an in vitro investigation[J].Mol Med Rep,2015,12(6):7992-8002.
[23]FOUAD H,SALEM H,ELLAKWA DE,et al.MMP-2and MMP-9 as prognostic markers for the early detection of urinary bladder cancer[J].J Biochem Mol Toxicol,2018.DOI:10.1002/jbt.22275.
[24]YU T,LIU L,LI J,et al.miRNA-10a is upregulated in NSCLC and may promote cancer by targeting PTEN[J].Oncotarget,2015,6(30):30239-30250.
[25]ZOGRAFOS L.Intravitreal anti-VEGF for the treatment of irradiation induced optic neuropathy and maculopathy[J].Acta Ophthalmologica,2018.DOI:10.1111/aos.13972_534.
[26]HAMILTON A J,SEID J,VERDECCHIA K,et al.Abscopal effect after radiosurgery for solitary brain metastasis from non-small cell lung cancer[J].Cureus,2018.DOI:10.7759/cureus.3777.
[27]PERUMAL E,SO YK,SUN S,et al.PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation ofβ-catenin and snail/slug in non-small cell lung carcinoma cells[J].Lung Cancer,2019.DOI:10.1016/j.lungcan.2019.01.013.
[28]WU Y,SONG Y,XIONG Y,et al.micro RNA-21(mi R-21)promotes cell growth and invasion by repressing tumor suppressor PTEN in colorectal cancer[J].Cell Physiol Biochem,2017,43(3):945-958.
[29]GAO W,LU X,LIU L,et al.miRNA-21:a biomarker predictive for platinum-based adjuvant chemotherapy response in pantents with non-small cell lung cancer[J].Cancer Biol Ther,2012,13(5):330-340.
[30]HE Y,HUANG C,LI L.miR-21 is a critical therapeutic target for renal fibrosis[J].Cell Biochem Biophys,2014,68(3):635-636.
[31]YANG M,SHEN H,QIU C,et al.High expression of miR-21 and miR-155 predicts recurrence and unfavourable survival in non-small cell lung cancer[J].Eur J Cancer,2013,49(3):604-615.
[2]BROWN NA,AISNER DL,OXNARD GR.Precision medicine in non-small cell lung cancer:current standards in pathology and biomarker interpretation[J].Am Soc Clin Oncol Educ Book,2018.DOI:10.1200/EDBK_209089.
[3]KUMARAKULASINGHE NB,VAN ZANWIJK N,SOORA.Molecular targeted therapy in the treatment of advanced stage non-small cell lung cancer:NSCLC[J].Respirology,2015,20(3):370-378.
[4]ZAMAY TN,ZAMAY GS,KOLOVSKAYA OS,et al.Current and prospective protein biomarkers of lung cancer[J].Cancers:Basel,2017.DOI:10.3390/cancers9110155.
[5]NAKAMURA H,NISHIMURA T.History,molecular features,and clinical importance of conventional serum biomarkers in lung cancer[J].Surg Today,2017,47(9):1037-1059.
[6]ZHENG Y,XIE J,JIANG F,et al.Inhibition of miR 21promotes cell apoptosis in oral squamous cell carcinoma by upregulating PTEN[J].Oncol Rep,2018,40(5):2798-2805.
[7]HUANG Y,HU Q,DENG Z,et al.MicroRNAs in body fluids as biomarkers for non-small cell lung cancer:a systematic review[J].Technol Cancer Res Treat,2014,13(3):277-287.
[8]LONG ZW,WU JH,CAI H,et al.miR-374b promotes proliferation and inhibits apoptosis of human GIST cells by inhibiting PTEN through activation of the PI3K/Akt pathway[J].Mol Cells,2018,41(6):532-544.
[9]YUAN Y,XU XY,ZHENG HG,et al.Elevated miR-21 is associated with poor prognosis in non-small cell lung cancer:a systematic review and meta-analysis[J].Eur Rev Med Pharmacol Sci,2018,22(13):4166-4180.
[10]LIU C,YANG Z,DENG Z,et al.Downregulated miR-144-3p contributes to progression of lung adenocarcinoma through elevating the expression of EZH2[J].Cancer Med,2018,7(11):5554-5566.
[11]TSAI CC,WU HH,CHANG CP,et al.Calycosin-7-O-beta-D-glucoside reduces myocardial injury in heat stroke rats[J].J Formos Med Assoc,2019,118(3):730-738.
[12]YANG J,JIA M,ZHANG X,et al.Calycosin attenuates MPTP-induced Parkinson’s disease by suppressing the activation of TLR/NF-κB and MAPK pathways[J].Phytother Res,2018,33(2):309-318.
[13]SUN H,YIN M,QIAN W,et al.Calycosin,a Phytoestrogen isoflavone,induces apoptosis of estrogen receptor-positive MG-63 osteosarcoma cells via the phosphatidylinositol 3-kinase(PI3K)/AKT/mammalian target of rapamycin(mTOR)pathway[J].Med Sci Monit,2018.DOI:10.12659/MSM.910201.
[14]DEY P,GHOSH RK.Fine-needle aspiration cytology of non-small cell lung carcinoma:a paradigm shift[J].Diagn Cytopathol,2019,47(4):351-358.
[15]HAJIASGHARZADEH K,SOMI MH,SHANEHBANDID,et al.Small interfering RNA-mediated gene suppression as a therapeutic intervention in hepatocellular carcinoma[J].J Cell Physiol,2019,234(4):3263-3276.
[16]LI M,AN W,XU L,et al.The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in human lung cancer cells[J].J Exp Clin Cancer Res,2019.DOI:10.1186/s13046-019-1064-8.
[17]LI YL,ZHANG XY,LENG Y,et al.Global protein expression analysis of molecular markers of DS-1-47,a component of implantation-promoting traditional chinese medicine[J].J Huazhong Univ Sci Technolog Med Sci,2016,36(6):910-915.
[18]ZHOU Y,LIU QH,LIU CL,et al.Calycosin induces apoptosis in human ovarian cancer SKOV3 cells by activating caspases and Bcl-2 family proteins[J].Tumor Biol,2015,36(7):5333-5339.
[19]CHEN J,HOU R,ZHANG X,et al.Calycosin suppresses breastcancer cell growth via ERβ-dependent regulation of IGF-1R,p38 MAPK and PI3K/Akt pathways[J].PLoSOne,2014.DOI:10.1371/journal.pone.0091245.
[20]CHEN J,ZHAO X,LI X,et al.Calycosin induces apoptosis by the regulation of ERβ/mi R-17 signaling pathway in human colorectal cancer cells[J].Food Funct,2015,6(9):3091-3097.
[21]QIU R,MA G,LI X,et al.Clinical case report of patients with osteosarcoma and anticancer benefit of calycosin against human osteosarcoma cells[J].J Cell Biochem,2018,120(6):10697-10706.
[22]CHENG XD,GU JF,YUAN JR,et al.Suppression of A549 cell proliferation and metastasis by calycosin via inhibition of the PKC-α/ERK1/2 pathway:an in vitro investigation[J].Mol Med Rep,2015,12(6):7992-8002.
[23]FOUAD H,SALEM H,ELLAKWA DE,et al.MMP-2and MMP-9 as prognostic markers for the early detection of urinary bladder cancer[J].J Biochem Mol Toxicol,2018.DOI:10.1002/jbt.22275.
[24]YU T,LIU L,LI J,et al.miRNA-10a is upregulated in NSCLC and may promote cancer by targeting PTEN[J].Oncotarget,2015,6(30):30239-30250.
[25]ZOGRAFOS L.Intravitreal anti-VEGF for the treatment of irradiation induced optic neuropathy and maculopathy[J].Acta Ophthalmologica,2018.DOI:10.1111/aos.13972_534.
[26]HAMILTON A J,SEID J,VERDECCHIA K,et al.Abscopal effect after radiosurgery for solitary brain metastasis from non-small cell lung cancer[J].Cureus,2018.DOI:10.7759/cureus.3777.
[27]PERUMAL E,SO YK,SUN S,et al.PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation ofβ-catenin and snail/slug in non-small cell lung carcinoma cells[J].Lung Cancer,2019.DOI:10.1016/j.lungcan.2019.01.013.
[28]WU Y,SONG Y,XIONG Y,et al.micro RNA-21(mi R-21)promotes cell growth and invasion by repressing tumor suppressor PTEN in colorectal cancer[J].Cell Physiol Biochem,2017,43(3):945-958.
[29]GAO W,LU X,LIU L,et al.miRNA-21:a biomarker predictive for platinum-based adjuvant chemotherapy response in pantents with non-small cell lung cancer[J].Cancer Biol Ther,2012,13(5):330-340.
[30]HE Y,HUANG C,LI L.miR-21 is a critical therapeutic target for renal fibrosis[J].Cell Biochem Biophys,2014,68(3):635-636.
[31]YANG M,SHEN H,QIU C,et al.High expression of miR-21 and miR-155 predicts recurrence and unfavourable survival in non-small cell lung cancer[J].Eur J Cancer,2013,49(3):604-615.