胶质瘤SHG44细胞p110α表达及对增殖和转移潜能影响机制研究
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摘要
目的 p110α是磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)的重要成员,与肿瘤的发生发展密切相关,但目前关于p110α与胶质瘤关系的研究甚少。本研究旨在探讨p110α在胶质瘤中的表达情况,对胶质瘤SHG44细胞增殖、侵袭迁移的影响及其可能的作用机制。方法采用蛋白质印迹法检测正常胶质细胞HEB和胶质瘤细胞SHG44中p110α、P-AKT、AKT、P-mTOR、mTOR蛋白水平的表达。采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测HEB和SHG44中p110α、AKT、mTOR的mRNA水平。分别检测p110α特异性抑制剂PIK75处理SHG44细胞后,p110α及其下游相关因子的蛋白表达和mRNA水平。采用细胞增殖检测试剂盒8(cell counting kit8,CCK8)法检测细胞增殖能力。采用划痕实验、Transwell侵袭实验检测p110α对SHG44细胞迁移和侵袭的影响。结果 SHG44细胞中p110α、P-AKT、AKT、P-mTOR、mTOR蛋白表达均高于HEB,均P<0.05,且p110α、AKT、mTOR的mRNA水平均高于HEB细胞,均P<0.01。使用PIK75处理SHG44后,p110α、P-AKT、AKT、P-mTOR、mTOR蛋白表达较处理前均降低,均P<0.05,p110α、AKT、mTOR的mRNA水平较处理前均降低,均P<0.01。CCK8实验结果表明,不同浓度的PIK75均能抑制细胞的增殖,并呈剂量依赖性,均P<0.05。划痕实验结果显示,在0.25μmol/L PIK75中SHG44细胞迁移率为(22.43±1.55)%,低于SHG44细胞组的迁移率(54.12±10.79)%,差异有统计学意义,t=5.035,P<0.01。0.25μmol/L PIK75在Transwell侵袭实验中显示能抑制SHG44穿膜细胞数为20.67±1.22,低于SHG44细胞组的44.33±1.90,差异有统计学意义,t=18.14,P<0.001。结论胶质瘤中p110α高表达激活PI3K/AKT/mTOR信号通路并促进肿瘤细胞的增殖、迁移和侵袭,抑制p110α表达可降低胶质瘤恶性生物学行为,p110α可能成为胶质瘤药物治疗的新靶点。
OBJECTIVE p110αis an important member of PI3 Kand is closely related to the occurrence and development of tumors.However,there is little research on the relationship between p110αand glioma.This study aimed to investigate the expression of p110αin glioma and its effects on proliferation,invasion and migration of the glioma cell SHG44,and to explore its possible mechanism.METHODS Western blot was used to detect the expressions of p110α,P-AKT,AKT,P-mTOR and mTOR in normal glial cell HEB and glioma cell SHG44.The mRNA levels of p110α,AKT and mTOR in HEB and SHG44 were detected by Real-time quantitative PCR(qRT-PCR).The protein expressions and mRNA levels of p110αand its downstream related factors treated with p110α-specific inhibitor PIK75 in SHG44 cells were respectively detected.Cell proliferation was detected by cell counting kit(CCK8).Cell cratch test and Transwell invasion test were used to detect the effects of p110αon migration and invasion of SHG44 cells.RESULTS The protein expressions of p110α,P-AKT,AKT,P-mTOR and mTOR in SHG44 cells were all significantly higher than those in HEB cells(all P<0.05),and the mRNA levels of p110α,AKT and mTOR were all significantly higher than those of HEB cells(all P<0.01).Compared with before treatment,the protein expressions of p110α,P-AKT,AKT,P-mTOR and mTOR decreased(all P<0.05)after SHG44 cells were treated with PIK75,which simultaneously companied with the mRNA levels of p110 a,AKT and mTOR were decreased(all P<0.01).The results of CCK8 experiment showed that PIK75 with different concentrations could inhibit cell proliferation of cells and presented dose-dependent manner(P<0.05).The results of scratch test showed that the migration rate of SHG44 cells in 0.25μmol/L PIK75 was(22.43±1.55)%significantly lower than that in the SHG44 cells group(54.12±10.79)%,t=5.035,P<0.01.At the same time,the number of SHG44 penetrating cells inhibited by 0.25μmol/L PIK75 in the Transwell invasion test was(20.67±1.22)significantly lower than that in the SHG44 cells group(44.33±1.90),t=18.14,P<0.001.CONCLUSIONS The high expression of p110αactivates the PI3 K/AKT/mTOR signaling pathway in glioma,which promotes the proliferation,migration and invasion of tumor cells.Inhibition of p110αexpression can reduce malignant biological behavior of glioma.p110αmay be a new target for glioma drug therapy.
OBJECTIVE p110αis an important member of PI3 Kand is closely related to the occurrence and development of tumors.However,there is little research on the relationship between p110αand glioma.This study aimed to investigate the expression of p110αin glioma and its effects on proliferation,invasion and migration of the glioma cell SHG44,and to explore its possible mechanism.METHODS Western blot was used to detect the expressions of p110α,P-AKT,AKT,P-mTOR and mTOR in normal glial cell HEB and glioma cell SHG44.The mRNA levels of p110α,AKT and mTOR in HEB and SHG44 were detected by Real-time quantitative PCR(qRT-PCR).The protein expressions and mRNA levels of p110αand its downstream related factors treated with p110α-specific inhibitor PIK75 in SHG44 cells were respectively detected.Cell proliferation was detected by cell counting kit(CCK8).Cell cratch test and Transwell invasion test were used to detect the effects of p110αon migration and invasion of SHG44 cells.RESULTS The protein expressions of p110α,P-AKT,AKT,P-mTOR and mTOR in SHG44 cells were all significantly higher than those in HEB cells(all P<0.05),and the mRNA levels of p110α,AKT and mTOR were all significantly higher than those of HEB cells(all P<0.01).Compared with before treatment,the protein expressions of p110α,P-AKT,AKT,P-mTOR and mTOR decreased(all P<0.05)after SHG44 cells were treated with PIK75,which simultaneously companied with the mRNA levels of p110 a,AKT and mTOR were decreased(all P<0.01).The results of CCK8 experiment showed that PIK75 with different concentrations could inhibit cell proliferation of cells and presented dose-dependent manner(P<0.05).The results of scratch test showed that the migration rate of SHG44 cells in 0.25μmol/L PIK75 was(22.43±1.55)%significantly lower than that in the SHG44 cells group(54.12±10.79)%,t=5.035,P<0.01.At the same time,the number of SHG44 penetrating cells inhibited by 0.25μmol/L PIK75 in the Transwell invasion test was(20.67±1.22)significantly lower than that in the SHG44 cells group(44.33±1.90),t=18.14,P<0.001.CONCLUSIONS The high expression of p110αactivates the PI3 K/AKT/mTOR signaling pathway in glioma,which promotes the proliferation,migration and invasion of tumor cells.Inhibition of p110αexpression can reduce malignant biological behavior of glioma.p110αmay be a new target for glioma drug therapy.
引文
[1]Lenting K,Verhaak R,Ter Laan M,et al.Glioma:experimental models and reality[J].Acta Neuropathol,2017,133(2):263-282.
[2]Lin L,Cai J,Jiang C.Recent advances in targeted therapy for glioma[J].Curr Med Chem,2017,24(13):1365-1381.
[3]Simond AM,Rao T,Zuo D,et al.ErbB2-positive mammary tumors can escape PI3K-p110αloss through downregulation of the Pten tumor suppressor[J].Oncogene,2017,36(43):6059-6066.
[4]Firoozinia M,Zareian Jahromi M,Moghadamtousi SZ,et al.PIK3CAgene amplification and PI3Kp110αprotein expression in breast carcinoma[J].Int J Med Sci,2014,11(6):620-625.
[5]Cui W,Cai Y,Wang W,et al.Frequent copy number variations of PI3K/AKT pathway and aberrant protein expressions of PI3Ksubunits are associated with inferior survival in diffuse large Bcell lymphoma[J].J Transl Med,2014,12(1):10-21.
[6]Guerreiro AS,Fattet S,Fischer B,et al.Targeting the PI3Kp110alpha isoform inhibits medulloblastoma proliferation,chemoresistance,and migration[J].Clin Cancer Res,2008,14(21):6761-6769.
[7]Lenting K,Verhaak R,Ter Laan M,et al.Glioma:experimental models and reality[J].Acta Neuropathol,2017,133(2):263-282.
[8]O’Donnell JS,Massi D,Teng MWL,et al.PI3K-AKT-mTOR inhibition in cancer immunotherapy,redux[J].Semin Cancer Biol,2018,48(2):91-103.
[9]Dey N,De P,Leyland-Jones B.PI3K-AKT-mTOR inhibitors in breast cancers:from tumor cell signaling to clinical trials[J].Pharmacol Ther,2017,175:91-106.
[10]刘源,王璐,马超,等.JTC-801抑制结肠癌增殖迁移和促进凋亡机制探讨[J].中华肿瘤防治杂志,2017,24(23):1635-1639.
[11]Kaur A,Sharma S.Mammalian target of rapamycin(mTOR)as apotential therapeutic target in various diseases[J].Inflammopharmacology,2017,25(3):293-312.
[12]Gini B,Zanca C,Guo D,et al.The mTOR kinase inhibitors,CC214-1and CC214-2,preferentially block the growth of EGFRⅧ-activated glioblastomas[J].Clin Cancer Res,2013,19(20):5722-5732.
[13]Duzgun Z,Eroglu Z,Avci CB.Role of mTOR in glioblastoma[J].Gene,2016,575(2):187-190.
[14]李堂月,吴淑华,李扬扬,等.结直肠锯齿状病变及癌变组织中PI3Kp110α、PI3Kp110β的表达及意义[J].临床与实验病理学杂志,2016,32(2):136-141.
[15]Yip WK,He PY,Abdullah MA,et al.Increased expression of phosphatidylinositol 3-Kinase p110αand gene amplification of PIK3CA in nasopharyngeal carcinoma[J].Pathol Oncol Res,2015,22(2):413-419.
[16]Lien EC,Dibble CC,Toker A.PI3Ksignaling in cancer:beyond AKT[J].Curr Opin Cell Biol,2017,45:62-71.
[17]许振,刘志强,郭庆枝,等.宫颈癌组织AKT和P-AKT表达与LMVD相关性及其对新辅助化疗效果预测意义[J].中华肿瘤防治杂志,2017,24(13):906-911.
[18]熊焰,曲琳琳,李东,等.非小细胞肺癌磷脂酰肌醇3激酶p110α蛋白高表达的临床意义及机制[J].中华肿瘤杂志,2017,39(10):737-743.
[19]Soler A,Figueiredo AM,Castel P,et al.Therapeutic benefit of selective inhibition of p110αPI3-kinase in pancreatic neuroendocrine tumors[J].Clin Cancer Res,2016,22(23):5805-5817.
[20]Wojtalla A1,Salm F,Christiansen DG,et al.Novel agents targeting the IGF-1R/PI3Kpathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma[EB/OL].PLoS One,2012,7(10):e47109[2018-04-09].https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047109.
[21]Weber GL,Parat MO,Binder ZA,et al.Abrogation of PIK3CAor PIK3R1reduces proliferation,migration,and invasion in glioblastoma multiforme cells[J].Oncotarget,2011,2(11):833-849.
[2]Lin L,Cai J,Jiang C.Recent advances in targeted therapy for glioma[J].Curr Med Chem,2017,24(13):1365-1381.
[3]Simond AM,Rao T,Zuo D,et al.ErbB2-positive mammary tumors can escape PI3K-p110αloss through downregulation of the Pten tumor suppressor[J].Oncogene,2017,36(43):6059-6066.
[4]Firoozinia M,Zareian Jahromi M,Moghadamtousi SZ,et al.PIK3CAgene amplification and PI3Kp110αprotein expression in breast carcinoma[J].Int J Med Sci,2014,11(6):620-625.
[5]Cui W,Cai Y,Wang W,et al.Frequent copy number variations of PI3K/AKT pathway and aberrant protein expressions of PI3Ksubunits are associated with inferior survival in diffuse large Bcell lymphoma[J].J Transl Med,2014,12(1):10-21.
[6]Guerreiro AS,Fattet S,Fischer B,et al.Targeting the PI3Kp110alpha isoform inhibits medulloblastoma proliferation,chemoresistance,and migration[J].Clin Cancer Res,2008,14(21):6761-6769.
[7]Lenting K,Verhaak R,Ter Laan M,et al.Glioma:experimental models and reality[J].Acta Neuropathol,2017,133(2):263-282.
[8]O’Donnell JS,Massi D,Teng MWL,et al.PI3K-AKT-mTOR inhibition in cancer immunotherapy,redux[J].Semin Cancer Biol,2018,48(2):91-103.
[9]Dey N,De P,Leyland-Jones B.PI3K-AKT-mTOR inhibitors in breast cancers:from tumor cell signaling to clinical trials[J].Pharmacol Ther,2017,175:91-106.
[10]刘源,王璐,马超,等.JTC-801抑制结肠癌增殖迁移和促进凋亡机制探讨[J].中华肿瘤防治杂志,2017,24(23):1635-1639.
[11]Kaur A,Sharma S.Mammalian target of rapamycin(mTOR)as apotential therapeutic target in various diseases[J].Inflammopharmacology,2017,25(3):293-312.
[12]Gini B,Zanca C,Guo D,et al.The mTOR kinase inhibitors,CC214-1and CC214-2,preferentially block the growth of EGFRⅧ-activated glioblastomas[J].Clin Cancer Res,2013,19(20):5722-5732.
[13]Duzgun Z,Eroglu Z,Avci CB.Role of mTOR in glioblastoma[J].Gene,2016,575(2):187-190.
[14]李堂月,吴淑华,李扬扬,等.结直肠锯齿状病变及癌变组织中PI3Kp110α、PI3Kp110β的表达及意义[J].临床与实验病理学杂志,2016,32(2):136-141.
[15]Yip WK,He PY,Abdullah MA,et al.Increased expression of phosphatidylinositol 3-Kinase p110αand gene amplification of PIK3CA in nasopharyngeal carcinoma[J].Pathol Oncol Res,2015,22(2):413-419.
[16]Lien EC,Dibble CC,Toker A.PI3Ksignaling in cancer:beyond AKT[J].Curr Opin Cell Biol,2017,45:62-71.
[17]许振,刘志强,郭庆枝,等.宫颈癌组织AKT和P-AKT表达与LMVD相关性及其对新辅助化疗效果预测意义[J].中华肿瘤防治杂志,2017,24(13):906-911.
[18]熊焰,曲琳琳,李东,等.非小细胞肺癌磷脂酰肌醇3激酶p110α蛋白高表达的临床意义及机制[J].中华肿瘤杂志,2017,39(10):737-743.
[19]Soler A,Figueiredo AM,Castel P,et al.Therapeutic benefit of selective inhibition of p110αPI3-kinase in pancreatic neuroendocrine tumors[J].Clin Cancer Res,2016,22(23):5805-5817.
[20]Wojtalla A1,Salm F,Christiansen DG,et al.Novel agents targeting the IGF-1R/PI3Kpathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma[EB/OL].PLoS One,2012,7(10):e47109[2018-04-09].https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047109.
[21]Weber GL,Parat MO,Binder ZA,et al.Abrogation of PIK3CAor PIK3R1reduces proliferation,migration,and invasion in glioblastoma multiforme cells[J].Oncotarget,2011,2(11):833-849.