TRIM22通过PI3K/AKT信号通路调控胶质母细胞瘤增殖、侵袭和迁移的研究
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摘要
目的:探讨基因TRIM22在人脑胶质母细胞瘤中的表达及对增殖、侵袭和迁移等生物能力的影响。方法:通过TCGA数据库分析胶质母细胞瘤组织和正常脑组织中TRIM22表达水平,采用实时定量PCR(qRT-PCR)和Western blot两种方法分别检测胶质母细胞瘤细胞系U87和U251中TRIM22 m RNA和蛋白表达水平。应用Lipofectamine 3000将2种TRIM22-si RNA分别转染至U87及U251细胞中,再分别通过q RT-PCR和Western blot检测胶质母细胞瘤细胞中TRIM22敲低效率。应用CCK-8法、细胞平板克隆形成实验来评估TRIM22敲低后对于细胞增殖能力的影响;应用细胞划痕实验和Transwell实验检测TRIM22敲低后细胞侵袭和迁移能力的变化。应用Western blot检测TRIM22敲低对PI3K/AKT信号通路蛋白和上皮间质化标记蛋白(N-cadherin、Vimentin、E-cadherin)表达的影响。结果:在U87及U251细胞中敲低TRIM22后,细胞增殖、侵袭和迁移都明显减弱,p-AKT(S473)、N-cadherin、Vimentin表达水平则明显降低,E-cadherin表达水平明显升高。结论:TRIM22可能通过调节PI3K/AKT信号通路调控胶质母细胞瘤增殖、侵袭和迁移能力。
Objective:To explore the effect of TRIM22 on proliferation,invasion and migration in glioblastoma. Methods:The expression levels of TRIM22 were analyzed in TCGA database and were examined in two glioblastoma cell lines(U87 and U251 cells)using q RT-PCR and Western blot. Two independent TRIM22 siRNAs were transfected into U87 and U251 cells,and the effect of TRIM22 depletion was confirmed. CCK8 assay and colony formation assay were carried out to assess the effect of TRIM22 depletion on glioblastoma cell proliferation. Transwell assay and wound healing assay were also performed to investigate the role of TRIM22 depletion on glioblastoma cell invasion and migration. The expression of p-AKT and epithelial-mesenchymal transition(EMT)-associated proteins including N-cadherin,Vimentin,E-cadherin were examined using Western blot after TRIM22 depletion. Results:Inhibition of TRIM22 expression markedly reduced the proliferation,invasion and migration of glioblastoma cells. Correspondingly,the expression of p-AKT(S473),N-cadherin and Vimentin were decreased,whereas the expression of E-cadherin was increased after TRIM22 depletion. Conclusion:TRIM22 is a crucial factor for regulating the proliferation,invasion and migration of glioblastoma cells through PI3K/AKT signaling.
Objective:To explore the effect of TRIM22 on proliferation,invasion and migration in glioblastoma. Methods:The expression levels of TRIM22 were analyzed in TCGA database and were examined in two glioblastoma cell lines(U87 and U251 cells)using q RT-PCR and Western blot. Two independent TRIM22 siRNAs were transfected into U87 and U251 cells,and the effect of TRIM22 depletion was confirmed. CCK8 assay and colony formation assay were carried out to assess the effect of TRIM22 depletion on glioblastoma cell proliferation. Transwell assay and wound healing assay were also performed to investigate the role of TRIM22 depletion on glioblastoma cell invasion and migration. The expression of p-AKT and epithelial-mesenchymal transition(EMT)-associated proteins including N-cadherin,Vimentin,E-cadherin were examined using Western blot after TRIM22 depletion. Results:Inhibition of TRIM22 expression markedly reduced the proliferation,invasion and migration of glioblastoma cells. Correspondingly,the expression of p-AKT(S473),N-cadherin and Vimentin were decreased,whereas the expression of E-cadherin was increased after TRIM22 depletion. Conclusion:TRIM22 is a crucial factor for regulating the proliferation,invasion and migration of glioblastoma cells through PI3K/AKT signaling.
引文
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[3]Stupp R,Mason WP,van den Bent MJ,et al.Radiothera-py plus concomitant and adjuvant temozolomide for glio-blastoma[J].N Engl J Med,2005,352(10):987-996
[4]Cammas F,Khetchoumian K,Chambon P,et al.TRIM in-volvement in transcriptional regulation[J].Adv Exp MedBiol,2012,770(1):59-76
[5]Cambiaghi V,Giuliani V,Lombardi S,et al.TRIM pro-teins in cancer[J].Adv Exp Med Biol,2012,770(1):77-91
[6]Duan Z,Gao B,Xu W,et al.Identification of TRIM22 as aRING finger E3 ubiquitin ligase[J].Biochem BiophysRes Commun,2008,374(3):502-506
[7]Saito-Kanatani M,Urano T,Hiroi H,et al.Identificationof TRIM22 as a progesterone-responsive gene in Ishikawaendometrial cancer cells[J].J Steroid Biochem Mol Biol,2015,154:217-225
[8]Sun Y,Ho GH,Koong HN,et al.Down-regulation of tri-partite-motif containing 22 expression in breast cancer is associated with a lack of p53-mediated induction[J].Bio-chem Biophys Res Commun,2013,441(3):600-606
[9]Zhan W,Han T,Zhang C,et al.TRIM59 Promotes the pro-liferation and migration of non-small cell lung cancercells by upregulating cell cycle related proteins[J].PLo SOne,2015,10(11):e0142596
[10]Liu L,Zhou XM,Yang FF,et al.TRIM22 confers poorprognosis and promotes epithelial-mesenchymal transitionthrough regulation of AKT/GSK3beta/beta-catenin signal-ing in non-small cell lung cancer[J].Oncotarget,2017,8(37):62069-62080
[11]Fresno Vara JA,Casado E,de Castro J,et al.PI3K/Aktsignalling pathway and cancer[J].Cancer Treat Rev,2004,30(2):193-204
[12]Jiang H,Shang X,Wu H,et al.Resveratrol downregulatesPI3K/Akt/m TOR signaling pathways in human U251 glio-ma cells[J].J Exp Ther Oncol,2009,8(1):25-33
[13]Han L,Yang Y,Yue X,et al.Inactivation of PI3K/AKTsignaling inhibits glioma cell growth through modulationof beta-catenin-mediated transcription[J].Brain Res,2010,1366(1):9-17
[14]Wei Y,Jiang Y,Zou F,et al.Activation of PI3K/Akt path-way by CD133-p85 interaction promotes tumorigenic ca-pacity of glioma stem cells[J].Proc Natl Acad Sci U S A,2013,110(17):6829-6834
[15]Wu YL,Maachani UB,Schweitzer M,et al.Dual inhibi-tion of PI3K/AKT and MEK/ERK pathways induces syner-gistic antitumor effects in diffuse intrinsic pontine gliomacells[J].Transl Oncol,2017,10(2):221-228
[16]Thiery JP.Epithelial-mesenchymal transitions in tumourprogression[J].Nat Rev Cancer,2002,2(6):442-454
[17]Yu JM,Sun W,Hua F,et al.BCL6 induces EMT by pro-moting the ZEB1-mediated transcription repression of E-cadherin in breast cancer cells[J].Cancer Lett,2015,365(2):190-200
[18]Roy BC,Kohno T,Iwakawa R,et al.Involvement of LKB1in epithelial-mesenchymal transition(EMT)of human lungcancer cells[J].Lung Cancer,2010,70(2):136-145
[19]Yoo YA,Kang MH,Lee HJ,et al.Sonic hedgehog path-way promotes metastasis and lymphangiogenesis via acti-vation of Akt,EMT,and MMP-9 pathway in gastric cancer[J].Cancer Res,2011,71(22):7061-7070
[20]Kong D,Sethi S,Li Y,et al.Androgen receptor splice vari-ants contribute to prostate cancer aggressiveness throughinduction of EMT and expression of stem cell markergenes[J].Prostate,2015,75(2):161-174
[21]Ye X,Brabletz T,Kang Y,et al.Upholding a role for EMTin breast cancer metastasis[J].Nature,2017,547(7661):E1-E3