miR-449b对GBM细胞替莫唑胺治疗敏感性的影响
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摘要
目的 miR-449b在多种肿瘤细胞中都处于低表达状态,是潜在的肿瘤抑制因子。本课题主要探究miR-449b表达对GBM细胞替莫唑胺(TMZ)治疗敏感性的的影响及内在的调控机制。方法选用两株GBM细胞,T98G和LN229,将miR-449b模拟剂转入这两种细胞中,采用MTT法、克隆集落法和免疫印迹法检测miR-449b表达对不同剂量TMZ处理后的GBM细胞的存活、增殖能力和凋亡水平的影响;同时检测miR-449b表达对替莫唑胺治疗下GBM细胞ATP水平的影响,以及将外源ATP脂质体转入后对细胞活力的影响。结果 MTT研究表明,增加miR-449b表达可降低替莫唑胺治疗下GBM细胞的存活能力,表现为肿瘤细胞活力下调、克隆集落形成能力减弱及Cleaved-PARP的蛋白水平增加;ATP检测结果发现,miR-449b表达可降低替莫唑胺应激下肿瘤细胞ATP水平,并且外源性ATP补充可明显增强替莫唑胺治疗下肿瘤细胞的活力。结论以上研究结果提示,miR-449b可通过加剧替莫唑胺治疗下GBM细胞ATP水平消耗,进而促进GBM细胞对替莫唑胺治疗的敏感性,有利于提高替莫唑胺对GBM的治疗效果及其在临床上的应用。
Objective Mir-449b, a member of the miR-449 family, has been shown to be down-regulated in a variety of human malignancies, and to be a potential tumor suppressor. The aim of this study was to study the role of miR-449 b in GBM cells sensitivity to TMZ, and the involved mechanism by which miR-449 b contributes GBM cells sensitivity to TMZ therapy.Methods Cell biology, molecular biology, pharmacology experiment methods were used in this study.Results ctopic expression of miR-449 b with specific mimics significantly enhanced the cytotoxicity of TMZ therapy in GBM cells, as indicated by decreases in GBM cells viability and colony formation, and increase in the amount of Cleaved-PARP. ATP assay showed that miR-449 b mimic greatly diminished ATP level, and exogenous ATP supplementation partially reduced the cytotoxicity effect of TMZ in GBM cells. Conclusion These results suggest that promoting ATP depletion contribute to the miR-449 b mediated TMZ-sensitivity.
Objective Mir-449b, a member of the miR-449 family, has been shown to be down-regulated in a variety of human malignancies, and to be a potential tumor suppressor. The aim of this study was to study the role of miR-449 b in GBM cells sensitivity to TMZ, and the involved mechanism by which miR-449 b contributes GBM cells sensitivity to TMZ therapy.Methods Cell biology, molecular biology, pharmacology experiment methods were used in this study.Results ctopic expression of miR-449 b with specific mimics significantly enhanced the cytotoxicity of TMZ therapy in GBM cells, as indicated by decreases in GBM cells viability and colony formation, and increase in the amount of Cleaved-PARP. ATP assay showed that miR-449 b mimic greatly diminished ATP level, and exogenous ATP supplementation partially reduced the cytotoxicity effect of TMZ in GBM cells. Conclusion These results suggest that promoting ATP depletion contribute to the miR-449 b mediated TMZ-sensitivity.
引文
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[37]Josson S,Sung S Y,Lao K,et al.Radiation modulation of micro RNA in prostate cancer cell lines[J].Prostate,2008,68(15):1599-1606.
[2]Mo R,Jiang T,Disanto R,et al.ATP-triggered anticancer drug delivery[J].Nature Database of Systematic Reviews,2011,3(3):CD007294.
[3]Bost F,Decoux-Poullot A G,Tanti J F,et al.Energy disruptors:rising stars in anticancer therapy?Oncogenesis,2016.5:188.
[4]Hart M G,Grant R,Garside R,et al.Chemotherapy wafers for high grade glioma[J].Cochrane,2011,16(3):CD007294.
[5]Kim C,Kim Y,Han J,et al.Survival benefit of levetiracetam in patients treated with concomitant chemoradiotherapy and adjuvant chemotherapy with temozolomide for glioblastoma multiforme.Cancer,2015.121(17):2926-2932.
[6]Fang C,Wang K,Stephen Z R,et al.Temozolomide nanoparticles for targeted glioblastoma therapy[J].ACS Appl Mater Interfaces,2015,7(12):6674-6682.
[7]Hegi M E,Liu L,Herman J G,et al.Correlation of O6-methylguanine methyltransferase[MGMT]promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity[J].J Clin Oncol,2008.26(25):4189-4199.
[8]Wang H,Cai S,Ernstberger A,et al.Temozolomide-mediated DNA methylation in human myeloid precursor cells:differential involvement of intrinsic and extrinsic apoptotic pathways.Clin Cancer Res,2013,19(10):2699-2709.
[9]Mallory A C,Reinhart B J,Jones R M W,et al.Micro RNAcontrol of PHABULOSA in leaf development:importance of pairing to the micro RNA 5'region[J].EMBO J,2004,23(16):3356-3364.
[10]Noonan E J,Place R F,Pookot D,et al.mi R-449a targets HDAC-1 and induces growth arrest in prostate cancer[J].Oncogene,2009,28(14):1714-1724.
[11]Kaila K,Saarikoski J,Voipio J.Mechanism of action of GABAon intracellular p H and on surface p H in crayfish muscle fibres[J].JPhysiol,1990:427:241-260.
[12]Wheeler G,Ntouniafousara S,Granda B,et al.Identification of new central nervous system specific mouse micro RNAs[J].FEBSLett,2006,580(9):2195-2200.
[13].Buurman R,Gürlevik E,Sch?ffer V,et al.Histone deacetylases activate hepatocyte growth factor signaling by repressing micro RNA-449 in hepatocellular carcinoma cells[J].Gastroenterology,2012,143(3):811-820 e1-15.
[14]Fang Y,Gu X,Li Z,et al.mi R-449b inhibits the proliferation of SW1116 colon cancer stem cells through downregulation of CCND1 and E2F3 expression[J].Oncol Rep,2013.30(1):399-406.
[15]Luo W,Huang B,Li Z,et al.Micro RNA-449a is downregulated in non-small cell lung cancer and inhibits migration and invasion by targeting c-Met[J].PLo S One,2013.8(5):64759.
[16]Yang X,Feng M,Jiang X,et al.mi R-449a and mi R-449b are direct transcriptional targets of E2F1 and negatively regulate p Rb-E2F1 activity through a feedback loop by targeting CDK6 and CDC25A[J].Genes Dev,2009.23(20):2388-2393.
[17]Alissa M,Aunica J,Bryar P J,et al.Micro RNAs-449a and-449b exhibit tumor suppressive effects in retinoblastoma.Biochem Biophys Res Commun,2013.440(4):599-603.
[18]?stling P,Leivonen S K,Aakula A,et al.Systematic analysis of micro RNAs targeting the androgen receptor in prostate cancer cells.Cancer Res,2011,71(5):1956-1967.
[19]Yang X,Feng M,Jiang X,et al.Two mi RNA clusters,mi R-34b/c and mi R-449,are essential for normal brain development,motile ciliogenesis,and spermatogenesis[J].Proc Natl Acad Sci U SA,2014,111(28):2851-2857.
[20]Chen L,Xu L,Wang G.,Regulation of MET-mediated proliferation of thyroid carcinoma cells by mi R-449b[J].Tumour Biol,2015,36(11):8653-8660.
[21]Sandbothe M,Buurman R,Reich N,et al.The micro RNA-449family inhibits TGF-beta-mediated liver cancer cell migration by targeting SOX4.J Hepatol,2017,66(5):1012-1021.
[22]Brodbelt A,Greenberg D,Winters T,et al.Glioblastoma in England:2007-2011[J].Eur J Cancer,2015,51(4):533-542.
[23]Cloughesy T F,Cavenee W K,Mischel P S.Glioblastoma:from molecular pathology to targeted treatment[J].Annu Rev Pathol,2014.9:1-25.
[24]Ostrom Q T,Gittleman H,Farah P,et al.CBTRUS Statistical Report:Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012[J].Neuro Oncol,2015,17 Suppl 4:1-62.
[25]Godquin B.Was Homer a surgeon?[J].Chirurgie,1990,116(2):136-143.
[26]Wen P Y,Kesari S.Malignant gliomas in adults[J].N Engl JMed,2008,359(5):492-507.
[27]Stupp R,Hegi M E,Mason W P,et al.Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study:5-year analysis of the EORTC-NCIC trial[J].Lancet Oncol,2009,10(5):459-466.
[28]Weller M,Bent M V D,Hopkins K,et al.EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma[J].Lancet Oncol,2014,15(9):395-403.
[29]Chinot O L,Wick W,Mason W,et al.Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glioblastoma[J].NEngl J Med,2014,370(8):709-722.
[30]Batchelor T T,Mulholland P,Neyns B,et al.Phase III randomized trial comparing the efficacy of cediranib as monotherapy,and in combination with lomustine,versus lomustine alone in patients with recurrent glioblastoma[J].J Clin Oncol,2013,31(26):3212-3218.
[31]Garzon R,Calin G A,Croce C M.Micro RNAs in Cancer[J].Annu Rev Med,2009,60:167-179.
[32]Liu S,Pan X,Yang Q,et al.Micro RNA-18a enhances the radiosensitivity of cervical cancer cells by promoting radiationinduced apoptosis[J].Oncol Rep,2015,33(6):2853-2862.
[33]Lynamlennon N,Reynolds J V,Marignol L,et al.Micro RNA-31 modulates tumour sensitivity to radiation in oesophageal adenocarcinoma[J].J Mol Med,2012,90(12):1449-1458.
[34]Yan D,Ng WL,Zhang X,et al.Targeting DNA-PKcs and ATMwith mi R-101 sensitizes tumors to radiation[J].PLo S One,2010,5(7):e11397.
[35]Lee K M,Choi E J,Kim I A.micro RNA-7 increases radiosensitivity of human cancer cells with activated EGFR-associated signaling[J].Radiother Oncol,2011,101(1):171-176.
[36]Moskwa P,Buffa F M,Pan Y,et al.mi R-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors.Mol Cell,2011,4(2):210-220.
[37]Josson S,Sung S Y,Lao K,et al.Radiation modulation of micro RNA in prostate cancer cell lines[J].Prostate,2008,68(15):1599-1606.