shRNA沉默AnnxinA2基因对放射抗拒鼻咽癌细胞迁移和侵袭的影响
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摘要
目的探讨sh RNA沉默Annxin A2基因对放射抗拒鼻咽癌细胞迁移和侵袭的影响。方法采用Fu GENE HD将Annexin A2 sh RNA转染放射抗拒的鼻咽癌CNE2(R743)细胞,q RT-PCR验证转染细胞中Annxin A2基因的表达,细胞划痕实验和Transwell实验观察下调Annxin A2基因表达及联合照射对鼻咽癌细胞迁移和侵袭的影响,Western blot检测细胞中基质金属蛋白酶2(MMP2)蛋白的表达。结果转染组细胞Annexin A2基因的m RNA相对表达量为(0.25±0.17),较对照组的(1±0.00)和转染对照组的(0.96±0.06)明显下降,差异有统计学意义(P<0.05)。细胞划痕实验显示,下调Annxin A2基因表达可抑制照射诱导的CNE2(R743)细胞的迁移能力(P<0.05)。Transwell实验结果显示,下调Annxin A2基因表达可抑制照射诱导的CNE2(R743)细胞的迁移和侵袭能力(P<0.05)。Western blot结果表明,下调Annxin A2基因表达能抑制照射诱导的MMP2蛋白的表达上调(P<0.05)。结论下调Annxin A2基因表达能抑制照射诱导放射抗拒的鼻咽癌CNE2(R743)细胞的迁移和侵袭能力,可能与MMP2蛋白表达下调相关。
Objective To investigate the effect of shR NA-mediated silencing of Annexin A2 and combined irradiation on the migration and invasion of radioresistant nasopharyngeal carcinoma cells. Methods Annexin A2 shR NA was transfected into CNE2(R743)cells using FuG ENE HD. The expression of Annexin A2 gene in transfected cells was verified by qR T-PCR. The effect of down-regulation of Annexin A2 expression combined with irradiation on the migration and invasion of nasopharyngeal carcinoma cells were determined by scratch assay and transwell chamber assay. The expression of MMP2 was detected by Western blot. Results The relative expression of Annexin A2 mR NA in transfected group(0.25±0.17)was significantly lower than in control group(1±0.00)and transfected control group(0.96±0.06),the difference was statistically significant(P<0.05). Cell scratches showed that down-regulation of Annexin A2 could inhibit the migration of CNE2(R743)cells induced by irradiation(P<0.05). Transwell assay showed that down-regulation of Annexin A2 expression inhibited the migration and invasion of CNE2(R743)cells induced by irradiation(P<0.05). Western blot analysis showed that down-regulation of Annexin A2 could inhibit the upregulation of MMP2 protein expression induced by irradiation(P<0.05). Conclusion Down-regulation of Annexin A2 can inhibit the migration and invasion of CNE2(R743)cells induced by irradiation,which may be related to the down-regulation of MMP2 protein expression.
Objective To investigate the effect of shR NA-mediated silencing of Annexin A2 and combined irradiation on the migration and invasion of radioresistant nasopharyngeal carcinoma cells. Methods Annexin A2 shR NA was transfected into CNE2(R743)cells using FuG ENE HD. The expression of Annexin A2 gene in transfected cells was verified by qR T-PCR. The effect of down-regulation of Annexin A2 expression combined with irradiation on the migration and invasion of nasopharyngeal carcinoma cells were determined by scratch assay and transwell chamber assay. The expression of MMP2 was detected by Western blot. Results The relative expression of Annexin A2 mR NA in transfected group(0.25±0.17)was significantly lower than in control group(1±0.00)and transfected control group(0.96±0.06),the difference was statistically significant(P<0.05). Cell scratches showed that down-regulation of Annexin A2 could inhibit the migration of CNE2(R743)cells induced by irradiation(P<0.05). Transwell assay showed that down-regulation of Annexin A2 expression inhibited the migration and invasion of CNE2(R743)cells induced by irradiation(P<0.05). Western blot analysis showed that down-regulation of Annexin A2 could inhibit the upregulation of MMP2 protein expression induced by irradiation(P<0.05). Conclusion Down-regulation of Annexin A2 can inhibit the migration and invasion of CNE2(R743)cells induced by irradiation,which may be related to the down-regulation of MMP2 protein expression.
引文
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[21]Kumar A,Collins HM,Scholefield JH,et al.Increased type-Ⅳcollagenase(MMP-2 and MMP-9)activity following preoperative radiotherapy in rectal cancer[J].British J Cancer,2000,82(4):960-965.
[22]Speake WJ,Dean RA,Kumar A,et al.Radiation induced MMP expression from rectal cancer is short lived but contributes to in vitro invasion[J].Eur J Surg Oncol,2005,31(8):869-874.
[23]Bharadwaj A,Bydoun M,Holloway R,et al.Annexin A2 heterotetramer:structure and function[J].Int J Mol Sci,2013,14(3):6259-6305.
[2]Lokman NA,Elder AS,Ween MP,et al.Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis[J].Oncotarget,2013,4(8):1199-1211.
[3]Zhang HJ,Yao DF,Yao M,et al.Annexin A2 silencing inhib its invasion,migration,and tumorigenic potential of hepa toma cell s[J].World J Gastroenterol,2013,19(24):3792-3801.
[4]Zhai H,Acharya S,Gravanis I,et al.Annexin A2 promotes glioma cell invasion and tumor progression[J].J Neurosci,2011,31(40):14346-14360.
[5]苏颖,何火聪,吴君心,等.si RNA沉默Annexin A2基因表达对鼻咽癌细胞放射敏感性的影响[J].中华放射肿瘤学杂志,2015,24(2):214-218.
[6]苏颖,吴君心,何火聪,等.鼻咽癌放射抗拒细胞株CNE-2(R743)的建立及其差异表达蛋白的初步分析[J].福建医科大学学报,2011,45(6):398-403.
[7]Lin S,Pan J,Han L,et al.Nasopharyngeal carcinoma treated with reduced-volume intensity-modulated radiation therapy:report on the3-year outcome of a prospective series[J].Int J Radiat Oncol Biol Phys,2009,75(4):1071-1078.
[8]Ong YK,Solares CA,Lee S,et al.Endoscopic nasopharyngectomy and its role in managing locally recurrent nasopharyngeal carcinoma[J].Otolaryngol Clin North Am,2011,44(5):1141-1154.
[9]Alonso-Alconada L,Santacana M,Garcia-Sanz P,et al.Annexin-A2 as predictor biomarker of recurrent disease in endometrial cancer[J].Int J Cancer,2015,136(8):1863-1873.
[10]Tristante E,Martínez CM,Jiménez S,et al.Association of a characteristic membrane pattern of annexin A2 with high invasiveness and nodal status in colon adenocarcinoma[J].Transl Res,2015,166(2):196-206.
[11]Yang SF,Hsu HL,Chao TK,et al.Annexin A2 in renal cell carcinoma:expression,function,and prognostic significance[J].Urol Oncol,2015,33(1):22e11-22e21.
[12]Liu X,Ma D,Jing X,et al.Overexpression of ANXA2 predicts adverse outcomes of patients with malignant tumors:a systematic review and meta-analysis[J].Med Oncol,2015,32(1):392.
[13]苏颖,何火聪,吴君心,等.鼻咽癌细胞放射敏感性的比较蛋白质组学研究[J].中华放射医学与防护杂志,2011,31(5):536-541.
[14]Nalla AK,Asuthkar S,Bhoopathi P,et al.Suppression of u PAR retards radiation-induced invasion and migration mediated by integrinβ1/FAK signaling in medulloblastoma[J].PLo S One,2010,5(9):e13006.
[15]Pickhard AC,Margraf J,Knopf A,et al.Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways[J].BMC Cancer,2011,11:388.
[16]高敏,张俊红,周云峰,等.X线照射人肺腺癌细胞后侵袭和转移能力变化及机制探讨[J].中华放射肿瘤学杂志,2013,22(2):163-166.
[17]Park CM,Park MJ,Kwak HJ,et al.Ionizing radiation enhances matrix metalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidylinositol 3-kinase/Akt signaling pathways[J].Cancer Res,2006,66(17):8511-8519.
[18]Mc Cawley LJ,Matrisian LM.Matrix metalloproteinases:multifunctional contributors to tumor progression[J].Mol Med Today,2000,6(4):149-156.
[19]Harada T,Arii S,Mise M,et al.Membrane-type matrix metalloproteinase-1(MT1-MMP)gene is overexpressed in highly invasive hepatocellular carcinomas[J].J Hepatol,1998,28(2):231-239.
[20]Hulboy DL,Rudolph LA,Matrisian LM.Matrix metalloproteinases as mediators of reproductive function[J].Mol Hum Reprod,1997,3(1):27-45.
[21]Kumar A,Collins HM,Scholefield JH,et al.Increased type-Ⅳcollagenase(MMP-2 and MMP-9)activity following preoperative radiotherapy in rectal cancer[J].British J Cancer,2000,82(4):960-965.
[22]Speake WJ,Dean RA,Kumar A,et al.Radiation induced MMP expression from rectal cancer is short lived but contributes to in vitro invasion[J].Eur J Surg Oncol,2005,31(8):869-874.
[23]Bharadwaj A,Bydoun M,Holloway R,et al.Annexin A2 heterotetramer:structure and function[J].Int J Mol Sci,2013,14(3):6259-6305.