TRIM28siRNA通过活化E2F转录因子1增加非小细胞肺癌PAa细胞对依托泊苷的敏感性
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摘要
目的探讨siRNA沉默TRIM28(tripartite motif containing 28)增强非小细胞肺癌(NSCLC)PAa细胞对依托泊苷的敏感性和可能的机制。方法应用RNA干扰技术沉默TRIM28基因在PAa细胞中的表达;TRIM28siRNA单独或联合依托泊苷处理PAa细胞后,应用MTT法和集落形成实验检测各组细胞的增殖;流式细胞术检测细胞凋亡;Western blotting方法检测各组细胞中E2F转录因子1(E2F1)的表达水平。结果在PAa中沉默了TRIM28的表达;TRIM28siRNA联合依托泊苷显著抑制PAa细胞的增殖和集落形成,明显诱导细胞凋亡;联合处理的PAa细胞中E2F1mRNA和蛋白水平的表达明显增加,同时抗凋亡蛋白Bcl-2表达减少。结论 TRIM28siRNA联合化疗药物依托泊苷对抑制NSCLC细胞生长有明显效果。
Objective To investigate the effect of small interfering RNA( siRNA)-mediated silencing of TRIM28( tripartite motif containing 28) on the sensitivity of non-small cell lung cancer cell( NSCLC) PAa to etoposide and explore the possible mechanism. Methods The expression of TRIM28 in PAa cell was silenced by RNA interference. MTT and colony formation assay were used to assess the inhibitory effect of TRIM28 siRNA alone or in combination with etoposide on the growth and proliferation of PAa cells. The apoptosis of different groups was detected by flow cytometry( FCM) and the expression of E2 F transcription factor 1( E2 F1) was evaluated by Western blotting. Results A PAa cell line silenced with the TRIM28 siRNA was established. TRIM28 in combination with etoposide significantly inhibited the proliferation and colony formation of PAa cells. TRIM28 siRNA in combination with etoposide induced apoptosis and increased the expression of E2 F1 at mRNA and protein levels. Conclusion TRIM28 siRNA in combination with etoposide has an obvious effect on the growth of NSCLC cells,suggesting a new clinical treatment strategy for NSCLC.
Objective To investigate the effect of small interfering RNA( siRNA)-mediated silencing of TRIM28( tripartite motif containing 28) on the sensitivity of non-small cell lung cancer cell( NSCLC) PAa to etoposide and explore the possible mechanism. Methods The expression of TRIM28 in PAa cell was silenced by RNA interference. MTT and colony formation assay were used to assess the inhibitory effect of TRIM28 siRNA alone or in combination with etoposide on the growth and proliferation of PAa cells. The apoptosis of different groups was detected by flow cytometry( FCM) and the expression of E2 F transcription factor 1( E2 F1) was evaluated by Western blotting. Results A PAa cell line silenced with the TRIM28 siRNA was established. TRIM28 in combination with etoposide significantly inhibited the proliferation and colony formation of PAa cells. TRIM28 siRNA in combination with etoposide induced apoptosis and increased the expression of E2 F1 at mRNA and protein levels. Conclusion TRIM28 siRNA in combination with etoposide has an obvious effect on the growth of NSCLC cells,suggesting a new clinical treatment strategy for NSCLC.
引文
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[23]Dong YB,Yang HL,Elliott MJ,et al.Adenovirus-mediated E2F-1 gene transfer sensitizes melanoma cells to apoptosis induced by topoisomeraseⅡinhibitors[J].Cancer Res,2002,62(6):1776-1783.
[24]Okamoto K,Kitabayashi I,Taya Y.KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction[J].Biochem Biophys Res Commun,2006,351(1):216-222.
[2]Maghfoor I,Perry MC.Lung cancer[J].Ann Saudi Med,2005,25(1):1-12.
[3]Hanna N,Neubauer M,yiannoutsos C,et al.PhaseⅢstudy of cisplatin,etoposide,and concurrent chest radiation with or without consolidation docetaxel in patients with inoperable stageⅢnonsmall-cell lung cancer:the Hoosier Oncology Group and US Oncology[J].J Clin Oncol,2008,26(35):5755-5760.
[4]Messerschmidt DM,de Vries W,Ito M,et al.Trim28 is required for epigenetic stability during mouse oocyte to embryo transition[J].Science,2012,335(6075):1499-1502.
[5]Barde I,Rauwel B,Marin-Florez RM,et al.A KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific control of mitophagy[J].Science,2013,340(6130):350-353.
[6]Pfeifer GP.Protein phosphatase PP4:Role in dephosphorylation of KAP1 and DNA strand break repair[J].Cell Cycle,2012,11(14):2590-2591.
[7]White DE,Negorev D,Peng H,et al.KAP1,a novel substrate for PIKK family members,colocalizes with numerous damage response factors at DNA lesions[J].Cancer Res,2006,66(24):11594-11599.
[8]Gilmore-Hebert M,Ramabhadran R,Stern DF.Interactions of Erb B4 and Kap1 connect the growth factor and DNA damage response pathways[J].Mol Cancer Res,2010,8(10):1388-1398.
[9]Hu C,Zhang S,Gao X,et al.Roles of Kruppel-associated box(KRAB)-associated co-repressor KAP1 Ser-473 phosphorylation in DNA damage response[J].J Biol Chem,2012,287(23):18937-18952.
[10]Lin LF,Li CF,Wang WJ,et al.Loss of ZBRK1 contributes to the increase of KAP1 and promotes KAP1-mediated metastasis and invasion in cervical cancer[J].PLo S One,2013,8(8):e73033.
[11]Addison JB,Koontz C,Fugett JH,et al.KAP1 promotes proliferation and metastatic progression of breast cancer cells[J].Cancer Res,2015,75(2):344-355.
[12]Fitzgerald S,Sheehan KM,O’grady A,et al.Relationship between epithelial and stromal TRIM28 expression predicts survival in colorectal cancer patients[J].J Gastroenterol Hepatol,2013,28(6):967-974.
[13]Yokoe T,Toiyama Y,Okugawa Y,et al.KAP1 is associated with peritoneal carcinomatosis in gastric cancer[J].Ann Surg Oncol,2010,17(3):821-828.
[14]Zhang L,Zhu C,Guo Y,et al.Inhibition of KAP1 enhances hypoxia-induced Kaposi’s sarcoma-associated herpesvirus reactivation through RBP-Jκ[J].J Virol,2014,88(12):6873-6884.
[15]Liu L,Zhao E,Li C,et al.TRIM28,a new molecular marker predicting metastasis and survival in early-stage non-small cell lung cancer[J].Cancer Epidemiol,2013,37(1):71-78.
[16]Wang C,Rauscher FJ 3rd,Cress WD et al.Regulation of E2F1function by the nuclear corepressor KAP1[J].J Biol Chem,2007,282(41):29902-29909.
[17]Meng RD,Phillips P,El-Deiry WS.p53-independent increase in E2F-1 expression enhances the cytotoxic effects of etoposide and of adriamycin[J].Int J Oncol,1999,14(1):5-14.
[18]White D,Rafalska-Metcalf IU,Ivanov AV,et al.The ATM substrate KAP1 controls DNA repair in heterochromatin:Regulation by HP1 proteins and serine 473/824 phosphorylation[J].Mol Cancer Res,2012,10(3):401-414.
[19]Wang C,Ivanov A,Chen L,et al.MDM2 interaction with nuclear corepressor KAP1 contributes to p53 inactivation[J].EMBO J,2005,24(18):3279-3290.
[20]Kamitani S,Ohbayashi N,Ikeda O,et al.KAP1 regulates type I interferon/STAT1-mediated IRF-1 gene expression[J].Biochem Biophys Res Commun,2008,370(2):366-370.
[21]Huang C,Martin S,Pfleger C,et al.Cutting edge:a novel,human-specific interacting protein couples FOXP3 to a chromatinremodeling complex that contains KAP1/TRIM28[J].J Immunol,2013,190(9):4470-4473.
[22]Nip J,Strom DK,Fee BE,et al.E2F-1 cooperates with topoisomeraseⅡinhibition and DNA damage to selectively augment p53-independent apoptosis[J].Mol Cell Biol,1997,17(3):1049-1056.
[23]Dong YB,Yang HL,Elliott MJ,et al.Adenovirus-mediated E2F-1 gene transfer sensitizes melanoma cells to apoptosis induced by topoisomeraseⅡinhibitors[J].Cancer Res,2002,62(6):1776-1783.
[24]Okamoto K,Kitabayashi I,Taya Y.KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction[J].Biochem Biophys Res Commun,2006,351(1):216-222.