ULF基因沉默对依托泊苷引起的非小细胞肺癌H1299细胞凋亡的影响
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摘要
目的探讨RNA干扰靶向抑制ULF的表达对化疗药物依托泊苷(etoposide)引起的非小细胞肺癌H1299细胞凋亡的影响。方法设计3条针对ULF基因的小干扰RNA(small interfering RNA,siRNA)片段及1条阴性对照siRNA,分别在慢病毒载体介导下感染H1299细胞。用real-time PCR及Western印迹法筛选出1条抑制效率最高的ULF序列,对有效干扰组和对照干扰组细胞进行etoposide处理,干扰对照细胞或沉默ULF的H1299细胞,用c-PARP做标记物检测H1299细胞的凋亡程度,MTT法检测H1299细胞增殖情况,流式细胞仪检测细胞周期分布。结果经感染ULF-shRNA-1序列的H1299细胞中ULFmRNA及蛋白表达降低最为明显,抑制率达80%,选择该条siRNA作为有效干扰组进行后续实验。Western blot结果显示etoposide处理后,有效干扰组H1299细胞凋亡水平显著增加,细胞增殖抑制率显著高于对照干扰组,并与etoposide剂量呈正相关,2组比较差异均有统计学意义(P<0.05)。流武结果显示有效干扰组G0/G1期细胞较对照组增加,S期细胞减少,差异有统计学意义(P<0.05)。结论 etoposide处理后通过干扰ULF蛋白表达可诱导H1299细胞的凋亡,抑制细胞增殖,使细胞出现S期阻滞,推测ULF基因可能成为癌症基因治疗的一个新靶点。
Objective To explore the effects of knocking down ULF gene on the apoptosis of non-small-cell carcinoma H1299 cell after treatment with etoposide.Methods Three ULF small interfering RNA(siRNA) sequences and one negative control siRNA sequence were designed and synthesized,and then individually transfected into H1299 cell via lentivirus.The interference efficiency of ULF-siRNA were screened by real-time PCR and Western blotting.Then the most target siRNA was used for apoptosis assay after treatment with etoposide,MTT assay for H1299 cell proliferation,flow cytometry for cell cycle distribution.Results The expression of ULF gene and its protein ULF were down-regulated in H1299 cell when transfected with ULFsiRNA,and ULF-siRNA-1 was the most effective one,which had the highest inhibition rate(80%) of ULF expression.Compared with negative control group,ULF-siRNA group showed an obvious apoptosis after treatment with etoposide,and the inhibition rate of was higher than control group,which was positively correlated with etoposide dose,the difference was statistically significant(P < 0.05).Flow cytometry showed that compared with the control group,G0/G1 cell cycle in ULF-siRNA group was increased,and S phase cells was decreased,the differences were all statistically significant(P <0.05).Conclusion Down-regulation ULF protein expression through treatment with etoposide can induce apoptosis of non-small-cell carcinoma H1299 cells,and inhibit cell proliferation,which lead to cell cycle arrest ULF gene may become the new target of gene therapy for cancer.
Objective To explore the effects of knocking down ULF gene on the apoptosis of non-small-cell carcinoma H1299 cell after treatment with etoposide.Methods Three ULF small interfering RNA(siRNA) sequences and one negative control siRNA sequence were designed and synthesized,and then individually transfected into H1299 cell via lentivirus.The interference efficiency of ULF-siRNA were screened by real-time PCR and Western blotting.Then the most target siRNA was used for apoptosis assay after treatment with etoposide,MTT assay for H1299 cell proliferation,flow cytometry for cell cycle distribution.Results The expression of ULF gene and its protein ULF were down-regulated in H1299 cell when transfected with ULFsiRNA,and ULF-siRNA-1 was the most effective one,which had the highest inhibition rate(80%) of ULF expression.Compared with negative control group,ULF-siRNA group showed an obvious apoptosis after treatment with etoposide,and the inhibition rate of was higher than control group,which was positively correlated with etoposide dose,the difference was statistically significant(P < 0.05).Flow cytometry showed that compared with the control group,G0/G1 cell cycle in ULF-siRNA group was increased,and S phase cells was decreased,the differences were all statistically significant(P <0.05).Conclusion Down-regulation ULF protein expression through treatment with etoposide can induce apoptosis of non-small-cell carcinoma H1299 cells,and inhibit cell proliferation,which lead to cell cycle arrest ULF gene may become the new target of gene therapy for cancer.
引文
[1]Hanahan D,Weinberg RA.The Hallmarks of cancer[J].Cell,2000,100(1):57-70.
[2]Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646-674.
[3]Vucic D,Dixit VM,Wertz IE.Ubiquitylation in apoptosis:a posttranslational modification at the edge of life and death[J].Nat Rev Mol Cell Biol,2011,12(7):439-452.
[4]Doco-Fenzy M,Landais E,Andrieux J,et al.Deletion 2q36.2q36.3with mμLtiple renal cysts and severe mental retardation[J].Eur J Med Genet,2008,51(6):598-607.
[5]Komander D,Clague MJ,Urbe S.Breaking the chains:structure and function of the deubiquitinases[J].Nat Rev Mol Cell Biol,2009,10(8):550-563.
[6]Park Y,Yoon SK,Yoon JB.TRIP12 functions as an E3 ubiquitin ligase of APP-BP1[J].Biochera Biophys Res Commun,2008,374(2):294-298.
[7]Park Y,Yoon SK,Yoon JB.The HECT domain of TKIP12 ubiquitinates substrates of the ubiquitin fusion degradation pathway[J].J Biol Chem,2009,284(3):1540-1549.
[8]Chen D,Shan J,Zhu WG,et al.Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses[J].Nature,2010,464(7288):624-627.
[9]史晨辉,王维山,李长俊,uPA-siRNA重组慢病毒载体感染兔软骨细胞对uPA和MMP-3表达的影响[J].重庆医学,2014,43(15):1892-1895.
[10]张娇,陈爱平,戚玉言,等.靶向EGFR的干扰RNA对卵巢癌耐药细胞凋亡的影响[J].中国肿瘤生物治疗杂志,2009,16(6):619.4623.
[11]许淑芬,刘磊,方艳秋.RNAi技术沉默IGFBP-2基因对人乳腺癌细胞株MCF-7增殖的影响[J].中国妇幼保健,2013,28(1):4719-4721.
[12]Jorgensen C,Apparailly F.Prospects for gene therapy ininflammatory arthritis[J].Best Pract Res Clin Rheuma-tol,2010,24(4):541-552.
[13]褚波,黄雪峰,唐云明.慢病毒载体及其应用进展[J].生物医学工程学杂志,2008,2(1):224-226.
[14]Sakuma T,Barry MA,Ikeda Y.Lentiviral rectors:basic to translational[J].Bio chem J,2012,443(3):603-608.
[15]Visse R,Nagase H.Matrix metalloproteinases and tissue inhibitors of metalloproteihases:structure,function,and biochemistry[J].Circ Res,2003,92(8):827-839.
[2]Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646-674.
[3]Vucic D,Dixit VM,Wertz IE.Ubiquitylation in apoptosis:a posttranslational modification at the edge of life and death[J].Nat Rev Mol Cell Biol,2011,12(7):439-452.
[4]Doco-Fenzy M,Landais E,Andrieux J,et al.Deletion 2q36.2q36.3with mμLtiple renal cysts and severe mental retardation[J].Eur J Med Genet,2008,51(6):598-607.
[5]Komander D,Clague MJ,Urbe S.Breaking the chains:structure and function of the deubiquitinases[J].Nat Rev Mol Cell Biol,2009,10(8):550-563.
[6]Park Y,Yoon SK,Yoon JB.TRIP12 functions as an E3 ubiquitin ligase of APP-BP1[J].Biochera Biophys Res Commun,2008,374(2):294-298.
[7]Park Y,Yoon SK,Yoon JB.The HECT domain of TKIP12 ubiquitinates substrates of the ubiquitin fusion degradation pathway[J].J Biol Chem,2009,284(3):1540-1549.
[8]Chen D,Shan J,Zhu WG,et al.Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses[J].Nature,2010,464(7288):624-627.
[9]史晨辉,王维山,李长俊,uPA-siRNA重组慢病毒载体感染兔软骨细胞对uPA和MMP-3表达的影响[J].重庆医学,2014,43(15):1892-1895.
[10]张娇,陈爱平,戚玉言,等.靶向EGFR的干扰RNA对卵巢癌耐药细胞凋亡的影响[J].中国肿瘤生物治疗杂志,2009,16(6):619.4623.
[11]许淑芬,刘磊,方艳秋.RNAi技术沉默IGFBP-2基因对人乳腺癌细胞株MCF-7增殖的影响[J].中国妇幼保健,2013,28(1):4719-4721.
[12]Jorgensen C,Apparailly F.Prospects for gene therapy ininflammatory arthritis[J].Best Pract Res Clin Rheuma-tol,2010,24(4):541-552.
[13]褚波,黄雪峰,唐云明.慢病毒载体及其应用进展[J].生物医学工程学杂志,2008,2(1):224-226.
[14]Sakuma T,Barry MA,Ikeda Y.Lentiviral rectors:basic to translational[J].Bio chem J,2012,443(3):603-608.
[15]Visse R,Nagase H.Matrix metalloproteinases and tissue inhibitors of metalloproteihases:structure,function,and biochemistry[J].Circ Res,2003,92(8):827-839.