【摘要】 【目的】应用慢病毒介导的RNA干扰(RNAi)技术,有效沉默鼻咽癌细胞CNE-2的CR-1基因表达,研究CR-1对鼻咽癌细胞生物学行为的影响。【方法】构建针对CR-1的siRNA慢病毒表达载体[含H1启动子和绿色荧光蛋白(GFP)]。利用包装细胞293FT生产慢病毒,感染鼻咽癌细胞CNE-2,流式筛选GFP阳性细胞。应用荧光定量PCR、Westernblot检测病毒感染后CNE-2细胞中CR-1基因的mRNA和蛋白表达,获得干扰CR-1基因的细胞群。并用MTT法、平板克隆形成实验及Boyden侵袭小室观察CR-1干扰后对CNE-2细胞生物学行为的影响。【结果】PCR和测序证实,成功构建了CR-1 shRNA的慢病毒载体pLVTHM-shCR-1。荧光定量PCR和Western blot结果表明,所获得的细胞中CR-1 mRNA及蛋白表达水平较阴性对照组、空白对照组均显著降低(P<0.05)。CR-1干扰后的细胞生长明显减慢,克隆形成能力减弱,侵袭运动能力下降。【结论】通过RNAi技术阻断CR-1的表达,可抑制CNE-2细胞的生长、增殖、迁徙,提示CR-1在鼻咽癌的发生、发展过程中起重要作用。更多还原

【Abstract】 【Objective】 To study the effect of CR-1 down-regulation by lentivirus-mediated RNA interference(RNAi) silencing technique on the biological features of nasopharyngeal carcinoma cell line CNE-2.【Methods】 A lentiviral vector for RNAi of CR-1,which contained H1 promoter and green fluorescent protein(GFP) was constructed.Package cells 293FT was used to produce virus stocks.Then CNE-2 cells were infected with the recombinant lentivirus and the cells with CR-1 knock-down were selected by FACS for GFP expression.CR-1 expression in the cells was determined by real-time reverse transcription-polymerase chain reaction(RT-PCR) and Western blot.The proliferative and invasive ability of CNE-2 cells were evaluated by MTT methods,the colony-forming unit assay and transwell migration assay,respectively.【Results】 PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of cripto-1(pLVTHM-shCR-1) was constructed successfully.Clones of CNE-2 cells infected with the recombinantlentivirus were selected and exhibited substantial knock-down of CR-1 mRNA and protein expression compared with the controlgroups.The proliferation,colony-forming ability and invasive ability of treated CNE-2 cells were inhibited in vitro.【Conclusion】The lentivirus-mediated shRNA of CR-1 is efficient in down-regulating CR-1 expression and diminishing the growth,proliferation and invasiveness of CNE-2 cells,suggesting that Cripto-1 might have an oncogene role in the tumorigenesis and progression of NPC.更多还原