慢病毒介导RNAi下调LMP2A基因表达抑制EB病毒相关胃癌细胞的体外生长

Silencing of LMP2A by Lentivirus-mediated RNAi Inhibits Growth of Epstein-Barr Virus-associated Gastric Carcinoma Cells in vitro

背景:EB病毒(EBV)与多种人类肿瘤,如Burkitt淋巴瘤以及鼻咽癌、胃癌等上皮性肿瘤相关。病毒编码的潜伏膜蛋白2A(LMP2A)存在于部分EBV相关胃癌(EBVaGC)中,与肿瘤发生、发展密切相关。目的:应用慢病毒介导的RNA干扰(RNAi)技术抑制LMP2A基因表达,探讨下调LMP2A对EBVaGC细胞体外生长的影响。方法:构建慢病毒载体pGCSIL-LMP2A-shRNA-LV和阴性对照载体并转染EBVaGC细胞株GT38,以real-time PCR和蛋白质印迹法检测慢病毒载体的抑制效率,CCK-8实验、克隆形成实验和流式细胞术检测GT38细胞的细胞生长、细胞周期和细胞凋亡。结果:GT38细胞转染LMP2A-shRNA-LV后,LMP2AmRNA和蛋白表达分别下调65.4%和50.8%,进而导致细胞体外增殖受抑,克隆形成能力降低,G0/G1期细胞比例增加,细胞凋亡率增高,与转染阴性对照慢病毒载体和未予转染慢病毒的GT38细胞相比,差异均有统计学意义(P<0.05)。结论:慢病毒介导的RNAi技术能高效抑制LMP2A基因表达,进而抑制EBVaGC细胞的体外生长,诱导G0/G1期阻滞,促进细胞凋亡。LMP2A可作为EBVaGC基因治疗的潜在靶点。

pstein-Barr virus(EBV)is associated with various human lymphoid and epithelial malignancies such as Burkitt’s lymphoma,nasopharyngeal carcinoma and gastric carcinoma.LMP2A,a virus-encoded latent membrane protein is expressed in a portion of EBV-associated gastric carcinoma(EBVaGC)and has been shown to be related with the tumorigenesis and progression of EBVaGC.Aims:To explore the effect of LMP2A silencing on growth of EBVaGC cells in vitro by using a lentivirus-mediated RNA interference(RNAi)to inhibit LMP2A gene expression.Methods:A lentivirus vector pGCSIL-LMP2A-shRNA-LV and a negative control vector were constructed and transfected into the EBVaGC cell line GT38.Real-time PCR and Western blotting were used to determine the inhibitory effect of the lentivirus vector;CCK-8assay,colony formation assay and flow cytometry were employed to assess the cell growth,cell cycle and apoptosis of GT38cells.Results:In GT38cells transfected with LMP2A-shRNA-LV,the expression level of LMP2A mRNA was decreased by65.4%,and that of LMP2A protein was reduced by50.8%;the cell proliferation was inhibited,the colony formation ability was suppressed,the percentage of cells in G0/G1phase and apoptotic rate were increased when compared with those transfected with negative control vector or without transfection(P<0.05).Conclusions:Lentivirus-mediated RNAi is effective for silencing LMP2A gene expression,subsequently inhibiting the growth of EBVaGC cells,inducing G0/G1phase arrest and enhancing cell apoptosis in vitro.LMP2A is supposed to be a potential target for gene therapy of EBVaGC.