GTP结合蛋白4在肝癌中的表达及作用的初步研究

摘要

目的：探讨GTP结合蛋白4（GTP binding protein 4，GTPBP4）在肝癌（hepatocelluar carcinoma，HCC）组织中的表达及沉默GT⁃PBP4对人肝癌HepG2细胞增殖和周期影响。方法：收集南昌大学第二附属医院2014年3月至2015年2月24例新鲜临床HCC组织及配对癌旁组织标本，采用Western blot检测GTPBP4在组织中的表达差异；在HepG2细胞中用慢病毒介导的RNAi干扰技术沉默该基因，运用荧光显微镜观察感染效率，Western blot、RT-qPCR检测沉默效果；CCK-8实验、流式细胞仪观察细胞增殖、细胞周期变化。结果：1）Western blot结果显示GTPBP4在21例（87.5%）HCC组织标本中明显高表达（P<0.0001）；2）用荧光显微镜观察显示慢病毒成功感染HepG2细胞后，发现90%细胞表达GFP；LV-GTPBP4-RNAi组GTPBP4在RNA水平、蛋白水平较对照组分别下降约70%和67%；3）GTPBP4基因沉默96 h后，LV-GTPBP4-RNAi组增殖能力抑制，抑制率约54.51%；LV-GTPBP4-RNAi组G0/G1期增多，S期减少，细胞周期发生停滞。结论：GTPBP4在HCC组织中高表达，利用RNAi干扰GTPBP4基因表达后，人肝癌HepG2细胞增殖能力明显下降，细胞周期停滞于G0/G1期，但是具体分子机制不明。%Objective:To investigate the protein expression of GTPBP4 in human hepatocelluar carcinoma (HCC) tissues and the influ-ence of GTPBP4 silencing by siRNA on the proliferation and cell cycle of HCC cell line Hep G2. Methods:Western blot analysis was per-formed to observe the protein expression of GTPBP4 in 24 cases of HCC tissues compared with their adjacent noncancerous liver tis-sues. Lentivirus-mediated RNA interference (RNAi) was used to silence GTPBP4 expression in Hep G2, and the infection efficiency was observed under a fluorescence microscope. The silencing effect was tested by Western blot and real-time PCR. After silencing the GT-PBP4 gene, cell proliferation was detected using CCK-8 assay, and the cell cycle was observed using flow cytometry. Results:(1) GT-PBP4 was overexpressed in 21 cases (87.5%) of HCC tissues (P<0.000 1). (2) After the lentivirus with GFP reporter infected the Hep G2 cells, 90%of the cells showed green fluorescence. LV-GTPBP4-RNAi effectively inhibited the expression of GTPBP4 at both mRNA (70%) and protein (67%) levels. (3) The proliferation ability of the LV-GTPBP4-RNAi group significantly decreased after 96 h (inhibition rate:54.51%). Flow cytometry showed that the LV-GTPBP4-RNAi group significantly increased at the G0/G1 phase, whereas the S phase de-creased and arrested at the G0/G1 phase. Conclusion:GTPBP4 overexpression in HCC tissues was associated with hepatocarcinogenesis and promoted tumor cell proliferation, but the specific molecular mechanisms remain to be investigated.