【摘要】 目的：探讨LRRFIP1表达上调对肝癌细胞Huh7的生物学行为影响，并初步探索其相关机制。方法：构建LRRFIP1过表达的稳定细胞株，实时荧光定量聚合酶链式反应（RT-qPCR）和蛋白免疫印迹法（western blotting）检测过表达效率，细胞增殖实验（CCK-8）检测过表达LRRFIP11对Huh7细胞增殖能力的影响；平板克隆形成实验检测过表达LRRFIP1对Huh7细胞克隆形成能力的影响；流式细胞术检测过表达LRRFIP1对Huh7细胞周期和凋亡的影响；划痕实验和Transwell实验检测过表达LRRFIP1对细胞迁移和侵袭能力的影响；检测与上皮间质转化（EMT）相关的蛋白E-cadherin、N-cadherin,Vimentin和Snail的表达；免疫共沉淀和蛋白质谱技术串联（CoIP-MS）法筛选可能与LRRFIP1互作蛋白。结果：过表达LRRFIP1促进了Huh7细胞的增殖（P<0.001）和克隆形成能力（P<0.001），抑制了细胞凋亡（P<0.01）；过表达LRRFIP1导致Huh7细胞G0/G1期比例减少，S期增加（P<0.001,P<0.01）。此外，过表达LRRFIP1组细胞的迁移（P<0.05）和侵袭（P<0.001）数量显著低于对照组；western blotting实验结果显示，与对照组相比，过表达LRRFIP1组细胞上皮细胞标志蛋白E-cadherin表达显著升高（P<0.001），间充质细胞标志蛋白N-cadherin和Vimentin表达显著减低（P<0.001,P<0.01）,EMT相关转录因子Snail蛋白表达显著降低（P<0.001）。最后，过表达LRRFIP1后我们采用CoIP-MS法筛选出80个可能与其互作蛋白，GO分析显示Huh7细胞株中下拉LRRFIP结合蛋白富集于27个生物过程、11个细胞组分和9种分子功能。KEGG通路注释显示这80种蛋白主要富集于ECM-受体相互作用信号通路；STRING和Cytoscape分析Hub基因。结论：LRRFIP1过表达促进了肝癌细胞Huh7的增殖和克隆形成能力，抑制其凋亡、迁移、侵袭能力；LRRFIP1可能与eEF1A1相互作用，从而影响肝癌的发生发展。更多还原

【Abstract】 Objective: To explore the effect of LRRFIP1 upregulation on the biological behavior of hepatocellular carcinoma cell Huh7, and to preliminarily explore its related mechanism. Methods: Stable cell line with LRRFIP1 overexpression was constructed, and the overexpression efficiency was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and western blotting. The effect of LRRFIP1 overexpression on the proliferation ability of Huh7 cells was detected by CCK-8; the effect of overexpression of LRRFIP1 on the clonogenic ability of Huh7 cells was detected by plate cloning experiment; the effect of overexpression of LRRFIP1 on cell cycle and apoptosis was detected by flow cytometry; the effect of overexpression of LRRFIP1 on the migration and invasion of Huh7 cells was detected by scratch assay and Transwell assay; western blotting was performed to detect the expressions of proteins E-cadherin, N-cadherin, Vimentin and Snail related to epithelial-mesenchymal transition(EMT).CoIP-MS was used to screen the proteins that might interact with LRRFIP1. Results: Overexpression of LRRFIP1 promoted the proliferation(P<0.001) and clonogenic ability(P<0.001) of Huh7 cells and inhibited cell apoptosis(P<0.01); overexpression of LRRFIP1 resulted in an decrease proportion in G0/G1 phase and S phase of Huh7 cells(P<0.001, P<0.01). In addition, the number of cell migration(P<0.05) and invasion(P<0.001)in the LRRFIP1 overexpression group was significantly lower than that in the control group. Western blotting results showed that compared with the control group, the expression of epithelial marker protein E-cadherin in the overexpressed LRRFIP1 group significantly increased(P<0.001), the expressions of mesenchymal marker proteins N-cadherin, Vimentin and the expression of EMT-related transcription factor Snail protein significantly decreased(P<0.001, P<0.01, P<0.001). Finally, after overexpression of LRRFIP1, CoIP-MS was used to screen80 proteins that might interact with LRRFIP1. GO analysis showed that the LRRFIP binding proteins in Huh7cell line was enriched in 27 biological processes, 11 cell components and 9 molecular functions. KEGG pathway annotation showed that these 80 proteins were mainly enriched in ECM-receptor interaction signal pathway;STRING and Cytoscape analyzed the Hub genes. Conclusion: LRRFIP1 overexpression can promote the proliferation and the clonogenic ability of Huh7 cells, inhibit their apoptosis, migration and invasion, and may interact with eEF1A1, thus affecting the occurrence and development of Hepatocellular carcinoma.更多还原