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目的探讨宫颈癌Siha细胞自噬过程中微管末端结合蛋白1(EB1)基因的表达变化。方法 Hank平衡盐溶液(HBSS)和秋水仙素处理体外培养的宫颈癌Siha细胞,分别采用实时定量PCR和Western blotting检测EB1基因、LC3和p62的表达,荧光显微镜检测特异性绿色荧光蛋白(GFP)-LC3以证实自噬体形成。结果宫颈癌Siha细胞随HBSS作用时间延长,LC3 mRNA、EB1 mRNA、LC3-Ⅱ蛋白和EB1蛋白的表达均呈时间依赖性增加,差异具有统计学意义(P<0.05)。EB1 mRNA和LC3 mRNA表达呈正相关,具有统计学意义(P<0.05)。p62mRNA和蛋白在HBSS作用后的表达呈时间依赖性降低,差异具有统计学意义(P<0.05),EB1 mRNA和p62mRNA表达呈负相关,具有统计学意义(P<0.05)。GFP-LC3结果显示,在HBSS作用12 h后,Siha细胞胞质中GFP-LC3强度和数目明显增加,含有GFP信号的细胞数量也明显增加。秋水仙素处理后,LC3、p62和EB1 mRNA及蛋白均未发生显著改变,差异无统计学意义(P>0.05),但此时几乎检测不到含GFP-LC3荧光信号的细胞。结论EB1在饥饿状态Siha细胞中高表达,此时伴随LC3表达增加、p62表达降低以及自噬体形成增加,而用秋水仙素破坏EB1赖以作用的微管时,上述现象消失,充分表明EB1参与细胞自噬的可能。
Objective To investigate the changes of the expression of microtubule-binding protein-1 (EB1) gene during autophagy in Siha cells. Methods Hanks balanced salt solution (HBSS) and colchicine treatment of cervical cancer Siha cells cultured in vitro were used to detect the expression of EB1 gene, LC3 and p62 by real-time quantitative PCR and Western blotting, fluorescence microscopy specific green fluorescent protein (GFP) LC3 to confirm autophagosome formation. Results The expression of LC3 mRNA, EB1 mRNA, LC3-Ⅱ protein and EB1 protein were increased in a time-dependent manner in HCC patients with Siha cells. The difference was statistically significant (P <0.05). EB1 mRNA and LC3 mRNA expression was positively correlated, with statistical significance (P <0.05). The expression of p62 mRNA and protein in HBSS decreased in a time-dependent manner, the difference was statistically significant (P <0.05). The expression of EB1 mRNA and p62 mRNA was negatively correlated with statistical significance (P <0.05). GFP-LC3 results showed that the intensity and number of GFP-LC3 in the cytoplasm of Siha cells increased significantly after treated with HBSS for 12 h, and the number of GFP-containing cells also increased significantly. After colchicine treatment, the mRNA and protein of LC3, p62 and EB1 did not change significantly (P> 0.05), but almost no cells with GFP-LC3 fluorescence signal were detected at this time. Conclusions EB1 is overexpressed in starvation Siha cells, which is accompanied by the increase of LC3 expression, the decrease of p62 expression and the increase of autophagosome formation. However, this phenomenon disappears when colchicine is used to destroy EB1-dependent microtubules, which indicates that EB1 is involved Cell autophagy may.