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目的探讨膜-细胞骨架联接蛋白ezrin对肝癌生长和运动转移能力的影响。方法选取4株肝癌细胞系(SF/SMMC7721、MHCC97-H、MHCC-1和HepG2)作为研究材料,针对ezrin基因设计小干扰RNA(siRNA)并通过脂质体转染入细胞。分别通过荧光实时定量聚合酶链反应(PCR)和Western印迹验证siRNA对ezrin在基因和蛋白水平的下调及下调作用随转染时间的变化。根据Western印迹的结果选取ezrin蛋白表达最低的时间点检测干扰后肝癌细胞生物学行为的改变。运用四甲基偶氮唑蓝(MTT)法检测肝癌细胞增殖能力的变化;流式细胞仪检测细胞周期;电子显微镜观察细胞伪足的形态和数量改变;transwell实验检测肝癌细胞运动和转移能力的变化。结果荧光实时定量PCR和Western印迹显示ezrin siRNA对ezrin在基因和蛋白水平均有显著的下调作用。下调ezrin蛋白可显著减慢4株肝癌细胞的生长速度,处于分裂期的肝癌细胞比例明显下降(SF/SMMC7721:28·07%下降到21·53%;MHCC97-H:24·94%下降到13·92%;MHCC-1:19·30%下降到13·2%;HepG2:7·73%下降到4·24%)。肿瘤细胞的运动侵袭能力下降,细胞伪足变短,且伪足的形成数量显著减少(SF/SMMC7721:20·8个/细胞±3·0个/细胞与13·2个/细胞±2·4个/细胞,P<0·05;MHCC97-H:18·4个/细胞±2·7个/细胞与14·0个/细胞±2·9个/细胞,P<0·01;MHCC-1:22·6个/细胞±3·5个/细胞与13·3个/细胞±1·9个/细胞,P<0·01;HepG2:31·0个/细胞±2·9个/细胞与17·8个/细胞±2·3个/细胞,P<0·01;每株计数5个细胞)。穿过人工基底膜的细胞数量也明显减少(SF/SMMC7721:49·9个±7·7个与31·9±5·2个,P<0·05;MHCC97-H:58·5个±4·2个与33·0个±3·3个,P<0·01;MHCC-1:57·6个±6·1个与28·3个±3·4个,P<0·01;HepG2:37·3个±3·0个与25·3个±2·3个,P<0·01;每株8个视野)。结论ezrin在肝癌生长和侵袭转移过程中发挥重要的作用,有可能成为抑制肝癌复发转移的关键分子。
Objective To investigate the effect of membrane-cytoskeleton attachment protein ezrin on the growth and motility of liver cancer. Methods Four hepatocellular carcinoma cell lines (SF / SMMC7721, MHCC97-H, MHCC-1 and HepG2) were selected as research materials. Small interfering RNA (siRNA) was designed for ezrin gene and transfected into cells by liposome. Fluorescence quantitative real-time polymerase chain reaction (PCR) and Western blotting were used to validate the down-regulation and down-regulation of ezrin at gene and protein level with the change of transfection time. According to the result of Western blotting, we selected the lowest ezrin expression time point to detect the change of biological behavior of hepatoma cells after interference. The proliferation of hepatocellular carcinoma cells was detected by MTT assay. The cell cycle was detected by flow cytometry. The morphological and quantitative changes of pseudopodia were observed by electron microscopy. The transwell assay was used to detect the cell motility and metastasis Variety. Results Fluorescence real-time quantitative PCR and Western blotting showed that ezrin siRNA had a significant down-regulation of ezrin at gene and protein levels. The down-regulation of ezrin protein could significantly slow down the growth of four hepatocarcinoma cells, and the proportion of hepatoma cells in the mitotic phase was significantly decreased (SF / SMMC7721: 28.07% to 21.53%; MHCC97-H: 24.94% to 13 · 92%; MHCC-1: 19 · 30% down to 13 · 2%; HepG2: 7 · 73% down to 4 · 24%). The motility of tumor cells was decreased, and pseudopodia became short and the number of pseudopodia was significantly reduced (SF / SMMC7721: 20.8 cells / cell ± 3.0 cells / cell and 13.2 cells / cell ± 2 · 4 cells / cell, P <0.05; MHCC97-H: 18.4 cells / cell ± 2.77 cells / cell and 14.0 cells / cell ± 2.9 cells / -1: 22.6 cells / cell ± 3.5 cells / cell and 13.3 cells / 1 ± 9 cells / cell, P <0.01; HepG2: 31.0 cells / cell ± 2.9 17.8 cells / cell ± 2.3 cells / cell, P <0.01; 5 cells per strain). The number of cells passing through the artificial basement membrane was also significantly reduced (SF / SMMC7721: 49.9 ± 7.7 and 31.9 ± 5.2, P <0.05; MHCC97-H: 58.5 ± 4 · 2 and 33 · 0 ± 3 · 3, P <0 · 01; MHCC-1: 57 ± 6 ± 6 · 1 and 28 · 3 ± 3 · 4, P <0 · 01 ; HepG2: 37 ± 3 ± 3.0 and 25 · 3 ± 2 · 3, P <0.01; 8 fields per plant). Conclusion ezrin plays an important role in the growth and invasion and metastasis of hepatocellular carcinoma and may be the key molecule that inhibits the recurrence and metastasis of hepatocellular carcinoma.