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目的干预人肝癌细胞系Huh-7的野生型p53诱导的磷酸酶1(Wip1)基因的表达,并观察其细胞生物学特性的变化。方法采用转染法,将促进Wip1基因表达的质粒和抑制Wip1基因表达的小干扰RNA(siRNA)分别转染Huh-7细胞。质粒转染的Huh-7细胞为质粒转染组,未经质粒转染的为未转染组,siRNA转染的为siRNA干扰组,用Negative Control siRNA转染的为NC组。转染后,行Western blot、CCK-8、细胞划痕、Transwell实验,检测各组细胞生物学特性的变化。结果质粒转染后,Huh-7细胞Wip1蛋白的表达上调,细胞增殖促进,增殖率在107.42%~176.36%之间。未转染组在0~24 h、~48 h、~72 h时的迁移距离分别小于质粒转染组:[(34.78±4.77)μm vs(61.60±2.02)μm,(33.98±3.48)μm vs(64.75±4.67)μm,(35.49±0.90)μm vs(67.89±4.23)μm,P均<0.01]。未转染组3次Transwell实验的细胞迁移数均少于质粒转染组:[(90.00±3.00)vs(178.67±5.77),(74.33±26.95)vs(209.00±32.91),(79.33±20.74)vs(208.67±73.24),P均<0.01]。siRNA转染后,Huh-7细胞Wip1蛋白的表达下调;细胞抑制率在12.37%~40.81%之间;NC组在0~24 h、~48 h、~72 h时的迁移距离分别大于siRNA干扰组:[(31.24±4.42)μm vs(16.04±6.21)μm,(31.33±2.13)μm vs(18.20±5.67)μm,(32.45±3.08)μm vs(18.99±5.32)μm,P均<0.01];NC组3次Transwell实验的细胞迁移数均多于siRNA干扰组:[(74.00±12.17)vs(33.00±10.44),(77.00±5.00)vs(32.67±2.08),(81.33±17.16)vs(29.30±2.51),P均<0.01]。结论促进Huh-7细胞Wip1基因表达后,其Wip1蛋白的表达上调,细胞增殖、迁移、侵袭能力增强。抑制Huh-7细胞Wip1基因表达后,其Wip1蛋白的表达下调,细胞增殖、迁移、侵袭能力被抑制。
Objective To investigate the expression of wild-type p53-induced phosphatase 1 (Wip1) gene in human hepatocellular carcinoma cell line Huh-7 and to observe the changes of its biological characteristics. Methods Huh-7 cells were transfected with plasmids that promote the expression of Wip1 gene and small interfering RNA (siRNA) that inhibited the expression of Wip1 gene, respectively. Plasmid-transfected Huh-7 cells were transfected with plasmids, untransfected plasmids without plasmids, siRNA transfected with siRNA, and NC transfected with Negative Control siRNA. After transfection, Western blot, CCK-8, cell scratch and Transwell assay were used to detect the changes of cell biological characteristics. Results After transfection, the expression of Wip1 protein in Huh-7 cells was up-regulated and the cell proliferation was promoted with the proliferation rates ranging from 107.42% to 176.36%. The migration distance of untransfected group at 0-24 h, ~ 48 h, ~ 72 h were less than that in plasmid transfected group [(34.78 ± 4.77) μm vs (61.60 ± 2.02) μm, (33.98 ± 3.48) μm vs (64.75 ± 4.67) μm, (35.49 ± 0.90) μm vs (67.89 ± 4.23) μm respectively, all P <0.01]. The number of cell migration in three Transwell experiments in the untransfected group was less than that in the plasmid transfected group: [(90.00 ± 3.00) vs (178.67 ± 5.77), (74.33 ± 26.95) vs (209.00 ± 32.91), (79.33 ± 20.74) vs (208.67 ± 73.24), all P <0.01]. After siRNA transfection, the expression of Wip1 protein in Huh-7 cells was down-regulated; the cell inhibition rate was between 12.37% and 40.81%; the migration distance in NC group was greater than that of siRNA at 0-24 hours, 48 hours and 72 hours respectively Group: (31.24 ± 4.42 μm vs 16.04 ± 6.21 μm vs 31.33 ± 2.13 μm vs 18.20 ± 5.67 μm vs 32.45 ± 3.08 μm vs 18.99 ± 5.32 μm respectively) ; The numbers of cell migration in NCT group were more than those in siRNA interference group in three times of Transwell experiments: (74.00 ± 12.17 vs 33.00 ± 10.44 vs 77.6 ± 2.08 vs 81.33 ± 17.16 vs 29.30 ± 2.51), all P <0.01]. Conclusion The expression of Wip1 protein in Huh-7 cells is upregulated after Wip1 gene expression and the ability of cell proliferation, migration and invasion is enhanced. After inhibiting the expression of Wip1 gene in Huh-7 cells, the expression of Wip1 protein was down-regulated and the ability of cell proliferation, migration and invasion was inhibited.