Effect and Mechanism of Twist1 Regulating Bmi1 on Invasion and Migration of Gallbladder Carcinoma Cells
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摘要:
目的 探讨Twist1对胆囊癌细胞侵袭转移的影响，以及对Bmi1、E-Cadherine、N-Cadherine的调控作用。 方法 构建Twist1 shRNA慢病毒载体感染GBC-SD细胞做为实验组，空载慢病毒感染GBC-SD细胞为阴性对照组，不做任何处理的GBC-SD细胞为空白对照组。细胞划痕及Transwell侵袭小室实验检测细胞的迁移及侵袭能力，Western blot及RT-PCR检测Twist1、Bmi1、E-Cadherine及N-Cadherine的蛋白及mRNA的相对表达量。 结果 与阴性组、空白组相比，实验组细胞划痕愈合度低、侵袭数少（P < 0.0001），而阴性组及空白组间细胞迁移率、侵袭数比较差异均无统计学意义（P > 0.05）。阴性组及空白组的Twist1、Bmi1、N-Cadherine的蛋白及mRNA的相对表达量明显高于实验组（P < 0.001），而E-Cadherine蛋白及mRNA的相对表达量则明显低于实验组（P < 0.0001），阴性组及空白组组间各蛋白及mRNA的相对表达水平比较差异无统计学意义（P > 0.05）。 结论 靶向沉默Twist1可以抑制胆囊癌细胞的迁移侵袭，其机制可能与抑制Bmi1的表达及抑制上皮间质转化过程相关。 Abstract:Objective To investigate the effect of Twist1 on invasion and metastasis of gallbladder carcinoma cells and the regulation of Bmi1, E-Cadherine andN-Cadherine. Methods Twist1 shRNA lentivirus vector was constructed to infect GBC-SD cells as the experimental group. The empty lentivirus-infected GBC-SD cells were the negative group, and the untreated GBC -SD cells were the blank group. Cell migration and invasion ability were detected by cell scratch and Transwell chamber invasion assay. Western blot and RT-PCR were used to detect the relative expression levels of Twist1, Bmi1, E-Cadherine and N-Cadherine. Results Compared with the negative and blank groups, the experimental group had a lower cell scratch healing degree and fewer invasion numbers （P < 0.0001）. There was no significant difference in cell migration rate and invasion number between the two control groups （P > 0.05）. The relative expression levels of Twist1, Bmi1 and N-Cadherine in the negative group and blank group were obviously higher than those in the experimental group （P < 0.001）. The relative expression levels of E-Cadherine protein and mRNA were obviously lower than those of the experimental group （P < 0.0001）. There was no significant difference in the relative expression levels of protein and mRNA between the negative group and the blank group （P > 0.05）. Conclusion Targeted silencing of Twist1 can inhibit the migration and invasion of gallbladder carcinoma cells, and the mechanism may be related to inhibiting the expression of Bmi1 and the process of epithelial-mesenchymal transformation. -
Key words:
- Gallbladder neoplasm /
- Twist1 /
- Bmi1 /
- Epithelial-mesenchymal transition /
- Invasion /
- Metastasis
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图 1 成功感染慢病毒并经过筛选后的细胞生长情况（10×）
A：Twist1组；B：NEG组。
Figure 1. Growth of cells successfully infected with lentivirus and screened （10×）
图 2 细胞划痕实验检测GBC-SD细胞的迁移能力（4×）
A：Twist1组0 h；B：NEG组0 h；C：GBC-SD组0 h；D：Twist1组48 h；E：NEG组48 h；F：GBC-SD组48 h；G：3组48 h细胞迁移率。ns为P > 0.05，****为P < 0.0001。
Figure 2. 2 Migration ability of BGC-SD cells detected by cell scratch assay （4×）
图 3 侵袭实验检测GBC-SD细胞的侵袭能力（10×）
A：Twist1组；B：NEG组；C：GBC-SD组；D：3组细胞穿膜细胞数，ns为P > 0.05，****为P < 0.0001。
Figure 3. Invasion assay to detect the invasion ability of BGC-SD cells （10×）
图 4 Western Blot检测各组细胞Twist1、Bmi1、E-Cadherine及N-Cadherine蛋白表达水平柱状图
Figure 4. Histogram of protein expression levels of Twist1，Bmi1，E-Cadherine and N-Cadherine in each group detected by Western Blot
图 5 Western Blot检测各组细胞Twist1、Bmi1、E-Cadherine及N-Cadherine蛋白表达水平
ns为P > 0.05，***P < 0.001，****P < 0.0001。
Figure 5. The expression levels of Twist1，Bmi1，E-Cadherine andN-Cadherine in each group were detected by Western Blot
图 6 qPCR检测各组细胞Twist1、Bmi1、E-Cadherine及N-Cadherine mRNA表达水平柱状图
ns为P > 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 6. mRNA expression columns of Twist1，Bmi1，E-Cadherine and N-Cadherine in each group were detected by qPCR
表 1 相关引物序列
Table 1. Sequence of relevant primers
基因 序列 碱基数 Twist1-F AGCTGAGCAAGATTCAGACC 20 Twist1-R CAGCTTGCCATCTTGGAGTC 20 Bmi1-F GACTCTGGGAGTGACAAGGC 20 Bmi1-R ACTGGAGTACTGGGGCTAGG 20 E-Cadherine-F GGCTGGACCGAGAGAGTTT 19 E-Cadherine-R GCTGTGGAGGTGGTGAGAG 19 N-cadherin-F GACTGGGTCATCCCTCCAATCAACT 25 N-cadherin-R GGGGCTTTGTCACCGACAGC 20 Gapdh-F CGCTGAGTACGTCGTGGAGTC 21 Gapdh-R GCTGATGATCTTGAGGCTGTTGTC 24 
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表 2 各组细胞Twist1表达情况（
$n\;=\;3$ ，$\bar x \pm s $ ）Table 2. Expression of Twist1 in each group （
$n\;=\;3$ ，$\bar x \pm s $ ）组别 Twist1 mRNA 蛋白 Twist1组 0.35 ± 0.02ab 0.35 ± 0.20ab NEG组 1.26 ± 0.23 0.95 ± 0.21 GBC-SD组 1.00 ± 0.48 1.01 ± 0.18 F 34.11 31.03 P 0.0005* < 0.0001* *P < 0.05；与NEG组比较，a P < 0.0001；与GBC-SD组比较，b P < 0.0001。
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表 3 各组细胞划痕愈合度及侵袭细胞数（
$n\;=\;20$ ，$\bar x \pm s $ ）Table 3. Scratch healing degree and number of cells invaded in each group （
$n\;=\;20$ ，$\bar x \pm s $ ）组别 细胞划痕愈合度（%） 穿膜细胞数（个） Twist1组 26.75 ± 19.89ab 34.95 ± 6.61ab NEG组 66.54 ± 7.37 327.00 ± 34.85 GBC-SD 63.37 ± 9.91 314.50 ± 32.08 F/χ² 147.6 40.39 P < 0.0001* < 0.0001* *P < 0.05；与NEG组比较，a P < 0.0001，与GBC-SD组比较，b P < 0.0001。
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