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ZEB2 Regulates the Migration and Invasion of PANC-1 Pancreatic Cancer Cells: An Experimental Study
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摘要:
目的 研究锌指E-box结合同源异型盒2基因（zinc finger E-box binding homeobox transcription factor-2, ZEB2）对胰腺癌PANC-1细胞增殖、集落形成、迁移和侵袭能力及上皮-间质转化（epithelial-mesenchymal transition, EMT）过程的影响。 方法 分析癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库中胰腺癌组织和癌旁组织中ZEB2的表达。将胰腺癌PANC-1细胞分为si-NC组、si-ZEB2组、pcDNA3.1组和pcDNA3.1-ZEB2组，采用qRT-PCR技术和Western blot法验证ZEB2敲降或过表达的有效性。CCK-8实验、集落形成实验、划痕实验和Transwell实验分别检测ZEB2对PANC-1细胞的增殖、集落形成、迁移和侵袭的影响。利用qRT-PCR技术和免疫荧光实验检测细胞中EMT标志物E-cadherin、vimentin的表达水平。通过STRING网站预测与ZEB2具有相互作用的蛋白。 结果 TCGA数据库分析显示，与癌旁组织相比，胰腺癌组织中ZEB2表达水平明显升高（P<0.05）。与si-NC组相比，si-ZEB2组PANC-1细胞的增殖、集落形成、迁移、侵袭能力减弱；与pcDNA3.1组相比，pcDNA3.1-ZEB2组PANC-1细胞的增殖、集落形成、迁移、侵袭能力增强（均P<0.05）。qRT-PCR技术和免疫荧光实验结果提示，与si-NC组相比，si-ZEB2组PANC-1细胞的上皮标志物E-cadherin mRNA表达升高，而间质标志物vimentin mRNA和蛋白表达降低；与pcDNA3.1组相比，pcDNA3.1-ZEB2组PANC-1细胞中上皮标志物E-cadherin mRNA表达减少，间质标志物vimentin mRNA和蛋白表达增加（均P<0.05）。STRING网站预测出10个蛋白与ZEB2作用密切。 结论 过表达ZEB2能够促进胰腺癌PANC-1细胞的迁移、侵袭和EMT进程。 Abstract:Objective To investigate the effects and mechanisms of zinc finger E-box binding homeobox transcription factor-2 (ZEB2) on the proliferation, colony formation, migration, and invasion abilities and the epithelial-mesenchymal transition (EMT) of PANC-1 cells, a human pancreatic cancer cell line. Methods Data on the expression of ZEB2 in pancreatic cancer tissues and paracancerous tissues from The Cancer Genome Atlas (TCGA) database were analyzed. PANC-1 pancreatic cancer cells were divided into si-NC group, si-ZEB2 group, pcDNA3.1 group, and pcDNA3.1-ZEB2 group. qRT-PCR and Western blot were conducted to confirm the effectiveness of ZEB2 knockdown or overexpression. CCK-8, colony formation, wound healing, and Transwell assays were conducted to examine the effects of ZEB2 on the proliferation, colony formation, migration, and invasion of PANC-1 cells. qRT-PCR and immunofluorescence assays were performed to examine the expression of E-cadherin and vimentin, the EMT markers, in the cells. Prediction of proteins interacting with ZEB2 was made through the STRING database. Results TCGA database analysis showed that the expression level of ZEB2 in pancreatic cancer tissues was significantly higher than that in adjacent tissues (P<0.05). Compared with those of cells in the control group, the proliferation, colony formation, migration, and invasion of cells in the si-ZEB2 group were decreased (P<0.05). Compared with those of cells in the pcDNA3.1 group, the proliferation, colony formation, migration and invasion of cells in the pcDNA3.1-ZEB2 group were increased (all P<0.05). According to the results of qRT-PCR and immunofluorescence assays, compared with those of the si-NC group, the expression of E-cadherin mRNA, an epithelial marker, in the si-ZEB2 group increased, while the expression of vimentin mRNA, an mesenchymal marker, and the protein decreased. Compared with those of the pcDNA3.1 group, the expression of E-cadherin mRNA in the PANC-1 cells of the pcDNA3.1-ZEB2 group decreased, while the expression of vimentin mRNA and the protein increased (all P<0.05). Analysis with the STRING database predicted that 10 proteins had close interaction with ZEB2. Conclusion Overexpression of ZEB2 promotes the migration, invasion, and the EMT process of PANC-1 pancreatic cancer cells. -
Key words:
- ZEB2 /
- Pancreatic cancer /
- PANC-1 cell /
- Proliferation /
- Migration /
- Invasion
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图 1 ZEB2 mRNA在胰腺癌组织和癌旁组织中的表达水平
Figure 1. The expression of ZEB2 mRNA in pancreatic cancer tissues and paracancerous tissues
TPM: transcripts per million; *** P<0.001, vs. normal group.
图 2 敲降组PANC-1细胞中ZEB2 mRNA（A）和蛋白（B）的检测结果
Figure 2. ZEB2 mRNA (A) and proteins (B) in PANC-1 cells of the knockdown group
** P<0.01, **** P<0.0001, vs. si-NC group, n=3.
图 3 过表达组PANC-1细胞中ZEB2 mRNA（A）和蛋白（B）的检测结果
Figure 3. ZEB2 mRNA (A) and proteins (B) in PANC-1 cells of the overexpression group
*** P<0.001, vs. pcDNA3.1 group, n=3.
图 4 ZEB2对PANC-1细胞增殖（A、B）和集落形成（C、D）能力的影响
Figure 4. Effect of ZEB2 on proliferation (A, B) and colony forming (C, D) ability of PANC-1 cells
* P<0.05, ** P<0.01, *** P<0.001, vs. si-NC or pcDNA3.1 groups, n=3.
图 5 ZEB2对PANC-1细胞迁移距离的影响
Figure 5. Effect of ZEB2 on the migration distance of PANC-1 cells
* P<0.05, ** P<0.01, vs. si-NC and pcDNA3.1 group, n=3.
图 6 ZEB2对PANC-1细胞迁移（A）和侵袭（B）数量的影响（结晶紫染色×100）
Figure 6. Effect of ZEB2 on the migration (A) and invasion (B) of PANC-1 cells (crystal violet staining×100)
* P<0.05, *** P<0.001, vs. si-NC and pcDNA3.1 groups, n=3.
图 7 ZEB2对PANC-1细胞中EMT标志物表达的影响（×200）
Figure 7. Effect of ZEB2 on the expression of EMT markers in PANC-1 cells（×200）
A-B: the qRT-PCR assay; C-D: the immunofluorescence assay. * P<0.05, ** P<0.01, *** P<0.001, vs. si-NC and pcDNA3.1 groups, n=3.
图 8 STRING分析ZEB2的PPI
Figure 8. PPI（protein-protein interaction） analysis of ZEB2 with STRING database
表 1 引物序列
Table 1. Primer sequences
Gene name Primer sequences E-cadherin (F) 5′-AGTCACTGACACCAACGATAAT-3′ E-cadherin (R) 5′-ATCGTTGTTCACTGGATTTGTG-3′ Vimentin (F) 5′-TCGTGAATACCAAGACCTGCTCAATG-3′ Vimentin (R) 5′-AATCCTGCTCTCCTCGCCTTCC-3′ ZEB2 (F) 5′-GAAGACAGAGAGTGGCATGTAT-3′ ZEB2 (R) 5′-GTGTGTTCGTATTTATGTCGCA-3′ GAPDH (F) 5′-CAGGAGGCATTGCTGATGAT-3′ GAPDH (R) 5′-GAAGGCCTGGGGCTCATTT-3′ 
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