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目的探讨抑制dachshund同源物1(DACH1)的表达对Capan-1胰腺癌细胞周期、凋亡、迁移及侵袭的影响。方法设计合成针对人DACH1基因的4条小干扰RNA(si RNA),退火形成双链短发夹RNA(shRNA),插入p Genesil-1载体中,构建成重组质粒,进行酶切及测序鉴定,通过脂质体介导转染入Capan-1细胞,利用荧光显微镜、反转录PCR和Western blot法检测其转染表达效率。藻红蛋白标记的膜联蛋白Ⅴ和7-氨基放线菌素D(annexinⅤ-PE/7AAD)双标记结合流式细胞术检测Capan-1细胞凋亡和流式细胞术检测细胞周期,TranswellTM侵袭、迁移实验检测Capan-1细胞侵袭迁移能力。结果成功构建并筛选出干扰质粒pshRNA-DACH1,将其转染Capan-1胰腺癌细胞后,成功下调Capan-1细胞DACH1水平,同时细胞凋亡明显增加,而对细胞周期无明显影响。下调DACH1表达后细胞侵袭和迁移能力均降低。结论敲低DACH1基因表达能显著促进胰腺癌细胞Capan-1凋亡,抑制其侵袭及迁移。
Objective To investigate the effect of inhibiting Dachshund Homologue 1 (DACH1) expression on the cell cycle, apoptosis, migration and invasion of Capan-1 pancreatic cancer cells. Methods Four small interfering RNAs targeting human DACH1 gene were designed and synthesized, annealed to form double-stranded short hairpin RNA (shRNA), inserted into p Genesil-1 vector and constructed into recombinant plasmids for enzyme digestion and sequencing. Transfection into Capan-1 cells was carried out by lipofectamine. The transfection efficiency was detected by fluorescence microscopy, reverse transcription PCR and Western blot. The apoptosis of Capan-1 cells was detected by flow cytometry double labeling combined with annexinⅤ-PE / 7AAD and the cell cycle was detected by flow cytometry. The invasion of TranswellTM Migration assay was used to detect the invasion and migration of Capan-1 cells. Results The plasmid pshRNA-DACH1 was successfully constructed and screened. After transfection to Capan-1 pancreatic cancer cells, the DACH1 level of Capan-1 cells was successfully decreased and the apoptosis rate of Capan-1 cells was significantly increased with no significant effect on the cell cycle. Down-regulation of DACH1 expression decreased cell invasion and migration. Conclusion Knockdown of DACH1 gene expression can significantly promote the apoptosis of Capan-1 cells and inhibit its invasion and migration.