敲低dachshund同源物1(DACH1)促进Capan-1胰腺癌细胞凋亡并抑制其侵袭和迁移

Knockdown of dachshund homolog 1( DACH1 ) promotes cell apoptosis and inhibits the invasion and migration abilities of Capan-1 pancreatic cancer cells

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 impact of the decreased expression of dachshund homolog 1 （ DACH1 ） on cell cycle, apoptosis, invasion and migration of Capan-I pancreatic cancer cells. Methods After four pairs of DACH1 siRNA were designed and synthesized, double-stranded short hairpin RNA （shRNA） were annealed and inserted into pGenesil-1 vector. The product was then confirmed by enzyme digestion and sequencing analysis. The recombinant plasmids were transfected into Capan-I cells via LipofectamineTM 2000. Fluorescence microscopy, reverse transcription PCR （RT-PCR） and Western blotting were used to detect the transfection efficiency. Cell apoptosis and cell cycle were tested by flow cytometry. TranswellTM assay was used to monitor the invasion and migration abilities of Capan-1 cells. Results Recombinant plasmid pshRNA-DACH1 was successfully constructed and transfected into Capan-I cells. After transfection, the expression of DACHI was reduced to some extent. Flow cytometry revealed that cell apoptosis was promoted in the pshRNA-DACH1 transfected group compared with control groups, whereas cell cycle had no significant differences among the groups. TranswellTM assay validated that the abilities of migration and invasion were inhibited in the pshRNA-DACH1 transfected group. Conclusion Knockdown of DACHI expression can remarkably enhance the cell apoptosis, restrain the proliferation, migration and invasion of Capan-I cells.