慢病毒介导Vimentin基因沉默抑制胰腺癌细胞侵袭和粘附的机制

Lentivirus-mediated Vimentin Gene Silencing Inhibits Invasion and Adhesion of Pancreatic Carcinoma Cells

目的应用慢病毒介导的shRNA基因沉默技术,有效沉默人胰腺癌细胞株Panc-1中Vimentin表达,并探讨下调Vimentin后对Panc-1细胞侵袭运动及粘附能力的影响及其机制。方法 Lv-VIM-GFP-shRNA慢病毒转染人胰腺癌细胞株Panc-1,Real-time PCR、Western blot和免疫荧光法检测慢病毒转染后Vimentin的沉默效率;Transwell小室体外运动侵袭实验及划痕实验比较细胞运动侵袭能力变化,细胞粘附实验检测细胞粘附能力的变化;Real-time PCR和Western blot检测肿瘤侵袭和粘附相关因子的表达,免疫荧光法观察转染前后细胞骨架蛋白F-actin和黏着斑激酶FAK的变化。结果 Lv-VIM-GFP-shRNA慢病毒转染Panc-1后能稳定下调其Vimentin的表达(P<0.01)。Transwell小室及划痕实验显示,下调Vimentin后能显著抑制Panc-1细胞的侵袭及迁移能力(P<0.01)。粘附实验结果显示,粘附30min后,转染Lv-VIM-GFP-shRNA组较对照组粘附CollagenⅣ和FN的细胞数量均减少(P<0.05)。下调Vimentin后对肿瘤侵袭及粘附相关因子的mRNA和蛋白水平无明显影响(P>0.05),但能引起细胞骨架F-actin的解聚以及黏着斑激酶FAK的重排。结论 Vimentin基因沉默后可能通过引起F-actin骨架解聚、黏着斑激酶FAK重排来降低细胞与细胞外基质的粘附及细胞运动能力,从而抑制胰腺癌细胞的侵袭和转移。

Objective To study the effect of vimentin down-regulation by lentivirus-mediated RNA interference（RNAi）on invasion and adhesion of human pancreatic carcinoma cells.Methods Vimentin expression in human pancreatic carcinoma Panc 1 cells was silenced by Lv-VIM-GFP-shRNA infection.The expression of vimentin in the cells was detected by qRT-PCR,Western blot and immunofluorescence staining.Transwell and Scratch-wound assays were used to measure the invasive and migratory capability of cells.The adhesion ability of infected Panc-1cells was evaluated by cell adhesion assay.The expression of tumor adhesion-related factors was detected by qRT-PCR and Western blot.The expression of F-actin and focal adhesion kinase（FAK） was detected by immunofluorescence staining.Results Vimentin was constitutively expressed in Panc-1 cells.Vimentin expression was silenced by Lv-VIM-GFP-shRNA infection（P〈0.01）.The invasive and migratory capability of cells infected with LvVIM-GFP-shRNA was obviously reduced（P〈0.01）.The adhesion of Lv-VIM-shRNA-infected cells to collagen Ⅳ and FN was obviously reduced（P〈0.05）.No significant effect on tumor adhesion-related factors was found after down-regulation of vimentin（P〈0.05）,but the expression of F-actin was decreased and immunofluorescence staining showed FAK rearrangement in Lv-VIM-GFP-shRNA group.Conclusion Suppression of vimentin expression may inhibit the invasion and adhesion of pancreatic cancer cells in vitro by reducing the adhesion of tumor cells to the extracellular matrix and their migration via depolymerization of F-actin cytoskeleton and rearrangement of FAK.