摘要
目的建立稳定转染含N-myc下游调节基因-1(NDRG1)的SW1990细胞株,探讨NDRG1对SW1990细胞侵袭与迁移能力的影响及其可能的分子机制。方法经脂质体介导将含有NDRG1重组表达质粒转染人胰腺癌细胞株SW1990,用G418筛选阳性细胞克隆,逆转录-聚合酶链反应(RT-PCR)和Western blot鉴定阳性细胞克隆;采用RT-PCR及Western blot检测阳性细胞克隆MMP-9及VEGF mRNA与蛋白表达;采用Transwell小室侵袭实验与划痕实验分别检测细胞侵袭及迁移能力。结果 N17克隆组、转空载体组及未转染组NDRG1 mRNA表达量分别为0.53±0.25、0.26±0.11、0.25±0.13;相应的MMP-9 mRNA表达量分别为0.33±0.18、0.71±0.45、0.76±0.42;相应的VEGF mRNA表达量分别为0.27±0.16、0.68±0.36、0.69±0.38;相应的NDRG1蛋白表达量分别为0.27±0.13、0.11±0.04、0.12±0.06;相应的MMP-9蛋白表达量分别为0.12±0.04、0.38±0.15、0.36±0.14;相应的VEGF蛋白表达量分别为0.15±0.08、0.39±0.21、0.40±0.25。较其他组,N17克隆组NDRG1 mRNA及蛋白表达量显著增加(P<0.01),而MMP-9、VEGF mRNA及蛋白表达量显著降低(P<0.01)。N17克隆组、转空载体组及未转染组微孔滤膜外侧细胞数分别为31.27±11.54、58.93±17.23、60.26±19.38,N17克隆组较其他组微孔滤膜外侧细胞数显著减少(P<0.01)。N17克隆组、转空载体组及未转染组细胞迁移距离分别为51.35±18.24、120.68±42.97、124.54±51.66,N17克隆组较其他组细胞迁移距离显著减少(P<0.01)。结论 SW1990细胞NDRG1表达上调后,细胞侵袭和迁移能力受到显著抑制,其作用机制可能与MMP-9及VEGF表达降低有关。
Objective To evaluate the effect and mechanism of N-Myc downstream-regulated gene 1 （NDRG1） on invasion and migration of pancreatic cancer cells. Methods pcDNA3.0-NDRG1 and pcDNA3.0 were transfected into SW1990 cells through lipofectamine and positive clones were screened by G418. RT-PCR and Western blot were performed to identify mRNA and protein expressions of NDRG1 in SW1990 cells respectively. The expressions of MMP-9 and VEGF in positive clones were detected by RT-PCR and Western blot. The cell invasion and migrating ability were detected by transwell migration assay and wound healing assay. Results The mRNA levels of NDRG1, MMP-9 and VEGF in N17 clone , pcDNA3.0 transfected and empty nutrient fluid groups were：NDRG1 0.53±0.25, 0.26±0.11, 0.25±0.13;MMP-9 0.33±0.18, 0.71±0.45, 0.76± 0.42;VEGF 0.27±0.16, 0.68±0.36, 0.69±0.38, respectively. Accordingly, protein levels were：NDRG1 0.27± 0.13, 0.11±0.04, 0.12±0.06;MMP-9 0.12±0.04, 0.38±0.15, 0.36±0.14;VEGF 0.15±0.08, 0.39±0.21, 0.40 ±0.25, respectively. Results showed that both mRNA and protein levels of NDRG1 were significantly increased in positive clone of N17, however the expressions of mRNA and protein of MMP-9 and VEGF were significantly decreased in N17 clone group, compared with the other two groups（P〈0.01）. The cells that invaded through the basement membrane filter in N17 clone, pcDNA3.0 transfected and control groups were 31.27±11.54, 58.93± 17.23, 60.26±19.38. Cell migration was significantly decreased in N17 clone group, compared with other two groups（P〈0.01）. Distance of cell migrating in N17 clone , pcDNA3.0 transfected and control groups were 51.35 ±18.24, 120.68±42.97, 124.54±51.66. The distance of cell migrating was significantly decreased in N17 clone group, compared with the other two groups（P〈0.01）. Conclusion Upregulation of NDRG1 expression significant reduced invasion and migrating ability of SW1990 cells, which might be due to the downregulation of the MMP-9 and VEGF expressions in the SW1990 cells.
出处
《中华临床医师杂志（电子版）》
CAS
2013年第14期129-132,共4页
Chinese Journal of Clinicians(Electronic Edition)
基金
江苏省"333工程"培养资金资助项目(200924)
江苏省自然科学基金资助项目(BK2010357)
