摘要
目的研究靶向沉默肿瘤转移抑制基因1(tumor metastasis suppressor gene-1,TMSG-1,亦称LASS2基因)对人乳腺癌MCF-7细胞体外侵袭能力的影响,初步探讨其作用机制。方法构建TMSG-1的特异性干扰片段,通过脂质体转染技术,将靶向TMSG-1的siRNA转染MCF-7细胞,运用实时荧光定量PCR检测转染后TMSG-1mRNA的表达,筛选有效的siRNA片段;选用最佳沉默片段siRNA-2和阴性对照siRNA-NC转染MCF-7细胞并设置空白对照组。采用Western印迹法和明胶酶谱法分析MCF-7细胞上清液中基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)的表达及生物活性;采用V-ATP酶活性检测试剂盒来测定细胞内V-ATP酶的活性;应用划痕修复实验检测MCF-7细胞体外迁移能力;Transwell体外侵袭实验检测细胞侵袭能力。结果实时荧光定量PCR检测结果发现,不同转染序列对TMSG-1基因的沉默效果不同,siRNA-2组和siRNA-3组中TMSG-1基因mRNA的量与对照组、siRNA-NC组和siRNA-1组比较,表达明显减少,差异有统计学意义(P<0.05),本实验选用siRNA-2组用于后续试验;Western印迹法检测结果显示,空白对照组、siRNA-NC组与siRNA-2组3组之间比较差异无统计学意义(P>0.05);明胶酶谱法检测结果显示:siRNA-2转染组细胞培养上清液中MMP-2的活性是空白对照组的1.76倍,差异有统计学意义(P<0.05),是siRNA-NC组的1.81倍,差异有统计学意义(P<0.05);细胞内V-ATP酶的活性检测结果显示:转染siRNA-2组V-ATP酶活性明显高于空白对照组和siRNA-NC组(P<0.05);划痕修复实验结果显示:siRNA-2转染组的迁移能力明显高于空白对照组和无关阴性对照siRNA组(P<0.05);Transwell侵袭小室实验检测结果显示,转染siRNA-2组细胞数目明显多于siRNA-NC组和空白对照组(P<0.05)。结论靶向沉默TMSG-1的siRNA能有效下调其表达,提高MCF-7细胞的体外侵袭能力。
Objective To explore the effects of small interference RNA（ siRNA） targeting tumor metastasis suppressor gene- 1（ TMSG- 1） and its mechanism on the invasion of human breast carci noma cell line MCF- 7. Methods MCF- 7 cells were transfected with siRNA mediated by lipofectamine 2000. The expression of TMSG- 1 was detected after transfection by real- time fluorogentic quantitative PCR（ RFQ-PCR） to screen the effective siRNA fragment. Then,MCF- 7 cells were cultured and divided into three groups： the blank control group,cells transfected with nonsense siRNA（ siRNA- NC） group,and cells transfected with the effective siRNA（ siRNA- 2） targeting TMSG- 1. The expression of matrix metalloproteinase 2（ MMP- 2） was analyzed by Western blotting. The activity of MMP- 2 was examined by zymography. Then,the activity of vacuolar ATPase（ V- ATPase） was detected by kit. Furthermore,the in vitro invasion capacity and migragation capacity of MCF- 7 cells was tested by wound migration assay and invasionassay. Results Real- time fluorescent quantitative PCR detection results showed that different sequences of TMSG- 1 gene transfection of silence effect were different,compared with that of control group,siRNA-NC group and siRNA- 1 group,the amount of mRNA in TMSG- 1 gene in siRNA- 2 group,siRNA- 3TMSG- 1,expression decreased significantly,the difference was statistically significant（ P〈0. 05）,siRNA- 2 group was selected as the follow- up test in the experiment; Western imprinting method detection results showed that there were no statistical significant differences among the blank control group,the siRNA- NC group and siRNA- 2（ P〈0. 05）; Gelatinases spectrometry detection results showed that the cell culture supernatant on the activity of MMP- 2 in the siRNA- 2 transfection group was 1. 76 times that of the blank control group,the difference was statistically significant（ P〈0. 05）,was 1. 81 times that of siRNA- NC group,the difference was statistically significant（ P〈0. 05）; V- atpas
出处
《湘南学院学报（医学版）》
2016年第3期1-6,共6页
Journal of Xiangnan University（Medical Sciences）
基金
湖南省教育厅青年基金资助项目(13B111)
