摘要
目的:研究靶向人源性长寿保障基因2(homosapiens longevity assurance homologue2,LASS2)的小干扰RNA(small interfering RNA,siRNA)对人肝癌细胞MHCC97-L体外侵袭能力的影响。方法:通过脂质体转染将靶向LASS2的siRNA转染MHCC97-L细胞,运用实时荧光定量PCR(real-time fluorogentic quantitative PCR,RFQ-PCR)检测转染后LASS2mRNA的表达,筛选有效的siRNA片段;运用BCECF氢离子敏感荧光探针检测MHCC97-L细胞跨膜运输氢离子的功能;采用Western印迹法分析MHCC97-L细胞及上清液中基质金属蛋白酶2(matrix metalloproteinase2,MMP-2)的表达情况;应用明胶酶谱法检测MHCC97-L细胞上清液中MMP-2的活性;Transwell法检测细胞体外侵袭能力。结果:筛选得到靶向LASS2的有效siRNA-1片段。MHCC97-L细胞转染LASS2siRNA-1后,其跨膜运输氢离子的能力增强,与空白对照组比差异有统计学意义(P<0.05);MMP-2蛋白的表达及分泌无明显变化,但分泌的MMP-2活性形式增加,与空白对照组和无关序列组比明显提高,差异有统计学意义(P<0.05);且细胞的体外侵袭能力明显高于无关序列组和空白对照组(P<0.05)。结论:靶向LASS2的siRNA能有效下调其表达,并能提高MHCC97-L细胞在转染LASS2siRNA后的体外侵袭能力。
Objective:To explore the effect of small interference RNA (siRNA) targeting homosapiens longevity assurance homologue 2 (LASS2) on invasion for human hepatocellular carcinoma cell line (MHCC97-L). Methods: MHCC97-L cells were transfected with siRNA mediated by lipofectamine 2000. The expression of LASS2 was detected after transfection by real-time fluorogentic quantitative PCR (RFQ-PCR) to screen the effective siRNA fragment. The trans-membrane transport of proton by MHCC97-L cells was determined by measuring extracellular pH (pHe) with pH-sensitive fluorescence probe BCECF. The level of MMP-2
protein in the supernatant was analyzed by Western blotting. The activity of MMP-2 was examined by zymography. Furthermore, the in vitro invasion capacity of MHCC97-L cells was tested using Transwell invasion assay. Results:Two effective siRNA fragments targeting LASS2,siRNA-1 and siRNA-2, were selected. After expression of LASS2 gene was suppressed by siRNA-1 in the MHCC97-L cells, the proton trans membrane secretion was increased. The difference was significant compared with empty control group (P〈0.05). There were no differences in the expression and synthesis of MMP-2 protein between siRNA-treated group and control groups. However activity of secreted MMP-2 was up-regulated and the in vitro invasion capacity was remarkably enhanced in siRNA-1 treated cells compared with the null sequence
group and the empty control group (P〈0.05). Conclusion:siRNA targeting LASS2 gene could enhance the cell invasion in vitro by facilitating proton secretion and up-regulating the activation
of MMP-2 in MHCC97-L cells.
出处
《肿瘤》
CAS
CSCD
北大核心
2009年第5期399-403,共5页
Tumor
基金
国家重点基础研究发展计划资助项目(编号:2009CB521803)
上海市国际科技合作基金资助项目(编号:08410700900)