靶向LASS2基因的小干扰RNA对人肝癌细胞侵袭能力的影响

Small interfering RNA targeting LASS2 gene enhances invasion capacity of hepatocellular carcinoma cells

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.