Huang Xiu-yan, Huang Zi-li, Xu Yong-hua, Huang Xin-yu, Zhou Jian, Ye Sheng-long, Tang Zhao-you, Zheng Qi
Department of General Surgery, Shanghai Jiaotong University, Shanghai 200233, China.
Zhonghua Gan Zang Bing Za Zhi. 2010 Dec;18(12):915-9. doi: 10.3760/cma.j.issn.1007-3418.2010.12.008.
To investigate the effects of lentivirus mediated siRNA targeting human metastasis suppressor 1 (MTSS1, MIM-B gene) gene on the invasive and metastatic potentials of hepatocellular carcinoma (HCC) MHCC97H cells.
The siRNA targeting MTSS1 was cloned into one lentivirus work vector. The work vector and three package plasmids were co-transfected into 293T cells with the help of lipefeetamine 2000. Lentivirus was collected in 72 hours and was added to the cultured MHCC97H cells. The total cell MIM-B mRNA and MIM-B protein were extracted and underwent real-time PCR and western-blot test respectively. Boden chamber assay was used to evaluate the invasive potential of MHCC97H cells. Gelatin zymography was used to detect matrix metalloproteinase-2 (MMP2) activity. Metastatic human HCC nude mice models were established by orthotopic implantation with a high metastatic potential human HCC cell line MHCC97H. Twenty-four nude mice bearing orthotopic xenografts were randomized into black control group, Lenti-GFP group and intervention group (Lenti-MTSS1 group) 14 days after orthotopic implantation (8 per group). The ultrasound-guided multi-point injection was performed on mice with borate buffered saline, Lenti-GFP and Lenti-MTSS1 respectively. Mice were sacrificed on day 35 for the examination of pulmonary metastasis. The SPSS 13.0 soft ware was applied to data analysis.
The small interfering RNA targeting MTSS1 was constructed successfully with a transfection efficiency of 97.0%, which produced a marked inhibition of invasive ability of MHCC97H cells through Matrigel, being 37.9+/-4.4, 37.4+/-5.3 and 26.6+/-4.6 in the black control group, Lenti-GFP group and Lenti-MTSS1 group (F = 26.695, P value is less than 0.01), respectively. MIM-B expression and MMP2 activity of intervention group were also significantly down-regulated as compared to the control group. The results of in vivo studies showed that the numbers of lung metastatic nodules were 6.5+/-2.6, 6.4+/-2.7 and 3.8+/-1.3 in the black control group, Lenti-GFP group and intervention group respectively with significant statistical difference (F = 3.637, P value is less than 0.05), accorded with tumor tissue MIM-B mRNA expression of 0.39+/-0.19, 0.38+/-0.10 and 0.16+/-0.11 respectively (F = 11.644, P value is less than 0.01) when comparison was made between control group and therapy group.
Small interfering RNA mediated by lentivirus inhibited MIM-B expression and resulted in inhibition of the invasive and metastatic potentials of MHCC97H cells, which may attributed, in part, the down regulation of MMP2 activity, and thus may provide a new molecular targeted therapy for HCC patients in the future.
探讨慢病毒介导的靶向人转移抑制因子1（MTSS1，MIM - B基因）基因的小干扰RNA（siRNA）对肝癌MHCC97H细胞侵袭和转移潜能的影响。
将靶向MTSS1的siRNA克隆到一种慢病毒工作载体中。借助脂质体2000将工作载体和三种包装质粒共转染至293T细胞。72小时后收集慢病毒并加入到培养的MHCC97H细胞中。提取细胞总MIM - B mRNA和MIM - B蛋白，分别进行实时荧光定量PCR和蛋白质印迹检测。采用小室侵袭实验评估MHCC97H细胞的侵袭潜能。用明胶酶谱法检测基质金属蛋白酶-2（MMP2）活性。通过原位接种高转移潜能的人肝癌细胞系MHCC97H建立人肝癌裸鼠转移模型。原位接种1年后，将24只荷瘤裸鼠随机分为空白对照组、慢病毒绿色荧光蛋白（Lenti - GFP）组和干预组（Lenti - MTSS1组）（每组8只）。分别用硼酸盐缓冲液、Lenti - GFP和Lenti - MTSS1对小鼠进行超声引导下多点注射。在第35天处死小鼠以检测肺转移情况。应用SPSS 13.0软件进行数据分析。
成功构建了靶向MTSS1的小干扰RNA，转染效率为97.0%，其显著抑制了MHCC97H细胞通过基质胶的侵袭能力，空白对照组、Lenti - GFP组和Lenti - MTSS1组的侵袭细胞数分别为37.9±4.4、37.4±5.3和26.6±4.6（F = 26.695，P值<0.01）。干预组的MIM - B表达和MMP2活性与对照组相比也显著下调。体内研究结果显示，空白对照组、Lenti - GFP组和干预组的肺转移瘤结节数分别为6.5±2.6、6.4±2.7和3.8±1.3，差异有统计学意义（F = 3.637，P值<0.05），对照组和治疗组肿瘤组织MIM - B mRNA表达分别为0.39±0. +0.19、0.38±0.10和0.16±0.11（F = 11.644，P值<0.01）。
慢病毒介导的小干扰RNA抑制MIM - B表达，导致MHCC97H细胞侵袭和转移潜能受到抑制，这可能部分归因于MMP2活性下调，从而可能为未来肝癌患者提供一种新的分子靶向治疗方法。
