体外RNAi对S100A4基因表达及其对骨肉瘤细胞侵袭、转移能力的影响

RNAi inhibit the expression of S100A4 gene and its effect on invasion and metastasis ability of osteosarcoma cell in vitro

关键词:  RNA干扰  骨肉瘤  S100A4  侵袭  转移

Key words:  RNA interference  osteosarcoma  S100A4  invasion  metastasis

摘要:

[目的] 应用RNAi技术抑制人骨肉瘤MG-63细胞S100A4基因的表达，并观察对MG-63细胞增殖及体外侵袭能力的影响。[方法] 合成针对S100A4的siRNA载体，并根据目的mRNA序列，设计3条RNA干扰靶序列及非特异性的siRNA。将培养细胞分为C组（空白对照组）；C1组（转染脂质体组）；C2组（转染非特异性的siRNA组）；S1、S2、S3组（转染特异性的siRNAⅠ、Ⅱ、Ⅲ组）。荧光倒置显微镜下观察转染情况， Real-time PCR方法检测转染前后S100A4基因表达变化，应用Western-Blot方法检测转染前后S100A4蛋白表达变化。筛选出转染效率最高的特异性siRNA组作为后续实验的实验组。后续实验分为正常细胞组，阴性对照组和实验组，应用MTT法和平板克隆形成实验检测细胞的增殖率变化，应用Transwell小室实验检测细胞的侵袭及转移能力变化。[结果] 倒置显微镜下C2组与S1、S2、S3组培养细胞的细胞浆均可见绿色荧光。S1、S2与S3组的S100A4 mRNA表达水平均有不同程度的下调，以S3组最为显著。Western-Blot检测S100A4蛋白表达结果与其相符。MTT法和平板克隆形成实验均显示S3组细胞增殖受到抑制，与正常细胞组、阴性对照组比较差异有显著性意义（P<0.05）。Transwell小室实验显示RNA干扰S100A4基因表达后肿瘤细胞的运动侵袭能力明显下降，穿过人工基底膜细胞数量明显减少，与正常细胞组、阴性对照组相比差异有显著性意义（P<0.05）。 [结论] 体外合成的S100A4特异性siRNA能够抑制骨肉瘤细胞系MG-63的S100A4表达，进而抑制细胞的增殖和体外侵袭能力。

Abstract:

[Objective] To observe its effect on proliferation and invasion capacity in vitro of MG-63 cells, by application of RNAi technology to inhibit the expression of S100A4 gene in human osteosarcoma MG-63 cells. [Methods] The siRNA vector of S100A4 was constructed. According to target mRNA sequence, three RNA interference and non-specific target sequence of siRNA were designed. The cultured cells were divided into Group C (control group), group C1 (liposome transfection group), group C2 (non-specific siRNA transfection group), groups S1，S2，S3 (transfected with specific siRNA Ⅰ， Ⅱ，Ⅲ). The situation of transfection was observed by fluorescence inverted microscope. The changes of S100A4 gene expression of the cells before and after transfection were detected by real-time PCR method. The changes of S100A4 protein expression before and after transfection were detected by Western-Blot. The specific siRNA with high transfection efficiency was selected and used as the transfection experiment group for the following tests. The following test was divided into normal cell group, negative control group and experimental group. The changes of cell proliferation were determined by MTT and flat colony-forming experiment. The changes of cell invasion and metastasis ability were determined by transwell chamber assay. [Results] Green fluorescence could be seen in cytoplasm of group C2 and group S1, S2, S3 by fluorescence inverted microscope. The expression levels of S100A4 mRNA in each specific siRNA transfection group were down regulated at various degrees. The group S3 was the most significant one. The expression levels of S100A4 protein of Western-blot detection were similar to the conclusions. MTT method and flat colony-forming experiments had shown that cell proliferation was inhibited in group S3. There was significant difference compared with the normal cells group and non-specific group （P<0.05）. Transwell chamber experiments showed that the invasive ability of the cells significantly decreased after RNA interference S100A4 gene expression, the cell number through artificial basement membrane decreased significantly, compared with the normal cell group and negative group（P<0.05）. [Conclusion] S100A4 siRNA-specific vector in vitro synthesis can effectively inhibit the expression of S100A4 in osteosarcoma MG-63cells, and cell proliferation and invasive ability in vitro.