siRNA沉默结肠癌转移相关基因1表达对人类卵巢癌细胞株侵袭转移的影响

Effects of reduced gene expression of metastasis-associated in colon cancer 1 by RNA interference on the invasion and metastasis of ovarian cancer cells

目的 检测结肠癌转移相关基因1（MACC1）在卵巢癌细胞株中的表达,并探讨用siRNA技术抑制其表达对卵巢癌细胞生物学行为的影响.方法 应用实时荧光定量PCR （RT-qPCR）及Western blot法检测OVCAR3、ES-2、SKOV3、HO-8910卵巢癌细胞株中MACC1的表达,合成MACC1特异性siRNA并转染OVCAR3细胞,利用RT-qPCR筛选并鉴定MACC 1基因有效沉默后,应用体外黏附实验、Transwell迁移及侵袭实验、体外血管拟态实验检测MACC1基因沉默后OVCAR3细胞的体外黏附、迁移、侵袭及血管生成能力的变化.结果 OVCAR3细胞较其他卵巢癌细胞株高表达MACC1.MACC1基因沉默后,OVCAR3细胞的体外黏附能力受到不同程度的抑制;Transwell迁移实验中,MACC1 siRNA干扰的OVCAR3细胞（干扰组）转入底层膜的细胞数为（245.5±12.8）个,低于阴性对照组和空白对照组[分别为（500.3±16.5）、（496.3±13.1）个,均P＜0.05]; Transwell侵袭实验中,干扰组转入底层膜的细胞数为（185.3±14.1）个,低于阴性对照组和空白对照组[分别为（405.7±9.1）、（416.3±11.5）个,均P＜0.05];体外血管拟态显示干扰组细胞多呈散在分布,连接减少,形成的完整结构少.结论 利用siRNA技术抑制MACC1基因表达可有效抑制卵巢癌OVCAR3细胞的体外转移和侵袭能力,MACC1有望成为卵巢癌治疗的靶基因.

Objective To examine the expression of metastasis-associated in colon cancer 1 （MACC1） gene in ovarian cancer cell lines and investigate its effect on biological behaviors of ovarian cancer cells. Methods The expression of MACC1 was examined by qRT-PCR and Western blot analysis in four ovarian cancer cell lines inculding OVCAR3, ES-2, SKOV3 and HO-8910. When the MACC1 was transfected to OVCAR3 cells, fluorogenic quantitative PCR was used to filter and identify MACC1 gene after the efficient silencing. Changes of adhesion in the cells were analyzed by an adhesion assay. Transwell migration and invasion assay and in vitro vascular mimicry assay were used to detect migration, invasion and angiogenesis of OVCAR3 cells in vitro. Results The expression of MACC1 gene was higher in OVCAR3 compared to other cell lines, qRT-PCR confirmed that the expression of MACC1 was silenced successfully after transient transfected MACCI-siRNA into OVCAR3 cells. After successful silencing the MACC1 expression, the adhesion ability was inhibited to some degree. In transwell migration assay, the numbers of cells in upper chamber passing through the membrane in transfected group were less than control groups （245.5±12.8, 500.3±16.5 and 496.3±13.1 respectively）, while in transwell invasion assay, the numbers of cells in upper chamber passing through the membrane in transfected group were less than the negative group and control group （185.3±14.1, 405.7-±9.1 and 416.3_±11.5 respectively）, both with markedly differences among the three groups. In tube formation assay, the distrubition of HUVECs was diffused with less junctions, and the average number of complete tubular structure was decreased in transfected group compared to the corresponding controls. Conclusion RNA interference inhibits the expression of MACC1 and effectively inhibits the metastasis and invasion abilities of ovarian cancer cells in vitro,and MACC1 is expected to become the target gene of ovarian cancer treatment.