小干扰RNA沉默基质金属蛋白酶-2基因对卵巢癌细胞恶性行为的影响

Inhibitory effects of RNA interference on expression of matrix metalloproteinase-2 gene and malignancy behaviours of ovarian cancer cells

目的探讨靶向基质全属蛋白酶．2（MMP-2）基因的RNA干扰（RNAi）技术对卵巢癌OVCAR．3细胞MMP-2的沉默作用，以及沉默MMP-2基因对OVCAR-3细胞生长、黏附、侵袭和迁移能力的影响。方法合成特异性靶向MMP-2基因的小干扰RNA（siRNA）并转染卵巢癌OVCAR-3细胞，以非特异性序列转染细胞作为阴性对照组，以培养液代替siRNA转染细胞作为空白对照组，采用实时荧光定量PCR和Westernblot分别检测转染后24、48和72hMMP．2mRNA和蛋白的表达水平，通过四甲基偶氮唑蓝（M1Tr）法绘制细胞生长曲线，利用Transwell、划痕实验检测细胞侵袭和迁移能力。结果与阴性对照组相比，OVCAR-3细胞在转染siRNA．MMP．2后24、48和72hMMP-2mRNA表达量分别下降了73．8％、78．8％和78．4％（均P〈0．05），蛋白表达量分别下降了72．6％、81．2％和76．4％（均P〈0．05），以48h作用最强；细胞生长曲线显示细胞生长无明显变化（P〉0．05）；60min和90min时的黏附抑制率分别为55．0％和44．8％（均P〈0．05）；细胞的侵袭和迁移能力分别下降29．7％和35．8％（均P〈0．05）。结论siRNA介导的MMP-2基因沉默能明显抑制OVCAR．3细胞的黏附、侵袭和迁移能力，而对其生长无明显影响，MMP-2基因有可能成为卵巢癌基因治疗的重要靶点。

Objective To investigate the inhibitory effects of RNA interference （RNAi） on the expression of matrix metalloproteinase-2 （MMP-2） gene and growth, adhesion, invasiveness and migration of ovarian cancer cells. Methods One specific target sequence of MMP-2 gene and one non-specific sequence （NC group） were chosen, the medium DMEM as blank group. After transfection of ovarian cancer OVCAR-3 cells, the RT-PCR and Western blot were used to detect mRNA and protein expression of MMP-2 gene, the growth ability was detected by MTT assay, the abilities of adhesion was detected by cell adhesion assay, the invasion and migration were detected by Matrigel invasion assay and wound healing assay. Results By contrast to the NC group, the mRNA expression was decreased by 73.8 %, 78.8 % and 78.4 %（P〈 0.05） in 24 h, 48 h and 72 h after transfection and protein expression was decreased by 72.6 %, 81.2 % and 76.4 %（P〈 0.05） respectively at the same time. The 48 h group had the most efficient inhibitory effect. Cell growth curve revealed that cell growth was not significantly inhibited （P 〉 0.05）. Adhesion was significantly reduced,the inhibitory rate was 55.0 % at 60 min and 44.8 % at 90 min （P 〈 0.05）,respectively. Invasion and migration were significantly reduced as well, the inhibitory rate on invasion and migration were 29.7 % and 35.8 %（P〈 0.05）, respectively. Conclusion siRNA mediated MMP-2 down-regulation in ovarian OVCAR-3 cells can inhibits its adhesion, invasion and migration, but do not significantly affect its growth, suggesting a important target to ovarian cancer gene-therapies.