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目的探讨以基质金属蛋白酶(MMP)9基因为靶向的 RNA 干扰(RNAi)技术对 MMP-9基因在卵巢上皮性癌(卵巢癌)细胞中表达的抑制作用,以及对卵巢癌细胞侵袭和黏附能力的影响。方法以 MMP-9基因为靶基因,在编码区选择4个不同的目标序列,分别为 Site1、Site2、Site3、Site4及1个非特异性序列 Site5,利用 PCR 方法构建小分子干扰 RNA(siRNA)表达元件,转染卵巢癌 HO-8910PM 细胞,并依据序列不同将细胞分组,分别为 Site1、Site2、Site3、Site4和Site5组,另以未转染的母代细胞作为对照组。利用 RT-PCR、蛋白印迹法检测 MMP-9 mRNA 及蛋白的表达,通过四甲基偶氮唑蓝法绘制细胞生长曲线,细胞侵袭重建基底膜实验检测细胞体外侵袭能力,通过细胞黏附实验检测细胞体外黏附能力。结果 Site1、Site2、Site3、对照组细胞 MMP-9 mRNA 的相对表达水平分别为0.64±0.06、0.47±0.07、0.55±0.10、0.91±0.08,蛋白的相对表达水平分别为0.30±0.09、0.27±0.08、0.37±0.12、0.63±0.09,与对照组细胞比较,Site1、Site2、Site3组 MMP-9 mRNA 和蛋白的相对表达水平均有不同程度的下降(P<0.05),针对 Site2序列的 siRNA 的基因抑制效果最好。细胞生长曲线显示,Site1、Site2、SitP3、Site4组细胞的生长不同程度的减慢。Site1、Site2、Site3组细胞的侵袭抑制率分别为50.0%、50.0%和37.5%,均高于对照组细胞(0),分别与对照组比较,差异均有统计学意义(P<0.05)。Site1、Site2、Site3、Site4组细胞60min 时的黏附抑制率分别为43.8%、48.8%、33.9%和24.2%,90min 时的黏附抑制率分别为41.6%、40.2%、35.1%和16.0%,黏附抑制率均高于对照组(0),差异有统计学意义(P<0.05)。结论卵巢癌 HO-8910PM 细胞中存在 RNAi 现象,MMP-9siRNA 能特异性地抑制 MMP-9基因的表达,使卵巢癌细胞的侵袭和黏附能力受到不同程度的影响。
Objective To investigate the inhibitory effect of matrix metalloproteinase (MMP) 9 gene targeting RNA interference (RNAi) technology on the expression of MMP-9 in ovarian epithelial carcinoma (ovarian cancer) cells and the invasion and adhesion to ovarian cancer cells The impact of ability. Methods MMP-9 gene was used as target gene and four different target sequences were selected in the coding region. Site1, Site2, Site3, Site4 and a nonspecific sequence Site5 were constructed. The expression of small interfering RNA (siRNA) Cells were transfected with HO-8910PM cells. The cells were divided into two groups according to the sequence, Site1, Site2, Site3, Site4 and Site5, and untransfected parental cells as control. The expression of MMP-9 mRNA and protein was detected by RT-PCR and Western blotting. The cell growth curve was drawn by MTT method. The invasiveness of the basement membrane was evaluated by cell invasion and invasion assay. The cell adhesion assay In vitro adhesion capacity. Results The relative expression levels of MMP-9 mRNA in Site1, Site2, Site3 and control cells were 0.64 ± 0.06, 0.47 ± 0.07, 0.55 ± 0.10 and 0.91 ± 0.08, respectively. The relative expression levels of protein were 0.30 ± 0.09 and 0.27 ± 0.08 , 0.37 ± 0.12 and 0.63 ± 0.09, respectively. Compared with the control group, the relative expression levels of MMP-9 mRNA and protein in Site1, Site2 and Site3 decreased to some extent (P <0.05) Inhibit the best. The cell growth curve showed that the growth of cells in Site1, Site2, SitP3 and Site4 slowed to some extent. The invasion inhibition rates of Site1, Site2 and Site3 were 50.0%, 50.0% and 37.5%, respectively, which were significantly higher than that of the control group (0) (P <0.05). The adhesion inhibition rates of Site1, Site2, Site3 and Site4 cells were 43.8%, 48.8%, 33.9% and 24.2% at 60min, respectively. The adhesion inhibition rates of Site1, Site2, Site3 and Site4 were 41.6%, 40.2%, 35.1% and 16.0% The inhibition rate was higher than that of the control group (0), the difference was statistically significant (P <0.05). Conclusion The RNAi phenomenon exists in ovarian cancer HO-8910PM cells. MMP-9 siRNA can specifically inhibit the expression of MMP-9 gene and affect the invasion and adhesion ability of ovarian cancer cells to varying degrees.