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目的探讨 HER-2小分子干扰 RNA(siRNA)对卵巢上皮性癌(卵巢癌)细胞侵袭和趋化能力的影响。方法选用已知的干扰磷酸甘油醛脱氢酶(GAPDH)的特异性位点作为阳性对照,采用蛋白印迹法检测卵巢癌细胞株 SKOV3细胞 GAPDH 蛋白和 HER-2蛋白的表达水平,以吸光度(A)值表示。然后在 HER-2基因的编码区内根据干涉位点的筛选原则,选择 HER-2 siRNA Ⅰ、HER-2siRNAⅡ、HER-2 siRNAⅢ3段靶序列,体外转录合成 HER-2 siRNA。实验分为4组:空白对照组、脂质体组、非特异性转染组(非特异性 siRNAⅢ)、特异性转染组(HER-2 siRNAⅢ),以β肌动蛋白(β-actin)作为内参照,用荧光实时定量 PCR 和蛋白印迹法检测转染前后 SKOV3细胞 HER-2 mRNA 和蛋白表达的变化;细胞侵袭实验和细胞趋化实验检测转染前后 SKOV3细胞体外侵袭和趋化能力的变化。结果 GAPDH siRNA 能明显抑制 SKOV3细胞内源性 GAPDH 蛋白的表达,空白对照及加入不同剂量(0.5、1.0、1.5、2.0μg)的 GAPDH siRNA 转染 SKOV3细胞后,其 GAPDH 蛋白的表达水平分别为0.6855±0.0259、0.5698±0.0275、0.4542±0.0296、0.3341±0.0178及0.1816±0.0180,各剂量间比较,差异有统计学意义(F=198.126,P<0.01)。HER-2 siRNAⅡ、HER-2 siRNAⅢ均有抑制 HER-2蛋白表达的作用,HER-2 siRNAⅡ、HER-2 siRNAⅢ转染后 SKOV3细胞中 HER-2蛋白的相对表达水平(分别为0.2162±0.1589、0.1562±0.0067),分别与空白对照组、非特异性转染组及转染 HER-2siRNA Ⅰ的 SKOV3细胞(分别为0.3674±0.0350、0.3839±0.0188、0.3532±0.0197)比较,差异均有统计学意义(F=69.461,P<0.01)。不同剂量(0.5、1.0、1.5、2.0μg)的 HER-2 siRNAⅢ转染后,SKOV3细胞中 HER-2 mRNA 的相对表达水平比较,差异有统计学意义(F=174.53,P<0.01);HER-2siRNAⅢ转染后第1、3、6天,SKOV3细胞中 HER-2 mRNA 的相对表达水平分别为0.0506±0.0017、0.0266±0.0011及0.0154±0.0020,不同时间点比较,HER-2 mRNA 的相对表达水平呈明显下降趋势(P<0.01);特异性转染组 SKOV3细胞的侵袭和趋化能力均明显低于非特异性转染组及空白对照组,差异均有统计学意义(F=53.707,P<0.01;F=11.361,P<0.01)。结论 HER-2 siRNA 对卵巢癌细胞 HER-2基因的抑制作用呈时间及剂量依赖性,HER-2 siRNA 能显著抑制细胞侵袭和趋化能力,这为卵巢癌的基因治疗提供了一种新策略。
Objective To investigate the effect of HER-2 small interfering RNA (siRNA) on the invasion and chemotaxis of ovarian epithelial carcinoma (ovarian cancer) cells. Methods The specific sites of interference GAPDH were selected as positive control. The expression of GAPDH protein and HER-2 protein in ovarian cancer cell line SKOV3 were detected by Western blotting. The absorbance (A ) Value that. HER-2 siRNAⅠ, HER-2siRNAⅡ and HER-2 siRNAⅢ3sequences were selected by HER-2 gene coding region according to the selection principle of the interference sites, and HER-2 siRNA was synthesized and transcribed in vitro. The experiment was divided into 4 groups: blank control group, liposome group, non-specific transfection group (non-specific siRNAⅢ), specific transfection group (HER-2 siRNAⅢ), β-actin The changes of HER-2 mRNA and protein expression in SKOV3 cells before and after transfection were detected by real-time quantitative PCR and Western blotting. The invasion and chemotactic ability of SKOV3 cells were detected by cell invasion assay and chemotaxis assay. Results GAPDH siRNA could significantly inhibit the expression of endogenous GAPDH protein in SKOV3 cells. The GAPDH protein expression levels of GAPDH siRNA transfected with GAPDH siRNA in different concentrations (0.5, 1.0, 1.5 and 2.0 μg) were 0.6855 ± 0.0259,0.5698 ± 0.0275,0.4542 ± 0.0296,0.3341 ± 0.0178 and 0.1816 ± 0.0180. There was significant difference between each dose (F = 198.126, P <0.01). The expression of HER-2 protein in SKOV3 cells transfected with HER-2 siRNAⅡ and HER-2 siRNAⅢwere all lower than that of HER-2 siRNAⅡ and HER-2 siRNAⅢrespectively (0.2162 ± 0.1589, 0.1562 ± 0.0067), which were significantly different from those of blank control group, non-specific transfection group and SKOV3 cells transfected with HER-2 siRNA Ⅰ (0.3674 ± 0.0350,0.3839 ± 0.0188,0.3532 ± 0.0197, respectively) F = 69.461, P <0.01). The relative expression levels of HER-2 mRNA in SKOV3 cells after transfection with different doses (0.5,1.0,1.5,2.0μg) of HER-2 siRNAⅢwere significantly different (F = 174.53, P <0.01); HER The mRNA expression levels of HER-2 in SKOV3 cells were 0.0506 ± 0.0017, 0.0266 ± 0.0011 and 0.0154 ± 0.0020 respectively at 1, 3 and 6 days after transfection. The relative expression of HER-2 mRNA at different time points (P <0.01). The invasion and chemotaxis ability of SKOV3 cells in the specific transfection group were significantly lower than those in the non-specific transfection group and the blank control group (F = 53.707, P <0.01; F = 11.361, P <0.01). Conclusion The HER-2 siRNA inhibits HER-2 gene in ovarian cancer cells in a time and dose-dependent manner. HER-2 siRNA can significantly inhibit cell invasion and chemotactic ability, which provides a new strategy for the gene therapy of ovarian cancer .