RNA干扰下调HE4基因表达对人卵巢癌SK-OV-3细胞增殖和侵袭的影响

目的应用RNA干扰技术下调人附睾蛋白HE4基因表达水平,观察HE4对人卵巢癌SK-OV-3细胞增殖和侵袭能力的影响。方法化学合成3对HE4特异性小分子干扰RNA(siRNA),脂质体法转染人卵巢癌细胞系SK-OV-3(HE4-siRNA组),同时以非特异序列siRNA转染的SK-OV-3细胞(阴性对照组)和正常培养的SK-OV-3细胞(正常对照组)为对照,于转染后48 h,采用实时荧光定量PCR技术(RT-qPCR)和Western blot方法分别检测HE4 mRNA及蛋白表达水平。采用CCK8试剂盒检测卵巢癌细胞增殖活性的变化,穿膜小室模型测定HE4对细胞侵袭能力的影响。结果与正常对照组相比,HE4-siRNA转染卵巢癌SK-OV-3细胞48 h后,HE4 mRNA的表达水平显著下降,仅为正常对照组的12.7%(P<0.01),与之相应HE4蛋白表达也显著下降,而转染非特异序列的阴性对照组与正常对照组之间的差异无统计学意义(P>0.05)。HE4-siRNA下调卵巢癌SK-OV-3细胞HE4表达后,细胞增殖受到明显抑制,细胞增殖活性仅为正常对照组的60%,阴性对照组细胞增殖未见明显变化。体外侵袭实验显示,HE4-siRNA组穿膜细胞数为每视野(21.8±2.86)个,显著低于正常对照组(187.4±11.17)个(P<0.01)和阴性对照组(177.8±9.76)个(P<0.01),而阴性对照组和正常对照组两组之间差异无统计学意义(P>0.05)。结论 HE4特异性siRNA能成功下调SK-OV-3细胞中HE4基因的表达,显著降低卵巢癌细胞增殖和侵袭能力,HE4有可能成为人卵巢癌侵袭转移防治的重要靶点。

Effect of HE4 gene silenced by siRNA on the proliferation and invasiveness of ovarian cancer cell SK-OV-3

Objective To investigate the effect of HE4 gene knocked down by small interfering RNA （siRNA） on proliferation and invasiveness of ovarian cancer cell SK-OV-3. Methods 3 pairs of synthetic siRNAs targeted HE4 gene were transiently transfected into SK-OV-3 cells by liposomal method （HE4-siRNA group）, siRNA of scrambled sequence were transfected at the same time as a negative control, and normal cultured SK-OV-3 without any transfection were used as normal control. HE4 expression level in SK-OV-3 cells was determined using real-time quantitative PCR method and Western blot. CCK8 assay was used to identify proliferation of SK-OV-3 cells. The invasive ability of SK-OV-3 cells was measured by transmembrane （Transwell） small chamber method. Results The HE4 mRNA level in HE4-siRNA group cells was significantly decreased to about 12.7% of that in the normal control group. Western blot results showed that the HE4 protein level was the same trend with the mRNA one. However, there existed no statistically significant differences in HE4 expression levels between negative control group and normal control group （P 〉 0.05）. The inhibitory percentage of cell proliferation was about 60% of that in normal control group since HE4 expression was knocked down using HE4-siRNA. There was no significant difference in cell proliferation between negative control group and normal control group. In the invasion assay, the number of cells that invaded through the artificial basement membrane in HE4-siRNA group was （21.8 ± 2.86）, which was significantly lower than those in normal group and negative control group （187.4 ±11.17） （P 〈 0.01） and （177.8 ± 9.76） （P 〈 0.01） respectively, while difference between the 2 control groups were not statistically significant （P 〉 0.05）. Conclusion Downregulation of HE4 expression by siRNA inhibits invasion and proliferation of ovarian cancer cell.