RNAi阻断乙酰肝素酶介导的卵巢上皮癌细胞系浸润转移体外实验研究

Inhibition of RNAi on HPSE mediated invasive and metastasis ability of ovarian cancer cell line in vitro

目的:探讨乙酰肝素酶shRNA抑制人卵巢癌细胞系乙酰肝素酶(HPSE)表达及对其侵润侵袭能力的影响。方法:针对HPSE mRNA的不同区域设计并构建不同的shRNA表达载体,脂质体法转染HPSE高表达的人卵巢癌细胞系SKOV3,实时定量PCR检验干扰效率,选择抑制率最高的一组细胞进行抗性筛选,获得稳定转染细胞株,分别用RT-PCR和Western blot检验HPSE mRNA和蛋白表达受抑制情况,分别采用四甲基偶氮唑蓝(MTT)法和集落形成实验测定细胞增殖能力,分别采用Matrigel Invasion,Transwell Migration和Adhesion Assay方法测定细胞体外侵袭、迁移和黏附能力。结果:用荧光定量PCR对不同shRNA干扰载体的干扰效果进行检测,结果显示,pGPU6/GFP/Neo-HPSE-1222组相对拷贝数为0.936±0.417,与其他3组相比,差异有统计学意义,抑制率为48.63%。获得的该组稳定干扰表达SKOV3细胞系,经RT-PCR和Western blot检验其HPSE表达明显减少。细胞生长曲线显示干扰组细胞倍增时间延长。干扰组与对照组集落形成率分别为0.0433±0.0451和0.0397±0.00687,差异无统计学意义(P>0.05)。迁移能力实验中,干扰组与对照组相比,差异无统计学意义(P>0.05)。干扰组细胞黏附能力高于对照组(P<0.05)。侵袭能力实验干扰组低于对照组(P<0.05)。结论:采用构建shRNA干扰载体对HPSE基因进行干扰,有效地抑制了HPSE基因的活性,降低了卵巢癌细胞侵袭能力,黏附能力增强。

Objective： To evaluate the inhibitory effect of heparanase shRNA on heparanase and the invasiveness of human ovarian cancer cell line. Method：Several shRNA vector were designed to inhibit various HPSE mRNA domains and transfected into SKOV3 cells in which HPSE is highly expressed by using liposomes. The heparanase mRNA and protein expressions of the transfected and the control cell lines were detected by fluorescence quantitative- PCR and Western blot method respectively. The proliferation ability of cells were detected by 4- methyl-tetrazolium（MTF） and colony-forming experiment. The cells capacity of invasion, migration and adhesion in vitro were tested by using Matrigel Invasion,Transwell Migration and MTT methods. Results：The fluorescence quantitative PCR showed that,the relative copy number pG- PU6/GFP/Neo-HPSE-1222 group was 0. 936±0. 417, which had significant difference compared with the other three groups. The inhibition rate was 48.63 %. The Western blot result alsoshowed the expression of HPSE in SKOV3 cells transfected with pGPU6/GFP/Neo-HPSE-1222 decreased significantly. The cell doubling time extended after transfection. The colony-forming rate were 0. 0433 ±0. 0451 and 0. 0397±0. 00687 in the interference group and the control group respectively（P〉0.05）. The interference group and the control group had no significant difference in migration ability（ P〉0.05 ）. Cell adhesion ability of interference group was higher than that of the control group （P〈0.05）. Invasion ability of the interference group was lower than that of the control group（P〈0.05）. Conclusion： shRNA interference I-IPSE gene vector can effectively inhibit HPSE gene activity, reduce the invasion and metastasis ability and enhance the adhesion ability of ovarian cancer cells.