摘要
目的:构建间皮素(mesothelin,MSLN)RNAi重组慢病毒质粒,探讨其对卵巢癌OVCAR-3细胞MSLN的表达及细胞增殖的影响。方法:根据MSLN基因信息,设计了4个小干扰序列和1个阴性对照序列,利用慢病毒质粒载体pRNAT-U6.2/Lenti构建了5个重组质粒并进行了慢病毒包装,分别为LV-MSLN-negative、LV-MSLN-shRNA2、LV-MSLN-shRNA3、LV-MSLN-shRNA4;感染OVCAR-3细胞后,Westernblotting和荧光免疫组化检测干扰效率,选择干扰效率高的质粒载体进行慢病毒大量包装;感染卵巢癌细胞OVCAR-3后,用细胞增殖实验和平板克隆形成实验检测细胞增殖的变化。结果:测序结果证明5种质粒载体的插入序列完全正确,鉴定证明慢病毒包装成功。慢病毒感染OVCAR-3细胞后,Westernblotting证实重组慢病毒LV-MSLN-shRNA4的干扰效率最高,对MSLN蛋白表达的抑制达90%。荧光免疫组化的共聚焦照片显示干扰组(OVC-shRNA)定位于细胞膜的MSLN蛋白表达明显弱于阴性对照组(OVC-neg)和空白对照组(OVC)。OVC-shRNA组细胞增殖[(11.2±1.3)×105]显著慢于OVC-neg组[(20.5±2.5)×105]和OVC组[(21.9±2.3)×105](P<0.05)。OVC-shRNA组克隆形成率为(15.2±2.1)%,明显低于OVC-neg组[(27.9±2.5)%]和OVC组[(28.8±3.1)%](P<0.05)。结论:成功构建MSLNRNAi重组慢病毒质粒LV-MSLN-shRNA4,它能有效抑制卵巢癌OVCAR-3细胞的MSLN表达及细胞增殖,为进一步研究MSLN应用于肿瘤基因治疗奠定了基础。
Objective：To construct a recombinant lentivirus plasmid of RNA interference targeting （MSLN） gene and to observe its effect on MSLN expression in human ovarian cancer cell line OVCAR-3 and its effect on cell proliferation. Methods： According to the Genbank information of MSLN, four RNA interfering sequences and a negative sequence were designed and inserted into plasmid pRNAT-U6.2/Lenti and 5 kinds of plasmids were packaged： LV-MSLN-negative,LV-MSLN-shRNA1, LV-MSLN-shRNA2, LV-MSLN-shRNA3, and LV-MSLN-shRNA4; and they were used to transfect OVCAR-3 cells. Western blotting and indirect immunofluorescence were then used to investigate the interfering efficiency. The plasmid with high interfering efficiency was packaged. The cell proliferation test and clone-forming test was used to assess the changes in cell proliferation. Results： DNA sequencing showed that the sequences of 5 recombinant lentivirus plasmids were correct. Lentivirus packaging was successfully done. Western blotting analysis confirmed that LV-MSLN-shRNA4 had the highest interfering efficiency （90%）. MSLN specifically bound to cytomembrane of OVCAR-3 cells. Expression of MSLN in the interfered cells （OVC-shRNA） was weaker than that in the control cells （OVC-neg,OVC）. OVC-shRNA cells（[11.2±1.3]×10^5） grew slowly compared to OVC-neg cells（[20.5±2.5]×10^5） and OVC cells（[21.9±2.3]×10^5） （P〈0.05）. There was a significant reduction in clone forming rate of OVC-shRNA cells （15.2±2.1）% in comparison with OVC-neg cells（[27.9±2.5]%） and OVC cells（[28.8±3.1]%）（P〈0.05）.Conclusions： We have successfully constructed MSLN RNAi recombination plasmid LV-MSLN-shRNA4, which can effectively inhibit MSLN expression and cell proliferation, which paves a way for studying MSLN function and gene therapy.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2008年第2期134-138,共5页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No30572093)
河北省自然科学基金资助项目(NoC2005000804)~~
关键词
间皮素
RNA干扰
慢病毒
卵巢肿瘤
细胞增殖
mesothelin
RNA interference
lentivirus
ovarian neoplasms
cell proliferation
