论文部分内容阅读
目的:构建组织蛋白酶L基因(cathepsin L,CTSL)RNAi的真核表达载体,探讨干扰CTSL基因表达对卵巢癌细胞侵袭和转移的影响。方法:设计并合成4对CTSL小干扰RNA(small interfering RNA,siRNA),转染卵巢癌A2780细胞,RT-PCR检测各转染细胞CTSL的表达,筛选沉默效果最好的siRNA序列。设计并合成CTSL-shRNA序列,与psilence4.1-CMV-neo载体连接,构建psilence4.1-CTSL表达载体。psilence4.1-CTSL转染A2780细胞,获得稳定转染克隆A2780-CTSL。RT-PCR和Western blotting验证CTSL基因干扰效果,MTT法和集落形成实验检测A2780细胞的增殖,流式细胞术检测A2780细胞周期,Transwell侵袭小室实验检测A2780细胞体外侵袭和迁移能力。结果:筛选出干扰效果最好的siRNA-CTSL-1202序列,并成功构建相应的CTSL-shRNA表达载体psilence4.1-CTSL。稳定转染psilence4.1-CTSL能沉默A2780细胞中CTSL的表达。沉默CTSL基因后可抑制A2780细胞的侵袭和转移,但不影响A2780细胞的增殖、细胞周期和黏附活性。结论:成功构建CTSL基因siRNA的真核表达载体,RNA干扰CTSL基因表达可抑制卵巢癌细胞的侵袭和转移。
OBJECTIVE: To construct an eukaryotic expression vector for cathepsin L (CTSL) RNAi and explore its effect on the invasion and metastasis of ovarian cancer cells by interfering with the expression of CTSL gene. Methods: Four pairs of small interfering RNAs (siRNAs) targeting CTSL were designed and synthesized. A2780 cells were transfected into ovarian cancer cells. The expression of CTSL was detected by RT-PCR and the siRNA sequence with the best silencing effect was screened out. The CTSL-shRNA sequence was designed and synthesized and ligated with psilence4.1-CMV-neo vector to construct psilence4.1-CTSL expression vector. A2780 cells were transfected with psilence4.1-CTSL to obtain stably transfected clone A2780-CTSL. The interference effect of CTSL gene was verified by RT-PCR and Western blotting. The proliferation of A2780 cells was detected by MTT assay and colony formation assay. The cell cycle of A2780 cells was detected by flow cytometry. The invasion and migration of A2780 cells were detected by Transwell invasion chamber assay. Results: The siRNA-CTSL-1202 sequence with the best interference effect was screened and the corresponding CTSL-shRNA expression vector psilence4.1-CTSL was successfully constructed. Stable transfection of psilence4.1-CTSL silences CTSL expression in A2780 cells. Silencing CTSL gene can inhibit the invasion and metastasis of A2780 cells, but not affect the proliferation, cell cycle and adhesion activity of A2780 cells. Conclusion: The eukaryotic expression vector of siRNA of CTSL gene was constructed successfully. RNA interference of CTSL gene expression can inhibit the invasion and metastasis of ovarian cancer cells.