摘要
目的观察RNAi沉默G250基因表达后,肾癌Ketr-3细胞生长速度和体外侵袭力的变化。方法据G250基因的序列合成shRNA并克隆至真核表达载体pSilencer2.1-U6-neo中,构建G250shRNA真核表达载体,将该载体转染至人肾癌Ketr-3细胞株中。采用RT-PCR、West-ernblot及间接免疫荧光法对干扰效率进行检测。MTT法观察干扰后细胞生长情况的变化,Tran-swell小室体外侵袭实验探讨干扰后细胞体外侵袭能力的变化。结果成功构建了G250shRNA的真核表达载体。RT-PCR、Western blot和间接免疫荧光检测表明转染G250shRNA真核载体的肿瘤细胞G250/β-actin的比值明显降低。细胞生长曲线结果显示Ketr-3-MNRi组与Ketr-3组和阴性对照组Ketr-3-NC相比较,肿瘤细胞生长减慢;Ketr-3-MNRi细胞其穿膜细胞数明显低于Ketr-3-NC及Ketr-3细胞。结论G250shRNA可以下调ketr-3细胞G250的过度表达,抑制肿瘤细胞生长,下调其侵袭能力,为探讨G250在肾癌的发病机制中的作用及生物学治疗肾癌提供实验基础和理论依据。
Objective To observe the silencing effect of G250 gene on Ketr-3 through its variation of cell growth curve and cell invasive ability. Methods According to the sequence of G250, the shRNA was designed, and then cloned into eukaryotic expression vector pSilencer 2. 1-U6-neo. RT-PCR,Western blot and indirect imrnunofluorescent assay （IIFA） were used to identify the silen- cing effect. MTT was used to study the difference of the cell growth curves between Ketr-3 ,Ketr-3- NC and Ketr-3-MNRi cells. Transwell was used to study the difference of the cell invasive ability between them. Results pSilencer 2. 1-U6 neo-shRNA was constructed successfully. The results of RT-PCR,Western blot and IIFA confirmed the silencing effect on G250 gene expression. The results of MTT showed that there was significant difference in cell growth curve between Ketr-3-MNRi and Ketr-3-NC, Ketr-3 ceils （P〈0.05）. The results of Transwell showed that the cell invasive ability differed significantly between Ketr-3-MNRi and Ketr-3 NC, Kerr-3 cells （P〈 0. 05）. Conclusions G250 shRNA can down-regulate the expression of G250 by RNAi, and then slow down the cell growth rate and attenuate cell invasive ability, which provides new ideas for the research on pathogenesis and treatment of renal cell carcinoma.
出处
《现代泌尿生殖肿瘤杂志》
2009年第3期169-173,共5页
Journal of Contemporary Urologic and Reproductive Oncology
基金
广东省科技计划项目(2007B030704002)
关键词
肾肿瘤
RNA干扰
基因
Kidney neoplasms
RNA interference
Genes
