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目的:构建表达真核细胞起始因子-4E(eukaryotic initiation factor 4E,eIF-4E)特异性siRNA的重组腺病毒载体,观察其对人卵巢癌细胞SKOV-3体外转移能力的影响。方法:应用基因重组技术将eIF-4E siRNA序列构建于腺病毒载体pLP-Ade-X,经包装细胞包装后得到高滴度重组腺病毒pLP-Ade-4EsiRNA(psiE)。将腺病毒psiE感染SKOV-3细胞,用定量PCR进行eIF-4E基因表达检测,然后应用transwell小室法观察对细胞侵袭和运动能力的影响,同时检测感染后细胞内VEGF、MMP-2、MMP-9蛋白表达。结果:病毒检测结果与预期相符,real-time PCR可检测到感染重组腺病毒psiE后SKOV-3细胞没有eIF-4E基因表达;病毒感染后transwell小室法检测到SKOV-3细胞的侵袭和运动能力均受到显著的抑制(均为P<0.01);此外病毒感染的SKOV-3细胞中VEGF、MMP-2、MMP-9蛋白表达降低。结论:封闭eIF-4E基因表达对人卵巢癌细胞SKOV-3的侵袭和运动都有抑制作用,其作用机制可能与VEGF、MMP-2、MMP-9表达相关。
OBJECTIVE: To construct a recombinant adenovirus vector expressing specific siRNA targeting eukaryotic initiation factor 4E (eIF-4E) and observe its effect on the ability of human ovarian cancer SKOV-3 cells to metastasize in vitro. Methods: The eIF-4E siRNA sequence was constructed on the adenovirus vector pLP-Ade-X by gene recombination technique and packaged into high titer recombinant adenovirus pLP-Ade-4E siRNA (psiE). The adenovirus psiE was transfected into SKOV-3 cells and the expression of eIF-4E gene was detected by quantitative PCR. The transwell chamber assay was used to observe the effect on cell invasion and exercise capacity. The expression of VEGF, MMP-2 and MMP- 9 protein expression. Results: The result of virus detection was in line with the expectation. The expression of eIF-4E gene in SKOV-3 cells was not detected by real-time PCR after infection with recombinant adenovirus psiE. The invasion and motility of SKOV-3 cells were detected by transwell assay (All P <0.01). In addition, the expression of VEGF, MMP-2 and MMP-9 decreased in virus-infected SKOV-3 cells. CONCLUSION: Blocking the expression of eIF-4E inhibits the invasion and motility of human ovarian cancer cell line SKOV-3, and its mechanism may be related to the expression of VEGF, MMP-2 and MMP-9.