外源性AKT1增强上皮性卵巢癌细胞侵袭和迁移能力的机制

探讨AKT1基因对上皮性卵巢癌SKOV3细胞迁移和侵袭能力的影响及相关分子机制。方法 针对AKT1基因，设计并构建shRNA质粒和真核表达质粒，双向调节SKOV3细胞中AKT1的表达，运用RT-PCR和western blot检测转染效率。运用Wound healing和Transwell-Matrigel方法检测转染前后细胞迁移和侵袭能力的变化。RT-PCR法检测与细胞运动侵袭相关分子CXCR4、VEGF、MMP-2、MMP-9和uPA在mRNA水平的表达变化。结果 成功构建AKT1基因的真核表达质粒pEF-1α-AKT1和靶向抑制AKT1基因的shRNA表达质粒pRNAT-AKT1。转染上皮性卵巢癌SKOV3细胞后，能有效调控p-AKT表达。参照未转染组和空载体转染组，外源性AKT1促进细胞迁移和侵袭，CXCR4、VEGF、MMP-2和uPA的mRNA表达水平升高。shRNA靶向抑制AKT1基因的表达可抑制细胞迁移和侵袭，CXCR4、VEGF、MMP-2和uPA的mRNA表达水平下降。结论 AKT1可能通过调控CXCR4、VEGF、MMP-2和uPA的转录水平来影响细胞侵袭和运动能力。

Mechanism of AKT1 Enhancing Migration and Invasion of Epithelial Ovarian Cancer Cells

Abstract：Objective To explore the effects of AKT1 gene on the invasion and migration of epithelialovarian cancer cells SKOV3 and its mechanism. Methods Recombinant plasmid carrying the full-lengthAKT1 cDNA and AKT1 shRNA plasmid were constructed. Plasmids were transfected into SKOV3 cells todual-directionally regulate AKT1 expression. RT-PCR and western blot were used to detect the transfectionefficiency. Wound healing assay was used to analyze cell migration. Transwell-Matrigel assay was usedto analyze cell invasion. RT-PCR was used to explore the expression of CXCR4, VEGF, MMP-2, MMP-9and uPA which were related with cell invasion and migration at mRNA level. Results A plasmid (pEF-1α-AKT1) containing full length of AKT1 cDNA and a specifi c AKT1-targeted shRNA expression plasmidwere constructed successfully, which could effectively regulate the activation of p-AKT after SKOV3 cellstransfected. Compared with untranfected cells or non-target shRNA transfected cells, up-regulation AKT1promoted cell migration and invasion, and increased the expression of CXCR4, VEGF, MMP-2, and uPA atmRNA level(P＜0.05). On the other hand, down-regulation AKT1 inhibited cell migration and invasion, anddecreased the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level(P＜0.05). Conclusion AKT1might impact cell migration and invasion by regulating the expression of CXCR4, VEGF, MMP-2, and uPA atmRNA level.