Mechanism of AKT1 Enhancing Migration and Invasion of Epithelial Ovarian Cancer Cells
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摘要: 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。Abstract: Abstract：Objective To explore the effects of AKT1 gene on the invasion and migration of epithelial ovarian cancer cells SKOV3 and its mechanism. Methods Recombinant plasmid carrying the full-length AKT1 cDNA and AKT1 shRNA plasmid were constructed. Plasmids were transfected into SKOV3 cells to dual-directionally regulate AKT1 expression. RT-PCR and western blot were used to detect the transfection efficiency. Wound healing assay was used to analyze cell migration. Transwell-Matrigel assay was used to analyze cell invasion. RT-PCR was used to explore the expression of CXCR4, VEGF, MMP-2, MMP-9 and 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 plasmid were constructed successfully, which could effectively regulate the activation of p-AKT after SKOV3 cells transfected. Compared with untranfected cells or non-target shRNA transfected cells, up-regulation AKT1 promoted cell migration and invasion, and increased the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level(P＜0.05). On the other hand, down-regulation AKT1 inhibited cell migration and invasion, and decreased the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level(P＜0.05). Conclusion AKT1 might impact cell migration and invasion by regulating the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level.
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Key words:
- Epithelial ovarian cancer /
- AKT1 /
- Signal pathway
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