摘要
背景与目的:10号染色体上磷酸酶和张力蛋白同源缺失的基因(phosphatase and tensin homologue deleted on chromosome 10,PTEN)是较为常见的抑癌基因之一。多项研究表明,其在子宫内膜组织中表达量降低,但是对子宫内膜细胞具体的功能及机制却不十分清楚。本研究旨在探讨PTEN对子宫内膜癌HEC-1B细胞迁移及侵袭能力的影响及相关机制,为子宫内膜癌治疗提供新靶点。方法:运用基因重组技术构建pIRES2-ZsGreen1-PTEN重组质粒;将重组质粒转染至子宫内膜癌HEC-1B细胞中,以转染pIRES2-ZsGreen1空载质粒为对照;运用体外划痕、Transwell迁移及侵袭实验分别检测过表达PTEN后细胞迁移和侵袭能力的变化;运用Western blot法检测过表达PTEN后细胞中PTEN、AKT、p-AKT及MMP-2蛋白表达量的变化。结果:PCR产物凝胶电泳显示一条1.2 kb大小的条带,与PTEN cDNA大小符合;基因测序结果与Genbank中PTEN cDNA的序列一致,表明质粒构建成功;转染后48 h荧光显微镜下见荧光且Western blot显示PTEN蛋白表达量增加,表明重组质粒在HEC-1B细胞中表达成功;体外划痕及transwell迁移实验表明,过表达PTEN后HEC-1B细胞的迁移能力减弱;体外侵袭实验显示,过表达PTEN后HEC-1B细胞的侵袭能力明显低于对照组及未处理组;Western blot结果表明,过表达PTEN会降低HEC-1B细胞中p-AKT蛋白表达下降,但是AKT蛋白量却不受影响,同时p-AKT下游的MMP-2蛋白表达量下降。结论:PTEN能明显抑制子宫内膜癌HEC-1B细胞的迁移和侵袭,这一作用可能是通过抑制AKT磷酸化,从而降低MMP-2蛋白的表达而实现的。
Background and purpose： Phosphatase and tensin homologue deleted on chromosome 10 （PTEN） gene is a kind of tumor suppressors, which has been reported to be underexpressed in endometrial carcinoma （EC） tissues by several reports. However, the biological effects and possible mechanisms of PTEN on EC have been known less. In this study, we tried to investigate the effects and possible mechanisms of PTEN on the invasion and migration of endometrial carcinoma cells and to provide a potential target for endometrial carcinoma therapy. Methods： The recombinant plasmid pIRES2-ZsGreenl-PTEN was rebuilt by gene recombination technology; The plasmid was transferred into HEC-1B cells and the cells transfected with pIRES2-ZsGreenl plasmid were used as control; The expression of PTEN was observed by fluorescence microscope and Western blot assay; Cell migration and invasion was determined by the wound healing assay, transwell migration and invasion assays respectively; The Western blot analysis was performed to detect the expression of ATP-dependent tyrosine kinase （AKT）, phosphorylated-AKT （p-AKT） and matrix metalloproteinase-2 （MMP-2）. Results： The agarose gel electrophoresis showed a stripe of 1.2kb which was same to PTEN cDNA; The sequence analysis showed the PCR products owned the same sequence with the coding region of PTEN cDNA in GenBank, suggesting the recombinant plasmid was constructed successfully; The green light of cells observed by fluorescence microscope and the Western blot analysis showed the expression of PTEN was upregulated in the cells transfected with the recombinant plasmid, suggesting the plasmid expressed successfully in HEC-1B cells; The wound healing assay as well as transwell migration assay showed ectopic expression of PTEN suppressed cell migration; The invasive capacity of HEC-1B cells was significantly decreased upon transfection with PTEN plasmid compared to control and untreated groups; Moreover, compared with the control groups, the expression of p-AKT and MMP-2 was
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2013年第10期813-820,共8页
China Oncology
