PLK3在前列腺癌细胞中的表达与定位

The Expression and Localization of PLK3 in Prostate Cancer

据报道,Polo样蛋白激酶3(polo-like kinase 3,PLK3)具有促进前列腺癌细胞的生长增殖及迁移能力。本研究旨在构建pCDNA3-FLAG-PLK3真核表达质粒,初步探究其在前列腺癌细胞系内的表达与定位,并检测前列腺癌细胞系中的内源PLK3蛋白水平。根据PLK3蛋白的编码序列设计合成PLK3蛋白编码序列的引物,以含有PLK3蛋白编码序列的质粒作为模板,并通过PCR扩增目的片段,再用限制性内切酶EcoRⅠ和XhoⅠ进行双酶切、连接并转化后,挑取单克隆菌落扩增并提取质粒,进行双酶切鉴定以及测序比对。将序列比对正确的重组质粒转染至CWR22Rv1及LNCaP细胞中,利用Western Blot实验检测重组质粒在CWR22Rv1和LNCaP细胞中的表达;以免疫荧光染色实验检测外源的FLAG-PLK3蛋白在细胞中的定位。此外,利用Western Blot实验检测了在五种前列腺癌细胞系中内源PLK3蛋白水平。本研究经过以上实验构建出了FLAG-PLK3真核表达质粒,并且验证其能够在前列腺癌细胞中表达;通过免疫荧光确定FLAG-PLK3蛋白在细胞中主要分布在细胞膜中,少量分布在细胞质;在检测的五种前列腺癌细胞系中,DU145中PLK3蛋白水平最高,CWR22Rv1中PLK3蛋白水平最低。本研究为后续深入探究PLK3蛋白在前列腺癌细胞系中的功能与机制提供了科学依据。

According to the reports that polo-like kinase 3 can promote the growth,proliferation and migration of prostate cancer cells,this study was aimed at constructing the recombinant mammalian cell expression plasmid of pCDNA3-FLAG-PLK3,identifing the expression and localization of this recombinant protein in prostate cancer cells.The primers were designed and synthesized according to the full-length coding sequence of Polo-like kinase 3(PLK3).Plasmid containing coding sequence of PLK3 was used as a template in PCR amplification,and the PCR product was digested by endonucleases EcoR Ⅰ and Xho Ⅰ.After ligation and transformation,monoclonal colony was picked and amplificated.Then,plasmid extraction was carried out,and the plasmid was confirmed by enzyme digestion and sequencing.Further,the recombinant plasmid with the correct sequence was transfected into CWR22 RV1 and LNCaP cells,and the expression of FLAG-PLK3 in CWR22 RV1 and LNCaP cells was detected by Western Blot experiments.Then immunofluorescence staining was performed to detect the localization of the exogenous FLAG-PLK3 in cells.In addition,Western Blot experiments was used to determine endogenous PLK3 protein expression in prostate cancer cell lines.The results showed that pCDNA3-FLAG-PLK3 plasmid was correctly constructed and expressed in prostate cancer cells.Exogenous FLAG-PLK3 was mainly distributed in the cell membrane,and also with a small amount in the cytoplasm.Endogenous expression of PLK3 protein was higher in DU145 cells,and lower in CWR22 Rv1 cells,than that of the other cell lines.This study provides an experimental basis for the further studies on the function and mechanism of PLK3 protein in prostate cancer cells.