过表达PNRC对MCF-7乳腺癌细胞生长的影响

目的：探讨核受体辅活化子（coactivator）PNRC（proline—rich nuclear receptor coregulatory protein）对乳腺癌细胞生长的影响。方法：以pACT2／PNRC为模板，采用PCR扩增全长PNRC编码序列，将BclⅠ和XhoⅠ消化的PCR产物插入pCMVtaq2c，构建pCMVtaq2c／PNRC真核表达质粒。酶切和测序鉴定后，采用脂质体转染法，分别将pCMVtaq2c／PNRC表达质粒和pCMVtaq2c空质粒转染MCF-7细胞，经G418筛选4周后，分别采用RT—PCR、Northern blot和Western Blot鉴定稳定表达PNRC的MCF-7细胞株。最后，采用MTT法和^3H-胸腺嘧啶掺入法观察了过表达PNRC的MCF-7乳腺癌细胞和pCMVtaq2c空质粒转染的MCF-7乳腺癌细胞生长速率和增殖的差异。结果：成功构建了PNRC的真核表达质粒pCMVtaq2c／PNRC，并筛选到稳定表达PNRC的MCF-7乳腺癌细胞株，转染PNRC的MCF-7细胞的生长速率和增殖明显慢于对照组细胞。结论：过表达的PNRC对MCF-7乳腺癌细胞的生长具有抑制作用。

Effect on growth of MCF-7 cells by PNRC over-expression.

Objective:To explore the effect on growth of MCF-7 cells by over-expressing nuclear receptor coactivator PNRC(proline-rich nuclear receptor coregulatory protein).Methods:To construct the recombinant plasmid pCMVtaq2c/PNRC,full length PNRC cDNA with Bcl I and Xho I amplified by PCR was digested and inserted into eukaryotic expression plasmid pCMVtaq2c.After identification of restriction endonuclease and sequencing,the recombinant plasmid and empty plasmid was respectively transfected into MCF-7 cells by lipofectAMINE.The stably transfected strain after G418 screening for 4 weeks was identified with RT-PCR,Northern blot and Western blot.The different growth rate and proliferation of MCF-7 cells over-expressing PNRC and empty plasmid transfected were detected by MTT.Results:The eukaryocytic expression plasmid pCMVtaq2c/PNRC was constructed successfully.MCF-7 cell strain stably expressing PNRC was screened and the growth rate and proliferation of MCF-7 cells transfected PNRC was obviously slower than that of control cells.Conclusion:PNRC over-expression could inhibit the growth of MCF-7 cells.