摘要
目的构建微小RNA（miR）-21的真核表达载体pEZX-eGFP—microRNA-21。将构建成功的pEZX—eGFP—microRNA-21瞬时转染至甲状腺乳头状癌细胞株TPC—l中，探讨转染前后细胞蛋白程序性细胞凋亡4（PDCD4）蛋白的表达差异及其机制。方法人工合成microRNA-21基因序列，构建成重组质粒pEZX—eGFP—microRNA-21并转染TPC—l细胞。将TPC一1细胞分为转染组、空载组、空白组，每组20个复孔。实时定量聚合酶链反应（Real—timePCR）、Westernblot分别检测转染后miR-21及PDCIM蛋白在各组细胞中的表达。结果经酶切和测序分析鉴定后证明重组质粒pEZX—eGFP—microRNA-21构建成功。转染组TPC一1细胞miR一21的表达水平明显高于对照组：加尾法、茎环法中分别是对照组的6．39倍（P〈0．01）、7．58倍（P〈0．01）。Westernblot显示转染组细胞PDCD4表达量明显降低（0．24±0．03，P〈0．05）。结论成功构建miR-21基因真核表达载体pEZX—eGFP—microRNA．2l。miR-21在甲状腺乳头状癌细胞TPC一1中高表达可能导致PDCIM蛋白低表达。
Objective To construct microRNA-21 eukaryotic expression vector pEZX-eGFP-mi- croRNA-21, and explore the differences of programmed cell death 4 （ PDCD4 ） protein expression and the mechanisms after transient transfection of pEZX-eGFP-microRNA-21 into papillary thyroid cancer cell line TPC-1. Methods miR-21 sequence was synthesized and cloned into pEZX-eGFP to construct recombinant plasmid pEZX-eGFP-microRNA-21. TPC-1 cells were divided into transfected group, the no-load group, blank group （n = 20 each ）. Transcription level of microRNA-21 was detected by using real-time PCR. Western blotting was used to detect the differences of PDCD4 protein expression. Results Recombinant plasmid pEZX-eGFP-microRNA-21 was successfully constructed. The mieroRNA-21 expression level in transfected group was significantly higher than in no-load group and blank group. Tailing method revealed the expression level in transfected group was 6. 39 times that of no-load and blank groups （P 〈 0. 01 ）, and stem-loop method 7. 58 times （P 〈 0. O1 ）. Western blotting showed that PDCD4 expression level in trans- fected group was significantly lower than other groups （0. 24 -＋ 0.03, P 〈 0.05 ）. Conclusion miR-21 ex- pression vector was constructed successfully. The high expression of miR-21 in TPC-1 ceils may lead to the low expression of PDCD4 protein.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第3期487-489,共3页
Chinese Journal of Experimental Surgery
