siRNA沉默CXCR4基因的实验研究

A study on siRNA suppressed target gene CXCR4 expression

目的:设计和构建趋化因子受体4(CXCR4)基因的小干扰RNA质粒,并初步验证其对靶基因的抑制作用。方法:选择高表达CXCR4受体的人乳腺癌细胞系T47D作为研究对象。将预先设计的3段siRNA分别连入质粒载体,然后转化大肠杆菌,经过克隆、扩增、纯化后转染到T47D细胞中,G418筛选稳定表达细胞株,采用流式细胞术从蛋白水平检测CXCR4的表达率,实时定量PCR从基因转录水平检测CXCR4基因的表达率。结果:流式细胞术检测结果显示,T47D细胞经特异性siRNA作用后CXCR4的表达率由69.00%分别下降到4.95%、11.30%、5.53%。实时相对定量PCR检测结果显示,siRNA作用的细胞其CXCR4基因转录的抑制率分别为95.67%、85.94%、98.25%。结论:siRNA能够引发人乳腺癌细胞系T47D的CXCR4基因沉默,为进一步探讨CXCR4基因在乳腺癌转移中的作用奠定前期实验基础,也为siRNA作为一种可能的治疗手段提供了理论依据。

Objective： To design and construct small interfering RNA （siRNA） expression plasmids and explore their effects on gene silence of CXCR4 gene. Methods： Human breast cancer cell line T47D was used as the experimental subjects, which could highly express CXCR4 gene. Three siRNA oligonucleotides targeting CXCR4 at different locations were synthesized and inserted into pSilencer^TM 3.1-H1 neo vector. Then the plasmids were transformed into E.coli in order to clone and amplify. After verified the inserted sequence by DNA sequencing, the positive reconstructs was purified and transfected into T47D cells. Finally, the expression of CXCR4 in the transfected cells was measured by flow cytometry and real-time relative PCR. Results： The expression rate of CXCR4 of T47D cells was decreased from 69.00% to 4.95% , 11.30%, 5.53% after transfected three siRNA. The result of real-time PCR revealed that the suppressive rates of CXCR4 were 95.67%, 85.94%, 98.25%. Conclusion： Small interfering RNA expression plasmids are constructed successfully, which paves the way for future studies of CXCR4 gene on breast cancer metastasis.