摘要
目的设计构建趋化因子受体4(CXCR4)的RNA干扰逆转录病毒载体,探讨干扰CXCR4表达对前列腺癌细胞侵袭能力的影响。方法用PCR扩增CXCR4特异性小干扰RNA(smallinterferingRNA,siRNA),转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的Pgensil-1质粒中,再将重组基因片段导入到逆转录病毒真核表达载体PLX-SN中,行限制性酶切及测序鉴定。重组逆转录病毒载体转染包装细胞PA317,获取病毒上清转染前列腺癌细胞,RT-PCR检测CXCR4mRNA表达。Transwell小室侵袭实验检测前列腺癌细胞体外侵袭能力的变化。结果通过酶切及测序鉴定,成功构建了PLXSN/EGFP-U6-siCXCR4。干扰质粒抑制CXCR4mRNA的表达,与空载体组比较,PC-3m、LNCaPsiRNA对CXCR4mRNA的抑制率分别为(84.26±10.20)%和(88.17±11.23)%。Transwell侵袭实验结果显示,干扰组微膜下孔细胞数PC-3m、LNCaP分别为(14.7±3.1)和(18.9±4.2)个,空载体组分别为(46.9±5.3)和(66.7±6.0)个,两者差异显著(P<0.05)。结论PLXSN/EGFP-U6-siCXCR4可以有效地抑制前列腺癌细胞CXCR4mRNA的表达,干扰CXCR4可以显著抑制前列腺癌细胞体外侵袭转移能力。
Objective To construct RNA interference retrovirus vector targeting CXCR4 gene and to investigate the effect of invasion of prostate carcinoma cells by interfering expression of CXCR4 gene. Methods The CXCR4 targeting siRNA gene was cloned by PCR. The PCR products were inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP. Then, the recombinant EGFP-U6-siCXCR4 fragment was sub-cloned into PLXSN, and evaluated by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected into prostate carcinoma cells. The expression of CXCR4 mRNA was detected by RT-PCR. The ability of invasion of prostate carcinoma cells was detected by Transwell experiment. Results The expression of CXCR4 mRNA in PC-3m and LNCaP was reduced by the recombinant PLXSN successfully, The inhibitory rate of the expression of CXCR4 mRNA was （84.26± 10.20）% and （88.17±11.23）%, respectively. The results of Transwell experiment indicated that the number of cells in down-pore of micro-membrane was 14.7±3.1 and 18.9±4.2 in the treated group, respectively, compared with 46.9± 5.3 and 66.7 ± 6.0 in the control group in PC-3m and LNCaP （P〈0.05）. Conclusion PLXSN/EGFP-U6-siCXCR4 can significantly inhibit the expression of CXCR4 mRNA, which obviously affects invasiveness of prostate carcinoma cells in vitro.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2006年第5期426-432,共7页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.30300348)
