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目的构建人前列腺特异性抗原(hPSA)启动子调控的趋化因子受体4(CXCR4)的RNA干扰逆转录病毒载体,并探讨其对激素依赖性前列腺癌LNCaP细胞CXCR4基因表达的靶向抑制作用。方法PCR扩增CXCR4特异性小干扰RNA(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,以hPSA启动子代替U6启动子,再将重组基因片段导入到逆转录病毒真核表达载体pLXSN中,进行限制性酶切鉴定。转染。PA317包装细胞,收获病毒上清,分别转染前列腺癌细胞PC-3m、LNCaP和人乳腺癌细胞MCF7。采用RT-PCR、Western blot检测CXCR4基因表达。Transwell小室侵袭实验检测前列腺癌细胞体外侵袭能力的变化。结果通过限制性酶切鉴定该重组质粒,成功构建了CXCR4的RNA干扰质粒pLXSN/EGFP-hPSA-siCXCRd。与空载体组比较,CXCRd-siRNA对LNCaP细胞CXCR4 mRNA和蛋白表达抑制显著,48 h抑制率分别为(81.53±10.22)%和(90.52±9.31)%;对PC-3m、MCF-7细胞CXCRd mRNA和蛋白表达无抑制作用。Transwell侵袭实验结果显示,LNCaP细胞干扰组微膜下孔细胞数为(139.9±14.2)个,空载体组为(348.4±36.4)个,两组差异有统计学意义(P<0.05);PC-3m、MCF-7细胞干扰组微膜下孔细胞数与空载体组比较,差异无统计学意义(P>0.05)。结论该逆转录病毒系统中,hPSA启动子调控的下游RNA干扰序列特异性地抑制激素依赖前列腺肿瘤细胞的CXCR4基因表达,具有明显的靶向性。hPSA启动子调控的靶向CXCRd基因的RNA干扰技术在激素依赖性前列腺癌的基因治疗中具有一定价值。
OBJECTIVE: To construct an RNA interference retrovirus vector targeting chemokine receptor 4 (CXCR4) regulated by human prostate specific antigen (hPSA) promoter and to investigate its targeted inhibitory effect on CXCR4 gene expression in hormone-dependent prostate cancer LNCaP cells . Methods The CXCR4 specific small interfering RNA (siRNA) was amplified by PCR and transferred into pGensil-1 plasmid with enhanced green fluorescent protein (EGFP) and promoter U6. The hPSA promoter was used to replace the U6 promoter. The recombinant gene fragment Introduced into the retroviral eukaryotic expression vector pLXSN restriction endonuclease identification. Transfection. PA317 packaging cells, harvested virus supernatant, were transfected with prostate cancer cells PC-3m, LNCaP and human breast cancer cells MCF7. CXCR4 gene expression was detected by RT-PCR and Western blot. Transwell Chamber Invasion Assay to Detect Proliferation of Prostate Cancer Cells in Vitro. Results The plasmid pLXSN / EGFP-hPSA-siCXCRd was successfully constructed by restriction enzyme digestion of the recombinant plasmids. CXCRd-siRNA inhibited the expression of CXCR4 mRNA and protein in LNCaP cells significantly compared with the control group (81.53 ± 10.22% vs. (90.52 ± 9.31)%, respectively) No inhibition of mRNA and protein expression. The results of Transwell invasion assay showed that there were (139.9 ± 14.2) cells in the micro-membrane of the LNCaP cell-mediated interference group and (348.4 ± 36.4) in the empty vector group, with significant difference between the two groups (P <0.05) There was no significant difference in the numbers of cells in the MCF-7 cells interfered with the 3m, MCF-7 cells compared with the empty vector group (P> 0.05). Conclusion The downstream RNA interference sequence regulated by hPSA promoter specifically inhibits the CXCR4 gene expression in hormone-dependent prostate cancer cells in this retrovirus system and has obvious targeting. hPSA promoter-mediated CXCRd gene-targeted RNA interference technology in hormone-dependent prostate cancer gene therapy has some value.