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目的:探讨趋化因子受体7(Chemokine receptor 7,CXCR7)的短发夹RNA(a small hairpin Ribonucleic acid,shRNA)慢病毒表达载体对人肝癌细胞HepG2中CXCR7表达的影响。方法:合成4个针对CXCR7靶基因序列的shRNA,分别与Age I和EcoRI酶切后的pGCSIL-RFP载体连接,构建CXCR7的shRNA慢病毒表达载体pGCSIL-RFP-CXCR7-shRNA;构建含有CXCR7互补DNA(complementary DNA,cDNA)的真核过表达载体pEGFP-N1-3FLAG-CXCR7,与pGCSIL-RFP-CXCR7-shRNA共转染HEK293T细胞,筛选具有显著敲减作用的CXCR7-shRNA;将具有显著敲减作用的pGCSIL-RFP-CXCR7-shRNA与慢病毒包装质粒共转染人胚肾细胞(Human embryonic kidney cells,HEK293T)产生慢病毒颗粒LV-CXCR7-shRNA,并将纯化的慢病毒颗粒感染人肝癌HepG2细胞,以逆转录聚合酶链反应(RT-PCR)和免疫印迹(Western blot)分别检测CXCR7信使核糖核酸(mRNA)和蛋白的沉默效果。结果:聚合酶链反应(Polymerase chain reaction,PCR)鉴定及测序结果表明成功构建4个CXCR7的shRNA慢病毒表达载体,并筛选出具有显著敲减作用的pGCSIL-RFP-CXCR7-shRNA2;包装含有CXCR7-shRNA2的慢病毒颗粒LV-CXCR7-shRNA2(病毒滴度为3×109TU/ml),感染HepG2细胞,CXCR7mRNA和蛋白的表达水平下调。结论:成功构建靶向CXCR7基因的shRNA慢病毒表达载体,可有效抑制人肝癌HepG2细胞CXCR7 mRNA和蛋白的表达。
AIM: To investigate the effect of a small hairpin RNA (shRNA) lentiviral vector expressing CXCR7 on the expression of CXCR7 in human hepatocellular carcinoma cell line HepG2. Methods: Four shRNAs targeting CXCR7 target gene were synthesized and cloned into pGCSIL-RFP vector after digestion with Age I and EcoRI to construct shRNA lentiviral vector pGCSIL-RFP-CXCR7-shRNA targeting CXCR7. The recombinant plasmid pEGFP-N1-3FLAG-CXCR7 was transfected into HEK293T cells by co-transfection with pGCSIL-RFP-CXCR7-shRNA. The knockdown of CXCR7-shRNA with significant knockdown The recombinant plasmid pGCSIL-RFP-CXCR7-shRNA was co-transfected with lentiviral packaging plasmid to generate human lentiviral particles LV-CXCR7-shRNA and human lentiviral particles HEK293T were transfected into HepG2 cells Cells were used to detect the silencing effects of CXCR7 messenger RNA (mRNA) and protein by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. Results: Polymerase chain reaction (PCR) identification and sequencing results showed that four CXCR7 shRNA lentiviral expression vectors were successfully constructed and pGCSIL-RFP-CXCR7-shRNA2 with significant knockdown effect was screened. The recombinant plasmid containing CXCR7 -shRNA2 lentivirus particles LV-CXCR7-shRNA2 (virus titer of 3 × 109TU / ml) infected HepG2 cells, CXCR7 mRNA and protein expression levels were down-regulated. Conclusion: The shRNA lentiviral vector targeting CXCR7 gene was successfully constructed and could effectively inhibit the expression of CXCR7 mRNA and protein in HepG2 cells.