CXCR7-shRNA靶向抑制结肠癌SW620细胞的体外实验研究  

In vitro experimental study of CXCR7-shRNA's targeted inhibition on human colon cancer cell SW620

作　　者：王鑫[1] 陈玲[1] 孙飞[1] 陆航[2] 

机构地区：[1]辽宁医学院附属第一医院急诊科,辽宁锦州121001 [2]辽宁医学院附属第一医院胃肠外科,辽宁锦州121001

出　　处：《西安交通大学学报（医学版）》2014年第4期460-464,480,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：辽宁省自然科学基金项目资助(No.201102124)~~

摘　　要：目的探讨慢病毒介导的CXCR7-shRNA转染人结肠癌细胞株SW620后对CXCR7蛋白表达的影响。方法①设计并合成CXCR7的3对shRNA序列及1对阴性对照序列,与pSilencerTM4.1系统合成构建重组慢病毒载体,转染HEK293T细胞包装病毒并检测滴度;②将3种重组慢病毒载体及阴性对照分别感染人结肠癌细胞SW620,RT-PCR检测CXCR7mRNA的表达情况,测定沉默效率,筛选沉默效率最高的一组CXCR7-shRNA作为后续实验的表达载体;③MTT法检测转染CXCR7-shRNA对SW620细胞生长增殖的影响;④细胞划痕实验检测转染CXCR7-shRNA对SW620细胞侵袭迁移能力的影响;⑤Western blot检测转染CXCR7-shRNA对SW620细胞蛋白表达情况。结果①测序证实3对慢病毒载体及1对阴性对照载体均包装成功,滴度分别3.16×108pfu/mL、4.27×108 pfu/mL、3.93×108pfu/mL和2.95×108pfu/mL;②3组慢病毒载体转染SW620细胞后,CXCR7mRNA的表达量均较阴性对照组明显降低(P<0.05),其中CXCR7-shRNA-1对CXCR7的抑制率明显高于其他两组(P<0.05);③CXCR7-shRNA-1转染SW620后,肿瘤细胞的增殖程度显著减少,与空白组相比有统计学差异(P<0.05);④SW620细胞在划痕24h后,空白对照组和实验组的细胞迁移指数(MI)分别为(49.92±6.41)%和(29.13±5.38)%,差异有统计学意义(P<0.05),划痕48h后,对照组与实验组的MI分别为(96.52±7.44)%和(72.03±8.29)%,差异有统计学意义(P<0.05);⑤CXCR7-shRNA-1转染SW620细胞后,与空病毒载体组、空白组相比,CXCR7蛋白表达量明显降低,具有统计学意义(P<0.05)。结论 CXCR7被沉默后可有效抑制SW620肿瘤细胞的增殖与迁移,这为后续研究以CXCR7/CXCL12生物学轴为靶点的肿瘤基因治疗打下良好基础。Objective To investigate the changes of CXCR7 protein expression after CXCR7-shRNA transfected into human colon cancer cell SW620 which is lentivirus-mediated.Methods ① We designed and synthesized three shRNA sequences and one negative control sequence of CXCR7.We synthesized and constructed recombinant lentiviral vector by pSilencerTM 4.1 system and CXCR7.We transfected the vector into HEK293T cell for packaging viruses and assaying titer.② We transfected the three different recombinant lentiviral vectors and one negative control vector into human colon cancer cell SW620.Then we detected the expression and silence efficiency of CXCR7 mRNA by RT-PCR.The group expressing the best silence efficiency of CXCR7-shRNA was selected as expression vector for the next experiment.③ We detected the change of SW620 cell proliferation after CXCR7-shRNA transfection by MTT.④ We detected the change of SW620 cell invasion and migration after CXCR7-shRNA transfection by cell scratching experiment.⑤ We detected the SW620 protein expression after CXCR7-shRNA transfection by Western blot.Results ① Sequencing confirmed that packaging the three different recombinant lentiviral vectors and one negative control vector was successful,with the titer being 3.16 × 108pfu/mL,4.27 ×108pfu/mL,3.93 × 108pfu/mL and 2.95 × 108pfu/mL,respectively.② Expression of CXCR7 mRNA was lower after transfection in three positive groups than in negative control group （P＜0.05）.The inhibition rate of CXCR7-shRNA-1 on CXCR7 was higher than that in the other two groups （P＜0.05）.③ The tumor cell proliferation was reduced significantly after CXCR7-shRNA-1 transfection into SW620 cell compared with control group （P＜0.05）.④ The migration index in control group and positive group 24 h after cell scratching experiment was （49.92 ±6.41） ％ and （29.13 ± 5.38） ％,respectively,with a significant difference （P＜0.05）.The migration index in the two groups 48 h after cell scratching experiment was （96.52 ± 7.44） �

关 键 词：结直肠癌 CXCR7 CXCL12 重组慢病毒载体 RNAi 

分 类 号：R735.3[医药卫生—肿瘤]