慢病毒介导的shRNA敲低FHL1的表达促进HeLa和HepG2细胞生长  

FHL1 knockdown mediated by lentiviral shRNA promotes the growth of He La and HepG2 cells

作　　者：范忠义[1] 徐小洁[2] 张晓梅[3] 王涛[2] 韩白玉 梁迎春[2] 叶棋浓[2] 焦顺昌[1] 

机构地区：[1]解放军总医院肿瘤内科,北京100853 [2]军事医学科学院生物工程研究所,北京100850 [3]解放军总医院消化内科,北京100853 [4]北京军区第264医院,山西太原030001

出　　处：《细胞与分子免疫学杂志》2015年第7期879-883,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：卫生部医药卫生科技发展基金(W2011BX055);国家自然科学基金(31100604;81101065;81472589);北京市科技新星计划(Z141102001814055);北京市自然科学基金(7132155);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的通过构建靶向四个半LIM结构域蛋白1（FHLl）基因的慢病毒介导的短发夹RNA（shRNA），观察FHLl表达降低对HeLa细胞和HepG2细胞生长的影响。方法构建FHLl基因的shRNA干扰病毒载体，将重组质粒转染人胚肾293T细胞，实时定量PCR（qRT-PCR）和Western blot法检测转染pLenti-H1 FHLlsh RNA对FHLl表达的抑制效果。慢病毒感染宫颈癌HeLa细胞和HepG2肝癌细胞，经2-3周嘌呤霉素筛选后得到FHLl稳定低表达的细胞，利用生长曲线实验和克隆形成实验检测FHLl基因表达降低后对细胞生长的影响，利用软琼脂实验检测对肿瘤细胞非锚定依赖性生长的影响。结果qRT-PCR和Western blot结果显示，构建的shRNA病毒载体能够明显抑制FHLl的表达；生长曲线实验和克隆形成实验显示，慢病毒介导的FHLlshRNA能够明显促进肿瘤细胞的生长；软琼脂实验结果表明，FHLl低表达能够促进肿瘤细胞的非锚定依赖性生长。结论构建的pLenti-H1 FHLl shRNA慢病毒载体可有效地抑制FHLl表达，明显促进HeLa细胞和HepG2细胞的增殖。Objective To investigate the effect of four-and-a-half LIM domain 1 （FHL1） knockdown by lentiviral-mediated shRNA on the growth of HeLa and HepG2 cells. Metheds pLenti-H1 FHL1 shRNA was cloned, and then transfered into HEK293T cells. The inhibitory effect of pLenti-H1 FHL1 shRNA on FHL1 gene was detected by Westem blotting and real-time quantitative PCR （qRT-PCR）. Lentivirus particles were packaged, added to HeLa and HepG2 cells, followed by puromycin treatment for 2 - 3 weeks to screen stable clones. The knockdown effect on FHLI expression in these cells was checked by Western blotting and qRT-PCR. Cell growth and colony formation analysis were performed to investigate the effect of FHL1 down-regulation on tumor cell growth. Soft agar analysis was used to analyze its effect on tumor cell growth. Results Western blotting and qRT-PCR revealed that the pLenti-H1 FHL1 shRNA apparently inhibited the expression of FHLI gene. Cell growth and colony formation assay showed that the lentiviral-mediated shRNA for FHL] gene significantly accelerated the tumor cell growth in HepG2 and HeLa cells. Soft agar analysis demonstrated that FHL1 shRNA increased the growth of tumor cells. Conclusion pLenti-Hl FHL1 shRNA could significantly accelerate tumor cel nhibitincl the expression of FHL1

关 键 词：FHL1基因 小发夹RNA 细胞生长 

分 类 号：Q786[生物学—分子生物学] R735.7[医药卫生—肿瘤]