敲减人HPIP基因表达抑制细胞生长增殖  

Knockdown of Human HPIP Gene Inhibits Cell Growth and Proliferation

作　　者：徐小洁[1] 王凌雪[1] 范忠义[1] 丁丽华[1] 张浩[1] 杨智洪[1] 李杰之[1] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《中国生物化学与分子生物学报》2011年第2期190-196,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：全军医药卫生科研基金(No.06J021);国家重大科学研究计划(No.2007CB914603);国家自然科学基金(No.30800205)~~

摘　　要：为了探讨人造血相关的PBX相互作用蛋白质基因(HPIP)在肿瘤发生发展中的生物学作用,构建了HPIP小干扰RNA(siRNA)的真核表达载体,验证其敲减效果并观察其对细胞生长增殖的影响.根据人HPIP的cDNA序列,设计了含有小发卡结构的寡核苷酸序列,将其克隆到siRNA表达载体上;将重组质粒转染人胚肾293T细胞,通过实时定量RT-PCR及Western印迹分析检测HPIP基因的表达水平;结晶紫实验及流式细胞技术检测敲减HPIP基因表达对细胞生长和增殖的影响,软琼脂实验检测对肿瘤细胞非锚定依赖性生长的影响.结果显示,构建的siRNA能够有效抑制HPIP基因的表达;结晶紫实验与细胞周期分析实验显示,siRNA介导的HPIP表达沉默导致细胞生长增殖的显著抑制,软琼脂实验结果表明,稳定转染HPIP siRNA能够抑制肿瘤细胞的锚定非依赖性生长.上述结果初步表明,HPIP siRNA能明显抑制肿瘤细胞的生长与增殖,可能是一个潜在的肿瘤治疗新靶点.To study the biological function of hematopoietic PBX-interacting protein（HPIP） gene in the development of tumor,the eukaryotic expression vector of HPIP small interfering RNA（siRNA） was constructed and investigated its effect on cell growth and proliferation.HPIP siRNA was designed and constructed based on human HPIP cDNA sequence using pSilencer 2.1-U6 neo vector.The expression of HPIP was examined by Western blot and Real-time RT-PCR analysis.The effect of HPIP siRNA on cell growth and proliferation were analyzed by violet assay and FACS.HPIP siRNA was successfully cloned to the target vector,and was able to knockdown the expression level of HPIP gene.Violet assay and FACS analysis showed that down-regulation of HPIP obviously suppress cell growth and proliferation.Soft agar assay demonstrated HPIP siRNA decrease the anchorage-independent tumor cell growth.Taken together,the eukaryotic expression vector of HPIP siRNA obviously inhibits the expression of HPIP and suppresses cell growth and proliferation,which might serve as a therapeutic target in tumor treatment.

关 键 词：造血相关的PBX相互作用蛋白质基因(HPIP) 小干扰RNA 细胞生长 细胞增殖 

分 类 号：Q71[生物学—分子生物学] R73[医药卫生—肿瘤]