PCBP1基因RNAi序列的鉴定及其干扰效应的初步研究

Verification of an RNAi sequence for PCBP1 gene silencing

以能与PCBP1mRNA特异结合的核苷酸片段(21个碱基对)构建shRNA真核表达载体并稳定转染Hela细胞,通过半定量RT-PCR、免疫印迹和间接免疫荧光实验证实转染细胞中PCBP1的mRNA水平和蛋白质表达水平均被显著抑制.接着,本研究通过测定稳定转染Hela细胞的生长曲线、血清依赖性生长曲线和软琼脂集落形成率,证明PCBP1的沉默能够显著抑制细胞的生长增殖能力以及克隆形成潜能,揭示PCBP1可能直接或间接参与细胞周期的调控.本研究成功鉴定了沉默PCBP1基因表达的有效干扰片段,为下一步利用该有效干扰片段进行体内研究奠定了基础.同时,本研究的结果将有助于深入认识PCBP1蛋白在细胞周期调控和细胞癌变中的作用机制.

A short hairpin RNA（shRNA）expression vector carrying a 21-nucleotide fragment targeting PCBP1 mRNA was constructed and stably transfected into Hela Cells.Semiquantitative RT-PCR,western blotting,and immunofluorescence assays were conducted to confirm that PCBP1 mRNA content and protein level were effectively reduced.Cell growth index under normal culture condition or after serum deprivation,and cloning efficiency in soft agar were detected for PCBP1-deleted cells.These data indicated that PCBP1 gene silencing inhibited significantly cell growth and proliferation and reduced potential to form tumors.The present study successfully identified an effective RNAi sequence for PCBP1 gene silencing,providing a basis for further study of PCBP1 functions in vivo.These data will facilitate understanding of action mechanism of PCBP1 in cell cycle regulation and tumor formation.