摘要
目的:构建靶向PRL-3基因的miRNA495和miRNA551a真核表达载体,观察其转染胃癌SGC7901细胞后对胃癌细胞PRL-3基因表达的影响.方法:设计并合成两条针对PRL-3基因的特异性miRNA495和miRNA551a干扰序列,分别与pcDNA6.2-GW/Em GFP-miR载体连接,转化大肠杆菌,纯化并鉴定后利用LipofectamineTM2000转染胃癌SGC7901细胞,实时定量PCR及Western blot技术鉴定重组体对PRL-3基因表达的干扰效果.结果:针对PRL-3基因的miRNA495和miRNA551a干扰质粒构建成功,胃癌SGC7901细胞分别转染两种质粒后miRNA495及miRNA551a的表达明显升高,并且明显抑制了PRL-3基因mRNA及蛋白的表达.结论:靶向PRL-3基因的miRNA495和miRNA551a真核表达载体构建成功,其可有效提高胃癌SGC7901细胞miRNA495和miRNA551a的表达,并抑制PRL-3mRNA及PRL-3蛋白的表达.
AIM： To construct miRNA495 and miRNA551a expression vectors targeting the PRL-3 gene, and to observe the effect of vector transfection on the expression of PRL-3 in gastric cancer SGC7901 cells. METHODS： Specific pre-miRNA495 and pre- miRNA551a sequences targeting the PRL-3 gene were designed, synthesized, and ligated with the pcDNA6.2-GW/EmGFP-miR vector. After se-quencing to confirm correct insertion, the recom- binant vectors were transfected into SGC7901 cells with LipofectamineTM 2000. The expression of PRL-3 in transfected cells was detected by real-time PCR and Western blot. RESULTS： The PRL-3 pre-miRNA495 and pre- miRNA551a recombinant plasmids were suc- cessfully constructed, and they could effectively promote the expression of miRNA495 and miR- NA551a and inhibit PRL-3 mRNA and protein expression in SGC7901 cells. CONCLUSION： The miRNA495 and miRN- A551a expression vectors for silencing the PRL-3 gene have been constructed successfully, which could effectively promote the expression of rniRNA495 and miRNA551a and inhibit PRL-3 mRNA and protein expression in SGC7901 cells.
出处
《世界华人消化杂志》
CAS
北大核心
2012年第34期3361-3365,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30901429~~
