RNAi沉默galectin-1表达对黏附LST-R1细胞生长的影响

Influence on adhesive LST-R1 cell growth mediated by inhibition galectin-1 expression using RNAi

目的采用RNA干扰(RNA interference,RNAi)技术,构建galectin-1基因特异siRNAs真核干扰载体,检测LST-R1细胞galectin-1表达变化,观察黏附LST-R1细胞增殖和凋亡改变。方法设计2对galectin-1基因特异性siRNAs,确定相应的shRNAs,通过基因克隆技术将其插入真核表达质粒pSUPER.puro,构建galectin-1 siRNA表达载体,与pcDNA6/TR共转染LST-R1细胞,嘌呤霉素(0.8μg/mL)进行阳性克隆筛选。四环素(4μg/mL)诱导48 h后,RT-PCR检测galectin-1 mRNA水平改变,免疫印迹和细胞免疫化学检测蛋白质水平的改变,观察黏附LST-R1细胞增殖和凋亡改变。结果成功构建发夹样galectin-1 siRNA真核表达载体p-shRNA1和p-shRNA2,建立相应稳定的p-shRNA1-LST、p-shRNA2-LST细胞系。RNAi下调galectin-1表达后,细胞生长曲线表明第3至7天黏附p-shRNA1-LST细胞增殖较p-LST和LST-R1细胞增加,而低浓度血清诱导的细胞凋亡则减少,依次为(3.77±0.34)%、(7.92±0.60)%和(7.67±0.75)%(P<0.001)。结论Galectin-1的下调表达可抑制黏附LST-R1细胞凋亡,同时促进黏附LST-R1细胞增殖,提示galectin-1可能参与了LST病变浅表性扩散特性的形成。

Objective To observe the changes of adhesive LST-R1 cell proliferation and apoptosis after knockdowning galectin-1 gene expression using RNA interfering （RNAi） methods. Methods Two pairs of small interfering RNAs （siRNA） targeting to galectin-1 were designed and the corresponding short hairpin interfering RNAs （shRNA） were inserted into pSUPER, puro to set up the interfering system which co-transfected LST-R1 cell with pcDNA6/TR using Lipo-fectamine^RM2000, and the positive cell clones were selected and cultured with 0.8 μg/mL puromycin. The changes of galectin-1 mRNA and protein were determinated by RT-PCR and Western blotting, respectively. Proliferation and apoptosis of adhesive LST-R1 cell were observed. Results Two interfering vectors targeting to galectin-I, p-shRNA1 and pshRNA2 were effective to knockdown galectin-1 expression and two corresponding transfected cell lines p-shRNA1-LST and p-shRNA2-LST were established. Cell growth curve showed an increased p-shRNA1-LST cell proliferation from day 3 to day 7 compared with p-LST cell and LST-R1 cell. Flow cytometry assay showed cell apoptosis induced by low concentration serum rates of adhsive p-shRNA1-LST cell was decreased compared with adhesive p-LST and LST-RI cells. They were （3.77 ±0.34）%, （7.92±0.60）% and （7.67 ±0.75）%, respectively （P〈0.001）.Conclusion The downregulating of galectin-1 can accelerate adhesive LST-R1 cell proliferation and inhibit cell apoptosis, which indicates that galectin-1 possibly takes part roles in the developing of the superficial spreading specificity of laterally spreading tumor.