Cyclin D1反义寡核苷酸对Tca8113/CDDP细胞基因转录表达的影响

The influence on expression of cyclin D1 by anti-sense oligonucleotide expression vector

目的:构建细胞周期蛋白D1(cyclin D1)的反义寡核苷酸真核表达载体,并检测其转染Tca8113/CDDP细胞系后的表达情况。方法:以Tca8113/CDDP细胞总RNA为模板,应用RT-PCR法扩增cyclin D1全长,通过克隆至pUCm-T载体中,测序完全正确;经IPTG诱导,可正确表达蛋白。上下游分别利用H ind III和EcoRI的酶切位点插入真核表达载体pcDNA3.1,测序鉴定;并构建表达cyclin D1反义寡核苷酸的2个重组载体,通过Lipofectam ine将其导入Tca8113/CDDP细胞系中,G418筛选获得阳性稳定表达细胞系;通过RT-PCR、免疫组化检测cyclin D1基因转录和蛋白表达。结果:成功构建pcDNA3.1-cyclin D1真核表达载体,分别命名为pcDNA3.1空载体(Co)、cyclin D1m RNA5’端为靶区的反义载体(C5’)、cyclin D1m RNA3’端为靶区的反义载体(C3’)、cyclin D1正义载体(CD1)。3’端反义寡核苷酸转染细胞后,cyclin D1m RNA表达水平降低58.8%,m RNA5’端反义寡核苷酸组无明显抑制。免疫组化结果显示,转导正义寡核苷酸组cyclin D1阳性表达为最高,转导m RNA3’端、m RNA5’端反义寡核苷酸组为弱阳性表达。结论:cyclin D1m RNA3’端反义寡核苷酸能明显抑制cyclin D1在Tca8113/CDDP中的表达,为进一步研究逆转Tca8113/CDDP耐药性提供了实验工具。

PURPOSE: To construct eurokaratic expression vectors of pcDNA3.1-cyclin D1 and pcDNA 3.1-Anti-cyclin D1, and evaluate their influence on the expression of cyclin D1 in stable transfected Tca8113/CDDP cells. METHODS: The full length of cyclin D1 and its antisense fragments were acquired from Tca8113/CDDP cells by RT-PCR. These cDNA fragments were inserted into pUCm-T vector and the full length sequenced. Induced by IPTG, the vector could express cyclin D1 protein in vitro. After double digested by Hind III and EcoRI at the two ends of these cDNA fragments, these cDNA fragments were inserted into pcDNA3.1 vector and transfected into Tca8113/CDDP cells with the help of Lipfectamin 2000. Stable cell lines were acquired after continually selected with G418 for 4 weeks. The expression of cyclin D1 was detected with RT-PCR and immunohistochemical staining. RESULTS: pcDNA3.1-cyclin D1, pcDNA3.1-Anti-5' and pcDNA3.1-Anti-3' vectors were constructed and named as pcDNA3.1(CO), pcDNA3.1-Anti-cyclin D1 5'(C5'), pcDNA3.1-Anti-cyclin D1 3'(C3') and pcDNA3.1-cyclin D1(CD1), respectively. The expression of cyclin D1 in Tca8113/CDDP cells transfected with C3' was significantly inhibited and the inhibition rate was 58.8%. While C5' could not inhibit the expression of cyclin D1. The protein level of cyclin D1 was strongly positive in Tca8113/CDDP-C0, Tca8113/CDDP-CD1 and Tca8113/CDDP-C5' cells. It was obviously repressed in Tca8113/CDDP-3' cells. CONCLUSION: We successfully constructed pDNA3.1-cyclin D1 vector and pcDNA3.1-Anti-cyclin D1 vectors. Detected with RT-PCR and immunohistochemical staining, the mRNA level and protein expression of cyclin D1 were significantly inhibited in C3' transfected Tca8113/CDDP cells,which may provide a good experimental tool for further studying in reversing the multidrug resistance of Tca8113/CDDP cells.