Id1 miRNA表达载体构建及转染乳腺癌细胞.docVIP

Id1 miRNA表达载体构建及转染乳腺癌细胞【摘要】 目的 构建正义、反义Id1真核表达载体，并成功转染乳腺癌MDA-MB-231细胞系，筛选稳定表达细胞株。方法 设计针对Id1基因的mRNA效的干扰序列。将干扰序列插入pcDNA6．2-GW/EmGFP-miR 表达载体，构建Id1 miRNA真核表达载体，脂质体转染法转染乳腺癌MDA-MB-231细胞系，杀稻瘟霉素筛选稳定表达的细胞株。结果 Id1 miRNA真核表达载体的构建后，经酶切及测序鉴定正确后。转染乳腺癌MDA-MB231细胞系。经过2周杀稻瘟霉素筛选，获得稳定表达的细胞系。经RT-PCR鉴定，转染Id1 miRNA真核表达载体的细胞系Id1表达水平低于对照组。未转染组与阴性对照转染组无明显差异。 【关键词】Id1； 真核表达载体； 乳腺癌 Construct the eukaryotic expression vector of Id1 miRNA and transfect into breast caner cell line XU Zi-zhi,WANG Chuan.Institute of Tumor Disease, Union Hospital, Fujian Medical University, Fuzhou 350001,China 【Abstract】 Objective To construct the miRNA eukaryotic expression vector of Id1 and successfully transfect the MDA-MB-231 cell lines of breast cancer and finally select the stabilized expressed cells lines. Methods To design miRNA array of Id1 gene. to insert the target gene into pcDNA6．2-GW/EmGFP-miR to construct the Id1 miRNA eukaryotic expression vector. The liposome infection protocol transfect the MDA-Mb231 cell lines of breast cancer. Use Blasticidin to select stabilized expressed cell lines. Results The method of RT-PCR helps to acquire the whole Id1 array for about 212bp long. After identifying by enzyme and measuring array, it transfect the MDA-MB231 cell lines of breast cancer. We acquire the stabilized expressed cell lines through 2W Blasticidin selection. As a result of RT-PCR identification, the level of Id1 of Id1 miRNA transfection cell lines is lowerer than that of the other two groups. While the level of Id1 is no difference between the negative control transfection cell lines and negative control cell lines group. 【Key words】 Id1；Eukaryotic expression vector；Breast cancer Id1(Inhibition of DNA binding/differentiation)蛋白是一类具有螺旋-环-螺旋(helix-loop-helix)结构的分子，是一组缺乏碱性DNA连接结构域的HLH因子。Id1可与一类具有bHLH结构的转录因子结合形成无功能的异源二聚体，从而抑制下游一些包含有E-box(CANNTG)或E样序列的靶基因的转录，发挥其显性负性调节功能。干扰了DNA与bHLH的结合，抑制细胞分化，促进细胞增殖, 因此Id蛋白被认为是分化和DNA结合的抑制剂。在组织细胞的发育分化及增殖中发挥重要作用。 研究发现Id家族的蛋白在许多肿瘤中的表达是上调的［1-4］，研究证实其参与肿瘤的