pEGFP-C1-miR-335重组质粒的构建及其在乳腺癌细胞中的表达

Construction of pEGFP-C1-miR-335 Recombinant Plasmid and Observation of its Expression in Breast Cancer Cells

目的构建携带融合型microRNA(微小RNA)基因miR-335的荧光真核表达质粒pEGFP-C1-miR-335,并观察其在乳腺癌细胞MDA-MB-231中的表达。为研究小RNA基因表达调控机制建立实验基础。方法应用基因重组技术,从人类基因组DNA中扩增miR-335的前体序列,经PCR引物加入酶切位点,酶切后插入荧光真核表达载体pEGFP-C1,构建miR9真核表达载体pEGFP-C1-miR-335,然后进行酶切和测序鉴定重组质粒的正确性。应用脂质体介导的转染技术将该质粒导入MDA-MB-231细胞,24 h后观察荧光蛋白表达情况,用Northern blot方法检测miR-335表达。结果酶切和测序鉴定证实插入miR-335前体片段序列正确。细胞转染24 h后,荧光显微镜下观察到GFP表达,60%左右转染细胞发出荧光。Northern blot检测到miR-335表达。结论成功构建pEGFP-C1-miR-335荧光真核表达载体,并可在乳腺癌细胞中有效表达。

Objective To construct an Enhanced green fluorescent protein （ EGFP ） - labled euk - aryotic expression plasmid of mieroRNA gene with miR - 335 and to detect its expression in breast cancer cell line MDA - MB - 231. Methods With the technology of gene re - arrangement, miR - 335 precursor sequence was amplified from the human genomic DNA, and was introduced into the restriction sites of pEGFP -C1. Then the correctness of the pEGFP- C1 -miR - 335 was evaluated by using restriction enzyme and sequencing analysis. The plasmid was transfected into the cell line MDA - MB -231 with lipefectin, the transient expression of GFP was observed under fluorescence microscope after 24 hours and detected by Northern blot. Results Correct construction of pEGFP - C1 - miR - 335 was identified by methods of restriction enzyme analysis and nueleotide sequence determination. A total of about 60% transfected cells emitted out green fluorescence under fluorescent microscope after 24 h of transfection. MiR - 335 gene expressed by the transfected cells were testified by Northern blot. Conclusions The recombinant eukaryotie expression vectors have been constructed successfully and effectively expressed in breast cancer cells, which may provide an experimental basis for research of microRNA.