稳定过表达miR-27b的结直肠癌细胞模型的建立及意义

Establishment of a colorectal cancer cell line with stable over-expression miR-27b mediated by lentivirus and its roles on cells behavior

目的利用慢病毒表达载体建立稳定过表达miR-27b的结直肠癌细胞系,探讨miR-27b对结直肠癌细胞生物学特性的影响及作用。方法构建miR-27b过表达重组慢病毒载体,经包装病毒后感染结直肠癌细胞SW480,利用流式细胞仪分选获得绿色荧光蛋白(green fluorescent protein,GFP)阳性细胞并荧光定量PCR检测miR-27b表达情况,建立稳定过表达miR-27b的结直肠癌细胞系;通过MTT法和Transwel检测过表达miR-27b后细胞体外增殖、侵袭能力的变化。结果稳定过表达miR-27b的结直肠癌细胞中miR-27b的表达水平是空载体对照细胞中miR-27b的5.02倍;与对照细胞相比,miR-27b过表达能够促进结直肠癌细胞的增殖和侵袭能力(P<0.05)。结论成功建立了稳定过表达miR-27b的结直肠癌细胞模型,为深入研究miR-27b在结直肠癌发生发展过程中的作用、调控靶基因和相应的作用机制奠定了基础。

Objective To establish a colorectal cancer（CRC） cell line with stable over-expression of has-miR-27 andexplore the roles of miR-27 b on CRC cells. Methods The DNA fragment of hsa-pre-miR-27 b that was amplified fromhuman genome by polymerase chain reaction（PCR） was digested and cloned into the p LVTHM vector. The recombinantplasmid p LVTHM – miR- 27 b was comfirmed by restriction endonuclease analysis and DNA sequencing. 293 T cells werecotransfected with p LVTHM –miR-27 b, ps PAX2 and Pmd2.G. After up-regulated miR-27 b expression, cell growth ability invitro was determined by dimethyl thiazolyl diphenyl tetrazolium（MTT） assay and Transwell analysis were performed. Results The confirmed recombinant plasmid p LVTHM/miR- 27 b by sequencing was transfected into 239 T cells to product thelentivirus p LVTHM/miR-27 b. Human CRC cells, SW480, were infected with the lentivirus p LVTHM/miR-27 b to product acell line SW480/miR- 27 b with stable over- expression of miR- 27 b. Ectopic over- expression of miR- 27 b promoted cellproliferation and invasiveness of SW480 cells. Conclusions The recombinant lentivirus vector p LVTHM/miR-27 b results inthe stable over-expression of miR-27 b in infected SW480 cells, which provides a useful cell model for further studies of theeffect of miR-27 b in CRC.