长非编码RNA基因Z38对人乳腺癌细胞BT549生物学特性影响.docVIP

长非编码RNA基因Z38对人乳腺癌细胞BT549生物学特性影响 　　摘 要 为探讨稳定干扰长非编码RNA基因Z38对乳腺癌细胞BT549的影响，向BT549细胞转染干扰Z38表达的慢病毒，经嘌呤霉素筛选获得稳定干扰的细胞，采用RT-PCR检测Z38的表达水平、MTT法检测细胞增殖及耐药性、克隆法检测细胞集落形成能力、碘化丙啶染色方法分析细胞周期、流式细胞术分析肿瘤干细胞标志物CD44和CD24的表达.结果表明，特异性的慢病毒能显著降低Z38表达水平，使细胞增殖能力、抗化疗药物能力及细胞集落形成能力显著降低，导致G2/M期细胞数目显著减少，细胞周期受阻，并且显著降低了CD44+/CD24-细胞群百分比而导致细胞的肿瘤干细胞特性降低. 　　关键词 Z38；人乳腺癌；细胞增殖；CD44+/CD24- 　　中图分类号 R737.9；R96文献标识码 A文章编号 1000-2537（2017）04-0034-06 　　Abstract The present study aimed at exploring the effect of stably interfering the long non-coding RNA gene Z38 on the biological behavior of human breast cancer cell line BT549. The lentiviral vector harboring RNAi sequence with the Z38 gene targeted was transfected into BT549 cells， and then screened by puromycin. The expression level of Z38 was determined by RT-PCR. Its cell viability and drug resistance were measured by MTT assay. The colony formation ability of cells was ascertained by cloning method. The cell cycle distribution was analyzed by propidium iodide staining. And the expression of CD44 and CD24 was examined by flow cytometry. Our results indicate that the expression level of Z38 was seriously inhibited by the specific lentiviral vector， which in turn effectively arrested the cell proliferation， drug resistance， colony formation ability， and cell number in G2/M stage. Moreover， our study shows that compared to the control group， the percentage of CD44+/CD24- cell was significantly decreased in the interference group， suggesting that the properties of cancer stem-like cells were markedly impacted. 　　Key words Z38； human breast cancer； cell proliferation； CD44+/CD24- 　　近年来，在原核生物、真核生物以及古细菌中发现许多ncRNA （non-coding RNA），它们在多个水平上调节基因的表达[1].人类基因组中有80%左右的基因能够被转录，而其中只有2%的序列编码蛋白质，超过90%是ncRNA[2-4]，说明ncRNA是基因组转录的主要产物.lncRNA（long non-coding RNA）是转录本超过200 bp，不具备开放阅读框的ncRNA序列.lncRNA的启动子相对更加保守，提示这些可能在生物体中发挥着重要的作用.lncRNA的异常表达与多种疾病相关，尤其是对肿瘤的发生、发展有着重要影响[5]. 　　RNA干扰（RNA interference，RNAi）是一种转录水平上的基因隔断技术，具有高效性、特异性、选择性、快速性和可操作性等优点，在基因治疗中应用广泛，是现今沉默某些目标致癌基因的常用手段[6].