维甲酸受体β基因RNAi表达质粒的构建及其对A549细胞增殖和分化的影响

Construction of Retinoic Acid Receptor β Gene Small Interfering RNA Expression Vector and its Regulation on Cell Proliferation and Differentiation in A549 Cells

目的:利用RNA干扰(RNAi)技术,构建针对维甲酸受体(RAR)β基因的小干扰RNA(siRNA)表达质粒,诱导RARβ基因沉默,并观察其对肺癌细胞A549株的细胞周期和增殖的影响。方法:依据设计siRNA的原则,针对人RARβ的mRNA序列,设计并合成编码siRNA的2条寡核苷酸序列,经退火成互补双链,再克隆到pSUPER-NEO-GFP真核表达载体中构建重组体pSUPER-RARβ,转染至A549细胞中,以空质粒和RARβ高表达质粒转染为对照,用Western印迹检测RARβ基因的表达,并采用MTT试验检测转染后细胞株的增殖和细胞分化情况。结果:表达人类RARβ基因的siRNA重组表达质粒构建成功;MTT试验结果表明,转染的A549-RARβ-si细胞增殖能力降低。结论:采用RNAi技术特异阻断RARβ基因表达,通过转染A549细胞,可使其细胞形态发生变化,并抑制其细胞生长。

Objective： To construct the small interfering RNA（siRNA） expression vector of retinoic acid receptor（RAR） β and to observe its influence on cell cycle and proliferation of A549 cells. Methods： According to the encoding sequence of mRNA of RARβ, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pSUPER-NEO-GFP siRNA expression vector. The recombinant plasmid pSUPER- RARβ was transfected into A549 cells, and RARβ expression was assayed by Western blot. The changes of cell cycle and cell proliferation were observed by MTT assay. Results： The siRNA expression vector of RARβ was successfully constructed. MTT assay indicated that A549-RARβ-si cells got a slower growth compared with normal A549 cells. Conclusion： These results suggest that RARβ gene have an effect on growth in A549 lung carcinoma cells. This study will facilitate further study of biological function and molecular mechanism of RARβ gene.