人长链非编码RNA基因H19克隆、表达载体构建及对MCF-7细胞增殖的影响

Constructing an expression vector for human lncRNA H19 and the effect of its overexpression on MCF-7 cell proliferation

目的克隆人长链非编码RNA H19基因,构建表达载体,研究H19表达对MCF-7增殖的影响作用。方法制备乳腺癌MCF-7细胞总RNA,RT-PCR扩增长链非编码RNA H19全长序列,分子克隆至pc DNA3.1(-)真核表达载体;分别转染HEK-293T和COS 7细胞并行Real-time q PCR验证载体构建是否成功;转染H19表达载体入MCF-7细胞株,转染0、24和48 h后行MTS检测H19表达,同时设计siRNA小分子片段干扰H19的表达,观察对MCF-7细胞增殖活性的影响。结果成功克隆和构建了h H19-pc DNA3.1(-)表达载体;转染入MCF-7细胞48h后,H19过表达,MTS检测H19表达载体组吸光度明显高于空载体组和空白对照组;而转染H19 siRNA小分子片段后能干扰其表达,同时抑制细胞增殖。结论 H19过表达能够促进乳腺癌MCF-7细胞增殖。

Aims To construct an expression vector of human lncRNA H 1 9,and to determine the effect ofH19 overexpression on MCF-7 cell proliferation.Methods Total RNA was extracted from MCF-7cells,and the full-length of H19 lncRNA was amplified by RT-PCR and subcloned into pc DNA3. 1（-） expression vector. The constructed H19 expression vector was transfected into HEK-293 T and COS-7 cells and the H19 lncRNA expression was evaluated by real-time PCR. Following the transfection of H19 expression vector into MCF-7 cells for 0,24 h and 48 h and H19 siRNA interference fragment into MCF-7 cells for 24 h,MCF-7 cell proliferation was determined by MTS assay. Results A h H19-pc DNA3. 1（-） expression vector was successfully constructed. At Forty-eighthours after the transfection with H19 expression vector in to MCF-7 cells,cell proliferation was significantly increased in the transfected group compared to those without transfection and to those transfected with a negative control vector,while twenty-four hours after the transfection with H19 siRNA interference fragment into MCF-7 cells,cell proliferation was significantly decreased in the transfected group compared to those transfected with a negative control vector. Conclusion Ectopic overexpression of H19 lncRNA can promote breast cancer MCF-7 cell proliferation.