肝癌细胞HepG2中lncRNA BDNF AS对BDNF基因表达调控作用的研究

The Regulatory Effects of lncRNA BDNF AS on the Expression of BDNF in Hepatocellular Carcinoma Cells

该文旨在探讨肝癌细胞HepG2中lncRNA BDNF AS(antisense brain derived neurotrophic factor)对BDNF基因表达的调控作用。将BDNF AS质粒、BDNF AS与BDNF基因完全互补序列缺失突变质粒瞬时转染肝癌细胞HepG2后采用q PCR方法检测肝癌细胞中lncRNA BDNF AS和BDNF mRNA水平;采用Western blot方法检测BDNF蛋白质水平;采用ELISA方法检测细胞培养上清中BDNF蛋白质水平;采用免疫荧光染色检测BDNF蛋白质水平;采用FISH方法检测lncRNA BDNF AS和BDNF mRNA的水平和亚细胞定位。研究结果显示,转染BDNF AS质粒的细胞中,lncRNA BDNF AS水平高于对照组(P<0.001),BDNF mRNA和蛋白质水平均低于对照组(P<0.001)。转染突变体质粒的细胞中,lncRNA BDNF AS水平高于对照组(P<0.05),低于BDNF AS组(P<0.001),BDNF mRNA和蛋白质水平低于对照组(P<0.05,P<0.001),高于BDNF AS组(P<0.05,P<0.001)。免疫荧光染色发现,转染BDNF AS质粒和转染突变质粒细胞的平均D值均低于同一视野中未转染的其他细胞(P<0.05,P<0.01)。FISH染色显示,转染BDNF AS质粒的细胞中,BDNF mRNA在细胞中分布的核/质比低于对照组(P<0.001);转染突变质粒的细胞中,BDNF mRNA在细胞中分布的核/质比低于对照组(P<0.05),高于lncRNA BDNF AS过表达的细胞(P<0.05)。该研究结果表明,肝癌细胞HepG2中lncRNA BDNF AS过表达下调BDNF mRNA和蛋白质水平,二者基因完全互补序列参与lncRNA BDNF AS对BDNF基因表达的调控作用。

The aim of this study is to investigate the regulatory effects of lncRNA BDNF AS（antisense brain derived neurotrophic factor） on the expression of BDNF in human hepatocellular carcinoma cells of HepG2. The BDNF AS plasmid or mutant plasmid with a loss of the overlap region between BDNF AS and BDNF gene was transiently transfected into HepG2 cells, and the levels of lncRNA BDNF AS and BDNF mRNA were detected by q PCR. The BDNF protein level was examined by Western blot, and cell supernatant was used to detect BDNF secretion by ELISA. The immunofluorescent staining was used to determine the expression of BDNF. FISH was used to observe subcelluar localization of lncRNA BDNF AS and BDNF mRNA. Our results showed that theexpression of lncRNA BDNF AS in BDNF AS group was higher than control group（P〈0.001）, and the levels of BDNF mRNA and protein were lower than control group（P〈0.001）. lncRNA BDNF AS level in mutant group was higher than control group（P〈0.05）, but lower than BDNF AS group（P〈0.001）. The levels of BDNF mRNA and protein were lower than control group（P〈0.05, P〈0.001）, and higher than BDNF AS group（P〈0.05, P〈0.001）. The mean D value of BDNF level by immunofluorescent staining in BDNF AS and mutant transfected cells were both lower than the rest other cells in the same field（P〈0.05, P〈0.01）. The distribution of BDNF mRNA represented by the value of nucleus/cytoplasm in BDNF AS group was lower than control group（P〈0.001）. That in mutant group was lower than control group（P〈0.05）, but higher than BDNF AS group（P〈0.05）. Our data suggested that overexpression of lncRNA BDNF AS inhibited the levels of BDNF mRNA and protein, and the completely complementary sequence was of critical importance for the regulatory effects of lncRNA BDNF AS on BDNF expression in HepG2 cells.