摘要
目的探讨miR－19b对晚期分化P19CL6细胞功能的影响及其作用机制。方法通过脂质体2000转染技术将miR－19b过表达质粒与空载质粒转染到P19CL6细胞中；二甲基亚砜（DMSO）诱导小鼠P19CL6细胞株和稳定表达miR－19b的细胞株进行分化；MTT法和流式细胞术检测miR－19b对细胞活力和凋亡的影响；Western印迹检测P19CL6细胞中Sox6的表达；ELISA法和Western印迹检测细胞分化晚期P19CL6凋亡相关蛋白（Bax, Bcl－2）的表达变化；使用TargetScan预测及双荧光素酶报告基因实验证实miR－19b与Sox6的靶向作用。结果miR－19b在分化的晚期（第10天起）表达量显著降低。miR－19b过表达显著促进细胞增殖和抑制细胞凋亡；且凋亡相关蛋白Bax表达显著降低，而Bcl－2表达增加，即凋亡蛋白Bax/Bcl－2比例降低；TargetScan预测及双荧光素酶报告基因实验证实Sox6是miR－19b的靶基因。结论在心肌细胞发育晚期，miR－19b可能通过靶向干扰Sox6基因的表达，促进P19CL6细胞的增殖抑制细胞的凋亡。
ObjectiveTo explore the effect of miR-19b on the function of P19CL6 cells and its molecular mechanism.MethodsOverexpression of miR-19b was carried out by transfecting miR-19b plasmid into the P19CL6 cells. MTT assay and flow cytometry were used to determine cell growth and apoptosis, respectively. Western blot was used to detect the expression level of Sox6 in P19CL6 cells. ELISA assay was used to detect the expression levels of apoptosis-related genes （Bax, Bcl-2） in P19CL6 cells at late-stage cardiac differentiation. Further online software TargetScan was used to predict the target genes of miR-19b and verified by dual luciferase reporter assay.ResultsOur data showed that overexpression of miR-19b in P19CL6 cells significantly increased the cell growth rates and the apoptosis inhibition rates. The ratio of apoptosis-related proteins （Bax/Bcl-2） was significantly reduced. Results from the TargetScan and dual luciferase reporter showed that Sox6 is the direct target of miR-19b.ConclusionsWe conclude that miR-19b might promote cell proliferation and inhibits cell apoptosis during the late-stage of cardiac differentiation by targeting Sox6 expression.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2018年第8期617-621,共5页
National Medical Journal of China
关键词
心肌细胞
细胞分化
细胞增殖
细胞凋亡
Cardiomyocytes
Cell differentiation
Cell proliferation
Apoptosis
