5-磷酸核糖醇转移酶fukutin抑制HeLa细胞中α-dystroglycan的分泌

Ribitol-5-phosphate transferase fukutin inhibits the secretion of α-dystroglycan in HeLa cells

目的探究5-磷酸核糖醇转移酶fukutin(FKTN)对HeLa细胞周期、凋亡、迁移以及α抗肌萎缩相关糖蛋白(α-DG)分泌的影响。方法构建质粒pcDNA3.1-α-DAG-HA,将pcDNA3.1-FKTN-3xFlag转染至HeLa细胞,提取总蛋白,Western blot法检测FKTN的过表达;过表达FKTN后利用流式细胞术检测细胞周期、凋亡情况;过表达FKTN后,采用细胞划痕实验检测细胞迁移速率,使用麦胚凝集素琼脂糖珠(WGA-agarose)富集培养液中的α-DG,然后进行Western blot检测。结果成功构建pcDNA3.1-α-DAG-HA真核表达质粒;FKTN在HeLa细胞中可过表达;过表达FKTN后与对照组相比,实验组细胞周期S期百分比下降(P<0.001),细胞凋亡率无变化(P>0.05);过表达FKTN后与对照组相比,实验组细胞迁移速率无变化(P>0.05),α-DG分泌量减少。结论过表达FKTN阻滞HeLa细胞周期,抑制α-DG分泌,但对细胞凋亡、迁移无影响。

Objective To investigate the effects of fukutin(FKTN)which is a membeRof ribitol-5-phosphate transferases on HeLa cell cycle,apoptosis,migration and the secretion ofα-dystroglycan(α-DG).Methods The plasmid pcDNA3.1-α-DAG-HA was constructed.HeLa cells were transfected with plasmid pcDNA3.1-FKTN-3xFlag,then the total protein was extracted foRWestern blot to determine the expression level of FKTN.Cell cycle and apoptosis were measured by flow cytometry afteRthe overexpression of FKTN.Following the overexpression of FKTN,wound-healing assay was performed to detect the cell migration rate,as the same time wheat germ agglutinin-agarose(WGA-agarose)was used to enrichα-DG in the cell cultures,thenα-DG was detected by Western blot.Results The eukaryotic expression plasmid pcDNA3.1-α-DAG-HA was constructed successfully.FKTN could be overexpressed in HeLa cells.AfteRthe overexpression of FKTN,the percentage of S phase of cell cycle in the experimental group decreased(P<0.001)and apoptosis rate unchanged(P>0.05)when compared with the control group.There was no change in the cell migration rate of experimental group(P>0.05),but afteRthe overexpression of FKTN,secretion ofα-DG decreased when compared with control group.Conclusion Overexpressing FKTN arrests cell cycle and inhibits the secretion ofα-DG in HeLa cells.Apoptosis and cell migration of HeLa cells are not affected by the overexpression of FKTN.