miR-194在增龄性胸腺萎缩中的表达及靶基因研究

摘要

Objective:To study the expression and interaction between miR-194 and PTPN12 in the process of age-related atrophy of thymus for clarifying the regulatory mechanism in the process of this disease.Methods:C57BL/6 mouse were divided into 4 groups as 1 month,6 months,10 months and 19 months old and each group has 6 cases.Thymus tissue was removed and thymic stromal cells were isolated.And thymus epithelial cells were washed out by CD45 antibody and LS column after anesthesia.Fluorescence quantitative real-time PCR and Western blot were used to detect the changes of miR-194 and PTPN12 gene expression in thymus epithelial cells with aging.miR-194 and PTPN12 luciferase reporter vectors were transfected into HEK293 cells,and the auto fluorescence values were detected at 24 h and 48 h,respectively in vitro.Results:The expression level of miR-194 decreased (P<0.05),while the expression level of PTPN12 mRNA increased (P<0.05) as the age increased.And the correlation between miR-194 and PTPN12 mRNA expression was found to be negative(P<0.05).In vitro,luciferase reporter gene results show that miR-194 has a direct effect on the 3'UTR region of PTPN12 gene and had the highest binding efficiency in 48 h.Conclusion:PTPN12 is one of the target genes of miR-194,which is involved in the aging process of thymus and is an important factor regulating the function of thymic ep-ithelial cells.%目的:检测增龄性胸腺萎缩过程中miR-194与PTPN12的表达水平变化,并分析二者相互作用,阐明其中的分子调节机制.方法:选用C57BL/6小鼠,分为4组:1月龄组、6月龄组、10月龄组和19月龄组,每组6只,雌雄各半.麻醉后取出胸腺组织,用CD45抗体与LS柱吸附洗脱,筛选出胸腺上皮细胞.实时荧光定量PCR与Western blot方法检测随年龄增长,胸腺上皮细胞中miR-194与PTPN12基因的表达变化趋势.体外实验共转染miR-194与PTPN12荧光素酶报告载体到HEK293细胞内,分别于24、48 h后检测自发荧光值.结果:随月龄增长,miR-194表达出现下调趋势(P<0.05),而PTPN12基因表达出现上调趋势(P<0.05),且二者呈负相关性(P<0.05).体外荧光素酶报告基因结果显示,miR-194与PTPN12基因3' UTR区域发生直接作用,并在48 h结合效率最高.结论:PTPN12是miR-194靶基因之一,参与了增龄性胸腺萎缩过程,是调节胸腺上皮细胞功能的重要因子.