叶酸缺乏的小鼠胚胎干细胞ES-D3中miR-302a、Bcl2L11表达观察及其靶向关系预测和验证  

Expression of miR-302a and Bcl2L11 and target gene prediction and verification in folate deficiency mouse ES-D3 cell line

作　　者：梁燕 曹丁丁 李媛媛[2] 刘卓[2] 吴建新[2] LIANG Yan;CAO Dingding;LI Yuanyuan;LIU Zhuo;WU Jianxin(Shandong Provincial Hospital Affiliated to Shandong First Medical University,Jinan 250021,China;不详)

机构地区：[1]山东第一医科大学附属省立医院,济南250021 [2]首都儿科研究所

出　　处：《山东医药》2020年第30期40-44,共5页Shandong Medical Journal

基　　金：北京市医院管理中心儿科学科协同发展中心专项经费(XTZD20180402)。

摘　　要：目的观察叶酸缺乏的小鼠胚胎干细胞系ES-D3中微小RNA302a(miR-302a)、Bcl2L11的表达变化,并对miR-302a与Bcl2L11的靶向关系进行预测和验证。方法取适量ES-D3细胞,置于缺乏叶酸的培养液中培养,分别于培养0、24、48、72 h(A、B、C、D组)时收集细胞,实时荧光定量PCR法检测miR-302a、Bcl2L11 mRNA,Western blotting法检测细胞Bcl2L11蛋白(BimEL、BimL及BimS蛋白)。生物信息学软件预测Bcl2L11与miR-302a的靶向结合位点。以小鼠3T3细胞基因组DNA为模板,设计3’UTR扩增引物,PCR扩增基因的3’UTR序列,将其克隆到pmiR-RB-REPORT双荧光素酶报告载体中,构建Bcl2L11-3’UTR野生载体;设计突变引物将靶标序列突变,构建突变载体,miRNA-302a mimics及Negative Control分别与Bcl2L113’UTR双报告基因野生载体及突变载体共转染,分别为1组(Bcl2L113’UTR野生型载体+Negative Control)、2组(Bcl2L113’UTR野生型载体+miR-302a mimics)、3组(Bcl2L113’UTR突变型载体+Negative Control)、4组(Bcl2L113’UTR突变型载体+miR-302a mimics),转染后测算各组报告基因hRluc的相对荧光值,验证miR-302a与Bcl2L11是否存在相互作用。结果A、B、C、D组细胞miR-302a相对表达量分别为1.00±0.20、0.65±0.32、0.50±0.22、0.43±0.24,与A组比较,D组miR-302a相对表达量下降(P<0.05);A、B、C、D组细胞Bcl2L11 mRNA相对表达量分别为1.00±0.00、1.22±0.19、1.82±0.37、3.49±0.45,与A组比较,D组Bcl2L11 mRNA相对表达量升高(P<0.05);A、B、C、D组细胞BimEL相对表达量分别为0.86±0.02、0.87±0.03、0.92±0.02、1.10±0.06,与A组比较,C、D组BimEL相对表达量升高(P<0.05);A、B、C、D组细胞BimL相对表达量分别为0、0.09±0.02、0.16±0.04、0.26±0.03,与A组比较,B、C、D组BimL相对表达量升高(P<0.05);A、B、C、D组细胞BimS相对表达量分别为0、0、0.11±0.04、0.18±0.04,与A组比较,C、D组BimS相对表达量升高(P<0.05)。miR-302a和Bcl2L11存在相互作用。1、2、3、4组报告基因hObjective To investigate the expression changes of miR-302a and Bcl2L11 and to explore the target gene prediction and verification in folate deficiency mouse ES-D3 cell line.Methods Proper number of mouse ES-D3 cells were cultured in folate-deficient culture medium,and were collected at predetermined time points:0,24,48,72 h(groups A,B,C,D).MiR-302a and Bcl2l11 expression changes(BimEL,BimL,BimS)were detected by real-time q PCR and Western blotting.Multiple target gene prediction software was used to predict the binding sites between Bcl2l11 and miR-302a.The amplification primers were designed and constructed according to the 3’UTR sequence of Bcl2l11.The fragment of the Bcl2l11-3’UTR sequence was cloned into the pmiR-RB-REPORT double luciferase reporter vector,and the wild vector was successfully constructed.Similarly,Bcl2l11 mutation primers were designed to construct the mutation vector.MiR-302a mimics and/or negative control were co-transfected with Bcl2L113’UTR double reporter wild vector and/or mutant vector,respectively.Therefore,the following four groups were constructed:group 1(Bcl2l113’UTR wild vector and negative control),group 2(Bcl2l113’UTR wild vector and miR-302a mimics),group 3(Bcl2l113’UTR mutation vector and negative control),and group 4(Bcl2l113’UTR mutation vector and miR-302a mimics).Changes on the relative fluorescence value of the hRluc reporter gene were analyzed to further confirm the interaction between miR-302a and Bcl2l11.Results The relative miR-302a mRNA expression in the groups A,B,C,and D was 1.00±0.20,0.65±0.32,0.50±0.22,0.43±0.24,respectively.Compared with the group A,miR-302a expression was down-regulated in the group D(P<0.05).The relative Bcl2l11 mRNA expression in the groups A,B,C,and D was 1.00±0.00,1.22±0.19,1.82±0.37,and 3.49±0.45,respectively.Compared with the group A,Bcl2l11 expression was up-regulated in the group D(P<0.05).The relative BimEL expression in the groups A,B,C,and D was 0.86±0.02,0.87±0.03,0.92±0.02,and 1.10±0.06,respectively.Compa

关 键 词：微小RNA 微小RNA302a 凋亡调节蛋白 Bcl2L11基因 叶酸缺乏 胚胎干细胞 细胞凋亡 胚胎发育 

分 类 号：R394.3[医药卫生—医学遗传学]