大鼠Cacnb1报告基因载体构建及miR-16靶向验证  

Construction of Reporter Vector for Rat Cacnb1 Gene to Verify Its miR-16 Target

作　　者：张小真[1] 石科 李桂玲[1] 杨亮 范沛[1] 

机构地区：[1]河南工业大学生物工程学院,中国河南郑州450001 [2]河南医学高等专科学校检验系,中国河南郑州451191

出　　处：《生命科学研究》2016年第4期309-313,352,共6页Life Science Research

基　　金：国家自然科学基金青年科学基金项目(31402026)

摘　　要：子宫平滑肌过度收缩可导致原发性痛经。钙通道在子宫平滑肌收缩过程中起关键作用,钙通道电压依赖性β1蛋白(voltage-dependent L-type calcium channel subunit beta-1,CACNB1)是其主要组成部分。为探讨mi R-16在子宫平滑肌细胞收缩过程中的调控功能,需验证大鼠Cacnb1基因是否为mi R-16的靶基因。首先,设计特异性引物,扩增大鼠Cacnb1基因3′UTR序列,将扩增片段克隆到pmi R-RB-REPORTTM双荧光素酶报告基因载体,同时构建大鼠Cacnb1基因3′UTR突变的载体;随后,包含正常Cacnb1基因3′UTR序列的载体和突变后的载体分别与mi R-16 mimics共转染293T细胞,检测相对荧光素酶活性变化。结果发现,mi R-16 mimics能够通过结合大鼠Cacnb1基因3′UTR序列显著抑制荧光素酶活性(n=3,P<0.001),而对大鼠Cacnb1基因3′UTR突变后的荧光素酶活性无抑制作用。因此,mi R-16能够负向调控大鼠Cacnb1基因的表达,故大鼠Cacnb1基因是mi R-16的靶基因。Primary dysmenorrhea can be caused by over-contraction of uterine smooth muscle （USM）. Calcium channel, playing crucial roles in USM contraction, is constituted by several key proteins, including voltage- dependent L-type calcium channel subunit beta-1 （CACNB1）. To elucidate the roles of miR-16 in regulating USM contraction, it is necessary to know whether rat Cacnb1 gene is a target of miR-16. Firstly, the 3＇UTR of rat Cacnb 1 was amplified by specifically designed primers and cloned to the dual luciferase reporter vector pmiR-RB-REPORT. Meanwhile, the vector with mutant 3＇UTR of rat Cacnbl was also constructed. Then, the constructed vectors were transfeeted in 293T cells with miR-16 mimics, respectively, followed by the as- say of relative luciferase activity. The results demonstrated that miR-16 mimics were able to significantly in- hibit the relative luciferase activity by targeting 3＇UTR of rat Cacnb 1 （n=3, P〈0.001）. However, no inhibitory activity was shown with the mutation of 3＇UTR of rat Cacnbl. It can be concluded that rat Cacnbl is able to be down-regulated by miR-16. Therefore, rat Cacnbl is the targeted gent of miR-16.

关 键 词：原发性痛经 钙通道 钙通道电压依赖性茁1蛋白(CACNB1) miR-16 载体构建 靶向验证 大鼠 

分 类 号：Q782[生物学—分子生物学]