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当前位置: 首页 > 文章 > 调节猪Smad3表达的miRNA鉴定 广东农业科学 2014,41 (23) 123-126+144
Position: Home > Articles > Identification of miRNA regulated Porcine Smad3 expression Guangdong Agricultural Sciences 2014,41 (23) 123-126+144

调节猪Smad3表达的miRNA鉴定

作  者：
李虹仪;张茂
单  位：
龙岩学院生命科学学院/预防兽医学与生物技术福建省高等学校重点实验室
关键词：
Smad3;miR-23a;荧光素酶
摘  要：
生物信息学预测猪Smad3为miR-23a的靶基因,为了研究猪Smad3与miR-23a之间的调节关系,人工合成miR-23a双链序列及Smad3基因3′UTR中mR-23a的结合位点序列,分别与带荧光的慢病毒质粒pshRNAcopGFPLentivector及萤光素酶质粒pPGL3-control连接,构建重组质粒LV-shRNA-miR-23a和pPGL3-controlSmad3-3′UTR。将两重组质粒共转CHO细胞,以慢病毒空质粒与pPGL3-control-Smad3-3′UTR共转作为对照,48h后收集细胞裂解液检测荧光素酶活性变化,提取细胞总RNA实时荧光定量检测荧光素酶基因mRNA表达变化。结果显示,试验成功构建了重组质粒LV-shRNA-miR-23a和pPGL3-control-Smad3-3′UTR。miR-23a能够显著抑制荧光素酶基因mRNA表达(P<0.05),并能通过抑制Smad3结合位点序列抑制其上游荧光素酶的活性(P<0.05)。初步证明,猪Smad3与miR-23a有直接的靶向关系。
译  名：
Identification of miRNA regulated Porcine Smad3 expression
作  者：
LI Hong-yi;ZHANG Mao;College of Life Science, Longyan University/Fujian Proveincial Key Laboratory of Preventive Veterinary Medicine and Biotechnology;
关键词：
Smad3;;miR-23a;;luciferase
摘  要：
Porcine Smad3 was predicted to be the target gene of miR-23 a by online websites. In order to verify the relationship between Smad3 and miR-23 a, double stranded miR-23 a sequences and its biding site to the 3' untranslated region of Smad3 were synthetized, and conjugated to psh RNA-cop GFP Lentivector and p PGL3-control respectively, to construct recombinant plasmids of LV-sh RNA-miR-23 a and p PGL3-control-Smad3-3′UTR. Then the CHO cell line was cotransfected with these two recombinant plasmids, taking cotransfected with psh RNA-cop GFP Lentivector and p PGL3-control-Smad3-3′ UTR as negative control. Cells were collected 48 h after transfection, then the luciferase activity was measured using a dual luciferase reporter assay system and the expression of Smad3 m RNA was measured by Quantitative Real-time PCR. The results showed that two recombinant plasmids were successfully constructed. Expression of Smad3 m RNA as well as the luciferase activity were suppressed by miR-23a(P<0.05), which proved that Porcine Smad3 was the target gene of miR-23 a.

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