| 李虹仪，张茂.调节猪Smad3 表达的miRNA 鉴定[J].广东农业科学,2014,41(23):123-126 |
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调节猪Smad3 表达的miRNA 鉴定 |
| Identification of miRNA regulated Porcine Smad3 expression |
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| DOI： |
| 中文关键词: Smad3 miR-23a 荧光素酶 |
| 英文关键词: Smad3 miR-23a luciferase |
| 基金项目:福建省自然科学基金（2014J05043） |
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| 中文摘要: |
| 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忆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-control-Smad3-3忆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，48 h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。 |
| 英文摘要: |
| Porcine Smad3 was predicted to be the target gene of miR-23a by online websites. In order to verify the relationship between Smad3 and miR-23a, double stranded miR-23a sequences and its biding site to the 3’untranslated region of Smad3 were synthetized, and conjugated to pshRNA-copGFP Lentivector and pPGL3-control respectively, to construct recombinant plasmids of LV-shRNA-miR-23a and pPGL3-control-Smad3-3’UTR. Then the CHO cell line was cotransfected with these two recombinant plasmids, taking cotransfected with pshRNA-copGFP Lentivector and pPGL3- 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 mRNA was measured by Quantitative Real-time PCR. The results showed that two recombinant plasmids were successfully constructed. Expression of Smad3 mRNA 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-23a. |
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