| 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. |
