Construction And Expression Detection Of Lentiviral Vector In Mir-200 Family And Its Target Gene Verification

PartⅠConstruction of miR-200b/c/429and its Spronge Lentiviral Vector and its lentivius packageObjective:Refer to Bibliographic retrieval and bioinformatics technique, we found miR-200b/c/429may regulate PRDM163’UTR to influence its expression. To study the miR-200b/c/429regulate Brown adipose tissue development, we first constructed lentivirus vectors containing the precursor of miR-200b/c/429independently and anti-mir-200b/c/429independently.Methods:500bp DNA fragments containing the precursor of miR-200b/c/429were cloned from mouse genomic DNA, and constructed lentivirus vectors. MiR-200b/c/429sponge were designed and synthetized to constructed lentivirus vectors containing anti-miR-200b/c/429. The constructed plasmid and two helper plasmids, PMD2.G and psPAX2, were transducted into293FT cells by liposome2000to package lentivirus.Results:Recombinant lentiviral vectors miR-200b/c/249and anti-miR-200b/c/249were successfully constructed indepentedly. Packing of lentivirus was validated by the expression of Green Fluorescent Protein.Conclusion:We cloned miR-200b/c/249lentiviral vectors and construct its spronge. Part ⅡExpression of miR-200b/c/429in WAT and3T3-L1and its function in regulating PRDM16Construction of miR-200b/c/429and its Spronge Lentiviral Vector and Its function in regulate its target PRDM16Objective:To study the miR-200b/c/429regulate Brown adipose tissue development, we constructed a recombinant vector containing PRDM16-3’UTR and dual luciferase reporter. And we detect the expression of miR-200b/c/429in HFD C57mouse and during3T3-L1differentiation, lentivirus vectors containing anti-mir-200b/c/429independently to infected3T3-L1, and detect the expression of PRDM16after8day3T3-L1differentiation.Methods:Q-PCR were used to detect the expression of miR-200b/c/429in Subcutaneous WAT of HFD C57BL/6mouse and during3T3-L1differentiation. We constructed a recombinant vector containing PRDM16-3’UTR and dual luciferase reporter and verified the target via dual luciferase reporter assay system in293FT cell, indicating miR-200s could interact with PRDM16. After lentivirus vectors containing anti-miR-200b/c/429down-regulated the expression of miR-200b/c/429, we found the expression of PRDM16were upregulated in day8of3T3-L1differentiation.Results:Recombinant lentiviral vectors miR-200b/c/249and anti-miR-200b/c/249were successfully constructed indepentedly. And anti-miR-200b/c/249can downreglated the expression of miR-200b/c/249. The recombinant vector containing PRDM16-3’UTR and dual luciferase reporter can be downregulated by mir-200b/c/249during cotransfection. miR-200b/c were downregulated in HFD Subcutaneous WAT. And miR-200b/c were upregulated in3T3-L1during8day’s differentiation. PRDM16were upregulated in anti-miR-200b/c/249lentiviral infected 3T3-L1.Conclusion:PRDM16were regulated by miR-200b/c/429in vitro. miR-200b/c/429were upregulated in3T3-L1differentiation and downregualted in HFD mouse Subcutaneous WAT.