Preparation For Recombinant Lentivirus Expressing Selected Rat SMAD3 SiRNA

Abstract:TGF-β(transforming growth factorβ)and ACTIVIN A, are two multiple function cytokines and the studies of their signaling mechanism become more and more hot. Recent research suggested that the functions of TGF-β/ACTIVIN A signaling Pathways include cell Proliferation, differentiation, cell migration and apoptosis.In different organs, this cytokines seemed to take part in various physiological and Pathological processes. As we know, the inflammation reaction, tissue repairing and embryonic development are all related to TGF-βsignaling. In some cases, the mutant of this pathways component could result in certain human diseases, especially in liver. For example, hepatitis, liver fibrosis, hepatocirrhosis, liver cancer and even liver regeneration were already proved to be concerned with TGF-βsignaling Pathway. And one goal of this research is to realize how the proteins in this pathway are organized and what kind of interaction network they build. The purpose of this study is to construct shRNA (short hairpin RNA) expression lentiviral particle targeting to rat SMAD3 protein. This study provides a powerful tool for further exploring a new gene therapy way of liver regeneration in the future.Objective:For the researching of the blockage of SMAD3 signal transduction and inhibit the apoptosis of hepatocytes and promote liver regeneration, a siRNA fragment targeting the rat SMAD3 mRNA was selected and to be constructed to a lentiviral plasmid for RNA interference (RNAi).The recombinant lentiviral plasmid was packaged to be a lentivirus which can stably express SMAD3 shRNA, and then these recombinant lentiviral plasmids were used to infect the rat liver cell line BRL-3A. Methods:Six pairs of double-stranded siRNAs were designed and synthesized according to the sequence of SMAD3 mRNA. The best one was selected by western blotting analysis, then its corresponding dsDNA was ligated into linearized plentilox 3.7 plasmid that be cut by Xhol and HpaI. Then the recombinant plasmid was transformed into competent cell DH5α. After sequence analysis for verification of the positive clones, the plasmid pll 3.7 SMAD3-shRNA was extracted and transfected into CaCl2-treated DH5 a cells to obtain the viral vectors containing the RNAi sequence. BRL-3A cells were infected with lentivirus can express SMAD3 shRNA. The lentivius was used to infect the rat hepatocyte line BRL-3A, and fluorescent were observed by fluorescence microscopy. Results:The best siRNA targeting SMAD3 mRNA was be selected successfully by western blotting analysis. The recombinant vector was successfully constructed as confirmed by sequence analysis. The virus was obtained, and then infect BRL-3A cells successfully. Conclusion:The recombinant lentiviral plasmid for RNAi of SMAD3 gene has been successfully constructed, which provides the powerful tools for further study of the role of SMAD3 gene in liver regeneration.