Overexpression of Nanog gene in mouse mesenchymal stem cells and its influence on NF-kappaB expression / 生物工程学报

ABSTRACT

The expression of NF-kappaB is considered to be involved in the progress of neurodegeneration. It has been reported that Nanog can suppress the expression of NF-kappaB. To inspect and verify this finding, we constructed lentivirus (LV) vector that overexpressed the Nanog gene, infected mouse mesenchymal stem cells (mMSCs), and examined the influence of Nanog overexpression on NF-kappaB gene expression. The plasmid pNL-Nanog-IRES2-EGFP was constructed by double digestion and genetic recombination. Sequencing results confirmed that our cloned Nanog gene in the PNL-Nanog-IRES2-EGFP plasmid was consistent with the sequence reported in the GenBank. The three plasmids pNL-Nanog-IRES2-EGFP, HELPER, and VSVG were cotransfected into 293T cells to produce LV particles. After co-transfection of the three lentiviral plasmids, green fluorescence was observed confirming successful transfection. The mMSCs were infected by the LV and the expression of Nanog was then also verified by the presence of green fluorescence. Nanog expression levels in the mMSCs were examined using Western blotting. Expression of NF-kappaB was also examined using RT-PCR and Western blotting, and in addition with fluorescent microscope after immunocytochemical staining. The levels of Nanog protein expression in Nanog-mMSCs were significantly increased, and the levels of NF-kappaB mRNA and protein expression in Nanog-infected mMSCs were significantly lower than those of Mock-mMSCs and the mMSCs control groups. Our findings suggest that mMSCs genetically modified to overexpress Nanog can lead to the suppression of NF-kappaB expression. This suppression of NF-kappaB could have important implications for the treatment of neurodegeneration, and hence further scientific investigations of these interactions will have significant impact on future clinical attempts to attenuate disease progression.