FUNCTIONAL INHIBITION OF NUCLEOSTEMIN GENE-ACOORDINATOR OF SELF-RENEWAL ABILITY-IN BONE MARROW DERIVED MESENCHYMAL STEM CELLS BY RNAI STRATEGY

The aim is to downregulate the expression level of NS as an important factor in sustaining stem cells and certain types of cancer cells self-renewal ability in bone marrow derived mesenchymal stem cells by RNAi strategy and investigate the effects of absence of NS in these cells. Double strand NS-specific and control siRNA oligos were designed and transfected into the cultured Rat bone marrow stromal stem cells by lipofection. Changes in gene expression pattern after downregulation of nucleostemin were investigated by RT-PCR immunocytochemistry and western blotting. Also changes in rate of cell proliferation and cell cycle profile were studied. The isolated cells had fibroblastic morphology and were able to differentiated to neuron-like cells as well as expressing stem cells and mesenchymal lineage specific genes. Expression of nucleostemin highly decrease upon trasfection by siRNA accompanied by reduction in the rate of cell proliferation driving cells into senescence and reducing the expression of cyclin D1 and survivin. Analysis Studies revealed that introduction of nucleostemin-specific siRNA into the cultured cell was successful and caused a proportional reduction in expression of NS. Growth curve and cell cycle profile analysis as well as expression profile of certain genes involved in self-renewal processes represent an upstream role for nucleostemin in controlling bone marrow mesenchymal stem cells self-renewal.