Depletion of a single nucleoporin, Nup107, induces apoptosis in eukaryotic cells

Abstract

Nuclear pores are large protein complexes that cross the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are about on average 2,000 nuclear pore complexes (NPCs) in the nuclear envelope of a vertebrate cell, but it varies depending on cell type and the stage in the life cycle. The proteins that make up the NPC are known as nucleoporins. In mammalian cells, Nup107 is the homolog of yeast Nup84p nucleoporin. Nup107 contains a leucine zipper motif in its carboxyl-terminal region and numerous kinase consensus sites, but does not contain FG repeats. Previously it was reported that NUP88 and NUP107 are over expressed in many types of cancers including colon, breast, prostrate, etc. In this study, we were interested in investigating the role of NUP107 in grade 4 Astrocytoma, i.e., Glioblastoma multiforme cultured cell line. We transfected human Astrocytoma cells with Nup107-specific siRNA duplexes. The level of mRNA for Nup107 was monitored by RT-PCR, 24, 48, and 72 h after the initial transfection. Nup107 mRNA was significantly diminished by 24 h after transfection and we took that as our incubation time. Next we studied the effect of this inhibition on cell viability. We did a Trypan Blue cell viability assay and it showed increased cell death in NUP107 transfected cells than untreated control. We further tried to analyze the nature of the cell death whether apoptotic or necrotic by doing apoptosis assays like ssDNA ELISA assay, Caspase-3, and Caspase-8 assays. All the assays showed that siRNA transfected cells are undergoing increased apoptosis than control.