Objective: To find novel isoform of PIK3IP1 and analyze their effects on cell viability, subcellular localization, and expression profile in cell lines.
Methods: RT-PCR was used to clone PIK3IP1 and its novel splicing isoform PIK3IP1-v1 from multi-tissue cDNA pool. By cell-based assays, we studied how PIK3IP1 and PIK3IP1-v1 affected the activity of Renila luciferase and morphological change in the HEK 293T cells. Furthermore, flow cytometry experiment was used to validate that overexpression of both splicing isoforms could induce cell apoptosis. Bioinformatics analysis was used to identify structural characteristics of these two splicing isoforms. By fluorescence microscopy assay, we identified their subcellular localization. RT-PCR was used to detect the expression of PIK3IP1 in the cell lines.
Results: PIK3IP1 and its novel splicing isoform PIK3IP1-v1 were cloned and constructed into the pcDNA-and pEGFP-expression plasmids. They both had signal peptide and transmembrane domain. Nevertheless, PIK3IP1-v1 was in absence of an extracellular Kringle domain. They could inhibit the activity of Renila luciferase and induce cell apoptosis. Simultaneously, both splicing isoforms are validated with subcellular localization on cell membrane and lowly expressed in many cell lines.
Conclusion: PIK3IP1-v1 is a novel splicing isoform of PIK3IP1. Both of them are located on cell membrane and can induce cell apoptosis.