Studying the Oncosuppressive Functions of PTENP1 as a ceRNA.

PTENP1 is a processed pseudogene of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). It functions posttranscriptionally to regulate PTEN by acting as a sponge for microRNAs that target PTEN. PTENP1 therefore functions as a competitive endogenous RNA (ceRNA), competing with PTEN for binding of microRNAs (miRNA) and thereby modulating PTEN cellular abundance. Studies of the overexpression of PTENP1 all confirm its oncosuppressive function to be mediated through the suppression of cell proliferation, induction of apoptosis, and inhibition of cell migration and invasion of cancer cells of differing types. These oncosuppressive functions are a direct consequence of miRNA binding by PTENP1 and the subsequent liberation of PTEN from miRNA induced suppression. In this chapter, we will focus initially on the description of a high efficiency transient transfection method to introduce and overexpress PTENP1 in the cell type of interest, followed by accurate methodologies to measure transfection efficiency by flow cytometry. We will then continue to describe two methods to analyze cell proliferation, namely the CCK-8 assay and Click-iT® EdU assay. Due to commonalities in the manifestation of the oncosuppressive effects of PTENP1, mediated through its role as a ceRNA, the methods presented in this chapter will have wide applicability to a variety of different cell types.