Overexpression of long non-coding RNA WT1-AS or silencing of PIK3AP1 are inhibitory to cervical cancer progression

Abstract

ABSTRACT Accumulating evidence demonstrate that long non-coding RNAs (lncRNAs) play an important role in regulating the biological function of cervical cancer cells. However, the regulatory role of lncRNA Wilms tumor 1 homolog antisense RNA (WT1-AS) in cervical cancer cells remains uncertain. In this study, we explored the participation of WT1-AS in cervical cancer by first using the reverse transcription quantitative polymerase-chain reaction (RT-qPCR) was to analyze the expression of WT1-AS and phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1) in cervical cancer tissues and cells. Dual-luciferase reporter gene assay, RNA pull-down/RNA immunoprecipitation (RIP) assays and Chromatin Immunoprecipitation (ChIP) assay were conducted to explore the interactions among WT1-AS, PIK3AP1, and SPI1. Gain- and loss-of-function approaches were carried out to determine the effects of lncRNA WT1-AS, PIK3AP1 on cell biological characteristics, followed by assays of cell proliferation, autophagy, and apoptosis abilities using, respectively, EdU, monodansylcadaverine (MDC) staining, and flow cytometry. Finally, we measured growth of xenograft tumors in nude mice. We found decreased expression of lncRNA WT1-AS and increased expression of PIK3AP1 in cervical cancer samples. Moreover, PIK3AP1 was negatively regulated by WT1-AS, which promoted apoptosis, but inhibited cell proliferation and autophagy of cervical cancer cells. Furthermore, WT1-AS inhibited PIK3AP1 expression by recruiting SPI1, and inhibited the progression of cervical cancer through the SPI1/PIK3AP1 axis in vivo and in vitro. In summary, lncRNA WT1-AS repressed the development of cervical cancer by reducing PIK3AP1 expression through an interaction with SPI1, which may suggest new therapeutic approaches for treating cervical cancer. Abbreviations: HPV, human papillomavirus; lncRNAs, Long non-coding RNAs; WT1-AS, wilms tumor 1 antisense RNA; HCC, hepatocellular carcinoma; SFFV, Spleen focus forming virus; SPI1, Spleen focus forming virus proviral integration oncogene 1; TF, transcription factor; PIK3AP1, Phosphoinositide-3-kinase adaptor protein 1; NCBI, National Center for Biotechnology Information; oe, overexpressed; sh-PIK3AP1, short hairpin RNA against PIK3AP1; RIPA, radioimmunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; HRP, horseradish peroxidase; IgG, immunoglobulin G; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; EP, Eppendorf; RIP, RNA-binding protein immunoprecipitation; CHIP, Chromatin immunoprecipitation; EdU, 5-ethynyl-2ʹ-deoxyuridine; PI, propidium iodide; MDC, Monodansylcadaverine; PFA, paraformaldehyde; SPF, specific pathogen free; TV, tumor volume; DLG1-AS1, discs large MAGUK scaffold protein 1 antisense RNA 1; TOB1-AS1, transducer of epidermal growth factor receptor-2.1 antisense RNA 1; LC3II, light chain 3 type II; LC3I, light chain 3 type I; IRF4, interferon regulatory factor 4