Abstract 2525: Identification of mRNA targets associated with PIWIL1 protein complex in lung cancer cells

Abstract

Abstract Introduction: In previous published work we have shown that RASSF1C promotes expression of PIWIL1 and PIWI interacting RNAs (piRNAs) in lung cancer cells, suggesting the hypothesis that that the RASSF1C-PIWI-piRNA pathway may promote lung cancer cell growth and progression. PIWIL1 protein-piRNA complexes have recently been shown to regulate mRNAs in mouse testes, suggesting that PIWIL1 plays an important role in mRNA cleavage to regulate specific targets involved in cellular growth. To investigate this hypothesis, we used a non-small cell lung cancer (NSCLC) cell model to identify candidate mRNAs targeted by the RASSF1C-PIWIL1-piRNA pathway through PIWIL1-protein complex co-immunoprecipitation and RNA sequencing. Methods: Cell lysates were prepared under non-denaturing conditions from the lung cancer cell line H1299 over-expressing RASSF1C (experimental sample) and H1299 over-expressing the vector backbone (control sample) using a co-immunoprecipitation kit (Pierce). The PIWIL1 2C12 monoclonal antibody (Wako Chemicals Inc., Richmond, VA) was used to pull down PIWIL1 protein using 1 mg of cell lysates from experimental and control samples. The RNA associated with the precipitated PIWIL1 protein complex was isolated using a total RNA isolation kit (Qiagen) and the isolated RNA was subjected to RNA deep sequencing and bioinformatic analysis. Results: Differential expression analysis based on read counts was carried out using a fold-change threshold of 2.0, maximum p-value of 0.05, and a minimum 1CPM, identified over 5505 genes that are differentially expressed when comparing read counts from H1299 cells over-expressing RASSF1C to H1299 cells over-expressing the vector back bone. Some of the genes identified with reduced mRNA levels correspond to tumor suppressors such as PTEN and p53 and apoptosis-inducing genes such as BAD and CASP3. Conclusions: Our study was designed to show that PIWIL-protein complexes can be pulled down from lung cancer cell lysates, and that PIWIL-protein complexes are likely to bind and regulate mRNA targets in lung cancer cells. We have identified interesting mRNAs that are potentially silenced by the PIWIL1-piRNA protein complex. Ongoing work is focused on validating some of the potential mRNA targets identified. This paves the way for conducting RNA-sequencing studies to identify specific piRNAs that associate with PIWIL1-protein complexes in the nucleus and cytoplasm. Citation Format: Yousef G. Amaar, Mark E. Reeves. Identification of mRNA targets associated with PIWIL1 protein complex in lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2525.