Abstract 4435: Functional role of four-and-a-half LIM protein 2 in the alternative splicing of cancer hallmark genes of hepatocellular carcinoma

Abstract

Abstract Four-and-a-half LIM protein 2 (FHL2) participates in various cellular processes, including regulation of cell survival, transcription and signal transduction. In this study, FHL2 specific antibody was used to immunoprecipitate endogenous FHL2 together with its binding partners from WRL68 and Hep3B liver cell lysates, followed by protein identification using mass spectrometry. The top significant functional clusters of identified proteins were related to the splicing process. To validating the pull down results, the interaction of FHL2 with ASF and U1A in WRL68 cell line was confirmed using co-immunoprecipitation. To study the possible effects of FHL2 on mRNA splicing, we knocked down FHL2 in WRL68, L02 and Hep3B cells and the transcriptomes of knocked-down cells were examined by RNA sequencing. We identified a global impact on regulating differential exon usage by knocking down FHL2. Subsequently, several alternatively spliced cancer hallmark genes were selected for validation using real-time PCR. We found that knocking down FHL2 could significantly promote the extra domain B (EDB) exon inclusion in fibronectin 1 (FN1) and the E3b exon inclusion in Rac1. In addition, knocking down FHL2 significantly decreased the percentage of the epithelial specific transcript CD44E while only very mildly decreased the standard CD44S transcript in total CD44 mRNA. Using an ab initio motif identification approach, the possible splicing factor binding motifs in the alternative spliced exons regulated by over-expressing and knocking down FHL2 were obtained. ASF was found to be the most common splicing regulatory protein. To further explore the functional relationship between FHL2 and ASF, we examined the effects of FHL2 and ASF on exon inclusion/skipping of endogenous EDB exon of FN1 in WRL68 cells. It was found that knocking down FHL2 using siRNA promotes inclusion of EDB while knocking down ASF using siRNA promotes skipping of EDB of FN1. When FHL2 and ASF were knocked down simultaneously, the effect of FHL2 on EDB exon skipping was abolished, implying that ASF mediates the effect of FHL2 on the EDB exon skipping. To explore the clinical relevance of this study, the expression of FHL2 and the EDB exon were determined in 40 paired HCC and normal tissues using real-time PCR. Results showed that the fold changes of EDB inclusion level and the FHL2 expression were significantly correlated. In summary, we have identified many alternative splicing-related protein interacting partners of FHL2 and the downregulated of FHL2 could affect the splicing pattern of many cancer hallmark genes, including CD44, Rac1 and FN1. Using FN1 as a model, we elucidated ASF as one of the mediators of the effect of FHL2 on alternative splicing. Taken together, FHL2 is probably involved in the pre-mRNA splicing in liver cells and modulate genes involved in cancer development through the perturbation of RNA splicing machinery. Citation Format: Stephen Kwok-Wing Tsui, Cyanne Ye Cao. Functional role of four-and-a-half LIM protein 2 in the alternative splicing of cancer hallmark genes of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4435.