Genome-scale activation screen reveals lncRNA HNF1A-AS1 as a novel therapeutic target for pancreatic cancer metastasis

Abstract

Background Long non-coding RNAs (LncRNAs) have emerged as pivotal biomarkers and regulators across various cancers. In pancreatic cancer (PC), however, the mechanisms underlying the expression and functional roles of lncRNAs remain inadequately elucidated. Methods CRISPR/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA)-pooled lncRNA libraries were used to screen for the critical lncRNAs regulating PC metastasis. The expression levels of lncRNA HNF1A-AS1 were quantified in PC cell lines and clinical samples using qRT-PCR. Investigations into HNF1A-AS1's impact on PC cell migration and invasion were conducted through both loss-of-function and gain-of-function approaches. A range of techniques, including fluorescence in situ hybridization (FISH), mRNA sequencing, RNA immunoprecipitation (RIP), bioinformatics analysis, dual-luciferase reporter assays, RNA pull-down assays, ChIP-PCR, and rescue experiments, were employed to unravel the competitive endogenous RNA (ceRNA) network regulated by HNF1A-AS1. Results The research identified HNF1A-AS1 as a novel and influential lncRNA that acts as a pro-metastatic factor in PC. Compared to normal controls, HNF1A-AS1 levels were significantly elevated in PC cell lines and tissue samples. Elevated HNF1A-AS1 expression correlated with increased lymph node metastasis and poorer overall survival in patients with PC. Knocking down HNF1A-AS1 substantially reduced metastasis, whereas its overexpression exacerbated it. Mechanistically, HNF1A-AS1 promotes an oncogenic splice switch from the standard isoform CD44s to the variant isoform CD44v (3–10), acting as a scaffold for the binding of CD44 pre-mRNA to U2SURP. The levels of HNF1A-AS1 and CD44v (3–10) serve as indicators of poor prognosis. Furthermore, SNAI2 was shown to specifically bind to the HNF1A-AS1 promoter, thereby activating its transcription. Antisense oligonucleotides (ASOs) targeting HNF1A-AS1 also significantly inhibited cancer metastasis. Conclusions SNAI2’s role in enhancing HNF1A-AS1 transcription underscores the critical function of HNF1A-AS1 in promoting PC metastasis through modulation of CD44 alternative splicing via U2SURP. Targeted silencing of HNF1A-AS1 presents a promising therapeutic avenue for patients with PC.