Alternative Splicing of Secretin Receptor (SCTR) and its low Expression Drive the Occurrence of Pancreatic Ductal Adenocarcinoma.

Objectives: This study aim to elucidate the mechanistic role of secretin receptor (SCTR) in pancreatic ductal adenocarcinoma (PDAC) pathogenesis by systematically characterizing its alternative splicing (AS) landscape.

Methods: This study systematically investigated the specific gene expression profiles of PDAC by integrating multi-omics data from TCGA and GTEx public databases. We collected formalin-fixed paraffin-embedded (FFPE) specimens and clinicopathological data from 65 PDAC patients. Immunohistochemical (IHC) analysis was performed to determine the differential expression of secretin receptor (SCTR) between PDAC and adjacent non-tumor (ATN) tissues, along with its clinicopathological correlations. RNA sequencing (RNA-seq) was conducted on 9 paired PDAC and ATN samples, followed by alternative splicing (AS) analysis using rMATs software to identify prognostic-related splicing events. IHC validation confirmed the protein-level expression patterns. Notably, through RT-qPCR and cloning/sequencing approaches, we unexpectedly discovered an intron 2-retained alternative splicing variant of SCTR, which may represent a novel PDAC-specific isoform.

Results: Public data shows that compared with other tissues, SCTR expression in pancreatic tissue and pancreatic cancer is higher, and SCTR expression in pancreatic cancer is lower (P<0.001). In pancreatic cancer, the high expression of SCTR is associated with better overall survival (P<0.01). IHC expression analysis of 65 PDAC retrospective samples showed that：the expression of SCTR protein in PDAC was significantly lower than that of ATN (P<0.001), and the expression of SCTR protein was correlated with distant metastasis (P<0.05), tumor stage (P<0.001) and Ki67 expression (P<0.05) in PDAC.The RNA-seq results of this study showed that in 6 groups of samples, the expression of SCTR in PDAC was lower than that in ATN, with 5 groups significantly down regulated (P<0.05). RNA-seq sequencing revealed 18 alternative splicing patterns of SCTR in PDAC and ATN, among which the number of three high-frequency alternative splicing events, namely exon 9 skipping, fragments of intron 2 retention, and alternative 3' splice site of exon 3, were differed among multiple pairs of PDAC and ATN samples(P<0.05) (≥3 groups). Simultaneously, it was found that SNP rs2587682 (c.194-1804 T>C) and the fragments of intron 2 retention of SCTR were associated with the occurrence of this alternative splicing event (P<0.01).RT-qPCR and cloning sequencing results verified the existence of alternative splicing, the retained fragment is located in intron 2 between exons 2 and 3 of SCTR, and the sequence is 114bp.The expression of intron 2 of SCTR in ATN (2.58±4.41) was higher than that in PDAC (0.16±0.12) (P<0.001).Moreover, the expression of intron 2 of SCTR in samples with genotype GG of rs2587682 (2.42±1.61) was significantly higher than that in samples with genotype AA (1.01±0.20) (P<0.05).

Conclusions: This study provides novel insights into identifying potential therapeutic targets for pancreatic cancer, highlighting that SCTR may serve as a promising prognostic and diagnostic biomarker, with its low expression significantly associated with poor clinical outcomes in PDAC.