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 clinicopathologic data from 65 PDAC patients. Immunohistochemical (IHC) analysis was performed to determine the differential expression of secretin receptor (SCTR) between PDAC and adjacent nontumor (ATN) tissues, along with its clinicopathologic 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 3 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.