The SF3B1R625H mutation promotes prolactinoma tumor progression through aberrant splicing of DLG1

Weiyan Xie,Yazhuo Zhang,Yulou Liu,Yiyuan Chen,Jing Guo,Dawei Wang,Chuzhong Li,Qiuyue Fang

doi:10.1186/s13046-022-02245-0

Abstract

BackgroundRecently, a hotspot mutation in prolactinoma was observed in splicing factor 3b subunit 1 (SF3B1R625H), but its functional effects and underlying molecular mechanisms remain largely unexplored.MethodsUsing the CRISPR/Cas9 genome editing system and rat pituitary GH3 cells, we generated heterozygous Sf3b1R625H mutant cells. Sanger and whole-genome sequencing were conducted to verify the introduction of this mutation. Transcriptome analysis was performed in SF3B1-wild-type versus mutant human prolactinoma samples and GH3 cells. RT-PCR and minigene reporter assays were conducted to verify aberrant splicing. The functional consequences of SF3B1R625H were evaluated in vitro and in vivo. Critical makers of epithelial-mesenchymal transition and key components were detected using western blot, immunohistochemistry, and immunofluorescence. Suppressing proteins was achieved using siRNA.ResultsTranscriptomic analysis of prolactinomas and heterozygous mutant cells revealed that the SF3B1R625H allele led to different alterations in splicing properties, affecting different genes in different species. SF3B1R625H promoted aberrant splicing and DLG1 suppression in both rat cells and human tumors. In addition, SF3B1R625H and knocking down DLG1 promoted cell migration, invasion, and epithelial-mesenchymal transition through PI3K/Akt pathway.ConclusionsOur findings elucidate a mechanism through which mutant SF3B1 promotes tumor progression and may provide a potent molecular therapeutic target for prolactinomas with the SF3B1R625H mutation.

Highlights

A hotspot mutation in prolactinoma was observed in splicing factor 3b subunit 1 (SF3B1R625H), but its functional effects and underlying molecular mechanisms remain largely unexplored
Our results suggested that SF3B1 mutations in prolactinoma stimulate PI3K/AKT signaling by downregulating Discs large 1 (DLG1), which was induced by aberrant splicing to enhance tumor invasion and migration
To investigate the biological role of the SF3B1R625H mutation in prolactinoma progression as well as similarities and differences between human and rats, we introduced the Sf3b1R625H missense mutation into the rat pituitary cell line GH3 using the CRISPR/Cas9 gene editing system (Fig. S1A)

Summary

Introduction

A hotspot mutation in prolactinoma was observed in splicing factor 3b subunit 1 (SF3B1R625H), but its functional effects and underlying molecular mechanisms remain largely unexplored. Some prolactinomas are characterized by tumor invasion, Alternative splicing is an essential step in the posttranscriptional regulation of gene expression. It is a complex process that diversifies the proteome by creating multiple proteins from the same gene [4, 5]. Half of the aberrantly spliced mRNAs are subject to nonsense-mediated decay (NMD), which causes gene and protein production to be downregulated [15]. The underlying molecular mechanisms of this SF3B1 mutation and its downstream cellular processes in prolactinoma remain unclear

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Conclusion