Epigenetic Dysregulation of the Expression of PRSS3 Splice Variants Increases the Heterogeneity of Transcripts and Functionality in Human Hepatocellular Carcinoma

Abstract Background Hepatocellular carcinoma (HCC) is one of the most lethal human tumors with extensive heterogeneity. Serine protease 3 (PRSS3) is an indispensable member of the trypsin family and has been implicated in the pathogenesis of several malignancies including HCC. However, paradoxical effects of PPRSS3 on carcinogenesis impede the utilization of its biomarker potential. We hereby systematically dissected the expression of four known splice variants of PRSS3 (PRSS3-SVs) and their functional relevance to HCC. Methods The expression and DNA methylation of PRSS3 transcripts and their associated clinical relevance in HCC were analyzed using several publicly available datasets and were validated using qPCR-based assays. Functional assays were performed on gain- and loss-of-function cell models, in which PRSS3 transcript constructs were separately transfected after PRSS3 expression was knocked out by CRISPR-Cas9 editing. Results PRSS3 expression was differentially decreased in HCC cell lines and tissues attributable to aberrant expression of PRSS3-SVs towards bipolarity, in which PRSS3-V2 and then -V1 were dominantly expressed whereas PRSS3-V3 and -V4 were rarely or minimally expressed. The expression of PRSS3-V2 or -V1 was reversely associated with site-specific CpG methylation and was distinct between lowly-expressed PRSS3-SVs with hypermethylation (mPRSS3Low) and highly-expressed PRSS3-SVs with hypomethylation (umPRSS3High). Function analysis revealed that PRSS3-V2 exhibited oncogenic functions distinct from a tumor-suppressive role of ectopic PRSS3-V1 or -V3, or PRSS3-V4 with inhibitory effects in PRSS3 knockout HCC cells. Clinically, aberrant expression of PRSS3-SVs was translated into divergent relevance in patients with HCC, in which significant epigenetic downregulation of PRSS3-V2 was seen in early HCC and was associated with favorable patient outcome. Conclusions Aberrant expression of divergent PRSS3-SVs disrupted by CpG methylation may integrate the effects of oncogenic PRSS3-V2 and tumor-suppressive PRSS3-V1, resulting in the molecular diversity and functional plasticity of HCC. Dysregulated expression of PRSS3-V2 by site-specific CpG methylation may have potential diagnostic value for patients with early HCC.