*Purpose: Alternative splicing (AS) gene regulation is at the interface of suppressing tumor cell growth and regulating cancer immunity. HIF-2α is a key transcriptional factor regulating the cellular response to hypoxia. CEACAM1 (CC1) is a transmembrane glycoprotein that undergoes extensive AS in response to oxidative stress. Both HIF-2α and CC1 play a role in liver transplantation (LT), though whether they function coordinately remains to be determined.
*Methods: Livers from floxed HIF-2α-deficient mice (C57BL6) were used in nuclear fractionation western blot and Immunohistochemistry (IHC) studies. Primary hepatocytes treated with siRNAs directed to HIF-2α were cultured in the presence or absence of hypoxia/reoxygenation (H/R) treatment. Isolated RNAs were subjected to RT-PCR using Exon-Junction primers to detect changes in AS isoforms.
*Results: Naïve HIF-2α-deficient livers showed extensive differences in autophagy signaling markers LC3B and P70S6K (*p=0.0489) and proinflammatory markers MCP1 (***, p=0.0003), CXCL2, and CXCL10. Importantly, we observed downregulation of the splicing isoform CC1, the short cytoplasmic tail (CC1-S), leading to the hypothesis that HIF-2α may control its AS. The RNA splicing factors hnRNP A1 (*p=0.0242) and PTBP1 (**p=0.0097), known AS factors of CC1, showed upregulation while novel proteins SRp20 (**p=0.0045), SRp55 (*p=0.021), and ASF/SF2, all showed significant downregulation in naïve HIF-2α-deficient livers. IHC staining of SRp55 showed dysregulated cytoplasmic localization suggesting HIF-2α may control global regulation of AS by controlling the function of RNA splicing proteins. To validate this hypothesis, mouse hepatocytes tested in -/+ siHIF-2α and -/+ H/R conditions showed downregulation of canonical HIF-2α gene product Epo, switching from a mRNA splice variant containing exons 1-6 in mock controls to a splice variant containing exons 2-5. To generalize these findings in vivo, we validated HIF-2α orchestrated the AS by inducing the exon skipping of variable exon 7 (generating CC1-S) and excluding the 5’UTR/exon 1 isoform present in PARP8 of ischemia-reperfusion (IRI) stressed livers.
*Conclusions: This study identifies a novel regulator of AS in IRI opening the possibility of identifying splice isoforms that play a specific role in attenuating hepatic IRI in OLT.

Kenneth Dery

UCLA

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