Abstract
BackgroundPreviously, a novel nested gene resided in intron 6 of Aff3, named as Aff3ir temporarily, was identified during the stem/progenitor cells (SPCs) differentiation towards endothelial cells (ECs). A 151 amino acid protein derived from Aff3ir open reading frame 2 (AFF3IR-ORF2) was found upregulated in heart and coronary vessels after transverse aortic constriction in mouse. AFF3IR-ORF2 shifted state of SPCs from proliferation to differentiation by retaining MCM3 in cytosol. It also protected ECs from H2O2-induced cell apoptosis, but the mechanism was unclear. The Aff3ir global knockout (HOM) mice were generated by deletion of the promoter region and the first three exons. Preliminary phenotype studies observed endogenous H2O2 increased in HOM heart.
Hypothesis and aims
The nested gene Aff3ir derived AFF3IR-ORF2 plays an important role in cardiovascular system via maintenance of redox homeostasis in endothelium. In this project, we aim to investigate the potential mechanisms involved in the protective role of AFF3IR-ORF2 in H2O2-induced ECs apoptosis in vitro and to study the phenotype of Aff3ir global knockout mice in vivo.
Methods and results
By retaining MCM3 in cytosol, AFF3IR-ORF2 might activate Keap1/Nrf2/ARE pathway. In mouse C166 ECs, overexpression of AFF3IR-ORF2 was demonstrated to further up-regulate expression of antioxidant protein haem oxygenase 1 (HO-1) under H2O2 treatment. The translocation of Nrf2 into nucleus induced by overexpressed AFF3IR-ORF2 was verified by immunofluorescence (IF) and western blot (WB). However, knockdown of Nrf2 could only partially block AFF3IR-ORF2 enhanced upregulation of HO-1. The direct interaction of AFF3IR-ORF2 with the promoter region of Hmox1 was confirmed by Chromatin immunoprecipitation (ChIP).
In vivo, the phenotype study was performed among WT, heterozygous (HET) and homozygous (HOM) mice. The breeding study showed high mortality rate in HOM×HOM offspring. Post-mortem examination revealed cardiopulmonary failure signed as enlarged right atrium, patent ductus arteriosus and dilated pulmonary vessels in dead HOM pups. The mortality rate of breast-feeding HOM mothers (5 out of 9) was unexpectedly higher too. Post-mortem examination revealed haemorrhages in lung and intestine.
During phenotype study, an unusual genotyping pattern was noticed that the deleted fragment was still detectable in the ear-clip DNA samples of some HOM pups.
Accordingly, the hypothesis that Aff3ir might be present in extrachromosomal DNA (ecDNA) was proposed. Preliminary fluorescence in situ hybridisation (FISH) was performed but the experimental conditions still needed to be optimized. In live HOM mice, the expressing pattern of AFF3IR-ORF2 was the same with that in WT. While in dead HOM pups, very few AFF3IR-ORF2 proteins were detected in liver, kidney and
brain.
Conclusion
The nested gene Aff3ir plays critical roles in mice survival, partially via its encoded protein AFF3IR-ORF2 in the maintenance of redox homeostasis.
| Date of Award | 1 Jun 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Lingfang Zeng (Supervisor) & Min Zhang (Supervisor) |