Abstract
Background
Pancreatic ductal adenocarcinomas (PDACs) is a malignant disorder and is the most common pancreatic cancer type. The malignant cells depend on the uptake of asparagine (Asn) for growth. The synthesis of Asn occurs through the enzyme asparagine synthetase (ASNS). Interestingly, ASNS is known as is direct target of nonsense-mediated RNA decay (NMD). We have previously reported that NMD major factor UPF1 mutations in the pancreatic tumors. However, the relationship between NMD and the level of ASNS is unknown.
Method
We constructed point mutations by site-specific mutagenesis. To evaluate NMD magnitude, we assessed the expression ratio of an exogenously expressed wild-type and mutated β-globin mRNA with N39 allele, and five known NMD targets. Then, reverse transcription-polymerase chain reaction (RT-PCR), RT-qPCR and western bolt to determine RNA or protein levels, after knockdown of endogenous UPF1 by small RNA interference in the cells.
Results
An RNA editing event (c.3101 A > G) at UPF1 transcripts resulting in an Asparagine (p.1034) changed to a Serine is found in one primary PDAC patient. The edited UPF1 increases the ability of degrading of NMD provoking transcripts, such as β-globin mRNA with N39 allele and 5 out of 5 known endogenous NMD substrate mRNAs, including ASNS. In addition, ASNS mRNA is subjected to NMD degradation by virtue of its possessing uORFs at the 5′UTR. A reduction of endogenous ASNS RNA and the increased protein expression level is found either in the PDAC patient or in the cells with edited UPF1 at c.3101 A > G relative to the controls.
Conclusions
This edited UPF1 found in the PDAC results in hyperactivated NMD, which is tightly correlation to elevated expression level of ASNS. The targeting of knockdown of ASNS may improve the antitumor potency in PDACs.
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All data generated or analyzed during this study are included in this article.
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Funding
This study was supported by the National Natural Science Foundation of China (No. 81871992).
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JH and ZW conception, design and analysis of data and performed the data analyses. SY contributed significantly to analysis and manuscript preparation. GL contributed to the conception of the clinic study. YL contributed to the whole study and wrote the manuscript.
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Hu, J., Wang, Z., Yang, S. et al. The edited UPF1 is correlated with elevated asparagine synthetase in pancreatic ductal adenocarcinomas. Mol Biol Rep 49, 3713–3720 (2022). https://doi.org/10.1007/s11033-022-07211-9
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DOI: https://doi.org/10.1007/s11033-022-07211-9