Department of Hepatopancreatobiliary Surgery, First Affiliated Hospital, Changhai Hospital, Naval Medical University, Shanghai, 200433, China.
Mol Biol Rep. 2022 May;49(5):3713-3720. doi: 10.1007/s11033-022-07211-9. Epub 2022 Feb 7.
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.
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.
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.
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.
胰腺导管腺癌（PDACs）是一种恶性疾病，也是最常见的胰腺癌类型。恶性细胞依赖于天冬酰胺（Asn）的摄取来生长。Asn 的合成是通过酶天冬酰胺合成酶（ASNS）进行的。有趣的是，ASNS 是无意义介导的 RNA 降解（NMD）的直接靶标。我们之前报道过，胰腺肿瘤中存在 NMD 主要因子 UPF1 突变。然而，NMD 与 ASNS 水平之间的关系尚不清楚。
我们通过定点诱变构建点突变。为了评估 NMD 的幅度，我们评估了外源性表达的野生型和突变型β-珠蛋白 mRNA 与 N39 等位基因的表达比，以及五个已知的 NMD 靶标。然后，通过小 RNA 干扰敲低细胞内内源性 UPF1 后，进行逆转录-聚合酶链反应（RT-PCR）、逆转录定量 PCR（RT-qPCR）和 Western blot 以确定 RNA 或蛋白质水平。
在一名原发性 PDAC 患者中发现 UPF1 转录本中的一个 RNA 编辑事件（c.3101 A > G），导致天冬酰胺（p.1034）突变为丝氨酸。编辑后的 UPF1 增加了降解 NMD 触发转录物的能力，例如带有 N39 等位基因的β-珠蛋白 mRNA 和 5 个已知的内源性 NMD 底物 mRNA，包括 ASNS。此外，由于 5'UTR 存在 uORFs，ASNS mRNA 受到 NMD 降解。与对照相比，在 PDAC 患者或在具有 c.3101 A > G 编辑的 UPF1 的细胞中，均发现内源性 ASNS RNA 减少和蛋白质表达水平增加。
在 PDAC 中发现的这种编辑后的 UPF1 导致 NMD 过度激活，与 ASNS 表达水平升高密切相关。ASNS 的靶向敲低可能会提高 PDAC 的抗肿瘤效力。
