Acetyltransferase NAT10 regulates the Wnt/β-catenin signaling pathway to promote colorectal cancer progression via ac4C acetylation of KIF23 mRNA

Abstract

Background N4-acetylcytidine (ac4C) as a significant RNA modification has been reported to maintain the stability of mRNA and to regulate the translation process. However, the roles of both ac4C and its ‘writer’ protein N-acetyltransferase 10 (NAT10) played in the disease especially colorectal cancer (CRC) are unclear. At this point, we discover the underlying mechanism of NAT10 modulating the progression of CRC via mRNA ac4C modification. Methods The clinical significance of NAT10 was explored based on the TCGA and GEO data sets and the 80 CRC patients cohort of our hospital. qRT-PCR, dot blot, WB, and IHC were performed to detect the level of NAT10 and ac4C modification in CRC tissues and matched adjacent tissues. CCK-8, colony formation, transwell assay, mouse xenograft, and other in vivo and in vitro experiments were conducted to probe the biological functions of NAT10. The potential mechanisms of NAT10 in CRC were clarified by RNA-seq, RIP-seq, acRIP-seq, luciferase reporter assays, etc. Results The levels of NAT10 and ac4C modification were significantly upregulated. Also, the high expression of NAT10 had important clinical values like poor prognosis, lymph node metastasis, distant metastasis, etc. Furthermore, the in vitro experiments showed that NAT10 could inhibit apoptosis and enhance the proliferation, migration, and invasion of CRC cells and also arrest them in the G2/M phase. The in vivo experiments discovered that NAT10 could promote tumor growth and liver/lung metastasis. In terms of mechanism, NAT10 could mediate the stability of KIF23 mRNA by binding to its mRNA 3’UTR region and up-regulating its mRNA ac4c modification. And then the protein level of KIF23 was elevated to activate the Wnt/β-catenin pathway and more β-catenin was transported into the nucleus which led to the CRC progression. Besides, the inhibitor of NAT10, remodelin, was applied in vitro and vivo which showed an inhibitory effect on the CRC cells. Conclusions NAT10 promotes the CRC progression through the NAT10/KIF23/GSK-3β/β-catenin axis and its expression is mediated by GSK-3β which forms a feedback loop. Our findings provide a potential prognosis or therapeutic target for CRC and remodelin deserves more attention. Graphical Abstract

Antibody-Free Fluorine-Assisted Metabolic Sequencing of RNA N4-Acetylcytidine

“…Notably, when cells were cultured with an inhibitor of NAT10, Remodelin (10 μM), 10,38,39 which has been applied to inhibit ac 4 C modification in vitro and vivo, 40,41 the Remodelin not only inhibited NAT10 but also increased sensitivity to cisplatin in bladder cancer and mouse xenografts. 41 As shown in Figure 3B, after cell treatment with the addition of Remodelin for 24 h, the Fac 4 C modification on RNA was notably weakened.…”

NAT10 Is Involved in Cardiac Remodeling Through ac4C-Mediated Transcriptomic Regulation

“…Our data also demonstrated that the preventive role of Remodelin is equivalent to adeno-associated virus serotype 9-NAT10 inhibition in Ang II-induced cardiac remodeling mice. Studies have reported Remodelin as a potential cancer drug therapeutic agent, 43,44 but its structural NAT10 binding information is not yet available. 45,46 After sufficient overexpression of NAT10 in mouse hearts, Remodelin failed to show its preventive effects after TAC challenge, indicating that Remodelin acts by inhibiting NAT10 (Figure S10F).…”

NAT10‐mediated ac4C modification promotes ectoderm differentiation of human embryonic stem cells via acetylating NR2F1 mRNA

“…RNA dot blotting was conducted using an anti‐ac 4 C antibody, as previously described, with few adjustments 14,17 . Briefly, TRIzol was used to separate the RNA from cells.…”

“…De novo motif analysis identified the typical CXXCXXCX ac 4 C sequence motif previously reported. 10,17 The results showed the CXXCXXCX ac 4 C motif was conservative among hESCs, NEP and ME cells (Figure 1E). Moreover, consistent with the results of previous studies, 18 the largest fraction of ac 4 C sites was within the coding sequence area (45%-49%), while the 5 0 -UTR (12%-13%) and 3 0 -UTR (2%-4%) regions of mRNAs were also enriched (Figure 1F).…”

“…RNA dot blotting was conducted using an anti-ac 4 C antibody, as previously described, with few adjustments. 14,17 Briefly, TRIzol was used to separate the RNA from cells. The Dynabeads™ mRNA Purification Kit was used to purify the mRNA and RNA was placed in denaturation solution (20X SSC solution: RNA = 1:1) at 95 C for 5 min, then quickly placed on ice for 2 min.…”