The LINC00623/NAT10 signaling axis promotes pancreatic cancer progression by remodeling ac4C modification of mRNA

Abstract

Background: Although a substantial increase in the survival of patients with other cancers has been observed in recent decades, pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest diseases. No effective screening approach exists.

Methods: Differential exosomal long noncoding RNAs (lncRNAs) isolated from the serum of patients with PDAC and healthy individuals were profiled to screen for potential markers in liquid biopsies. The functions of LINC00623 in PDAC cell proliferation, migration and invasion were confirmed through in vivo and in vitro assays. RNA pulldown, RNA immunoprecipitation (RIP) and coimmunoprecipitation (Co-IP) assays and rescue experiments were performed to explore the molecular mechanisms of the LINC00623/NAT10 signaling axis in PDAC progression.

Results: A novel lncRNA, LINC00623, was identified, and its diagnostic value was confirmed, as it could discriminate patients with PDAC from patients with benign pancreatic neoplasms and healthy individuals. Moreover, LINC00623 was shown to promote the tumorigenicity and migratory capacity of PDAC cells in vitro and in vivo. Mechanistically, LINC00623 bound to N-acetyltransferase 10 (NAT10) and blocked its ubiquitination-dependent degradation by recruiting the deubiquitinase USP39. As a key regulator of N4-acetylcytidine (ac4C) modification of mRNA, NAT10 was demonstrated to maintain the stability of oncogenic mRNAs and promote their translation efficiency through ac4C modification.

Conclusions: Our data revealed the role of LINC00623/NAT10 signaling axis in PDAC progression, showing that it is a potential biomarker and therapeutic target for PDAC.

Fig. 1

Exosomal LINC00623 is upregulated in PDAC and has clinical significance. a Workflow of screening for exosomal diagnostic biomarkers in the serum of patients with PDAC and healthy individuals. b Flowchart showing the process used to narrow down the upregulated exosomal lncRNAs in the serum of patients with PDAC compared to that of healthy individuals. c The levels of circulating exosomal LINC00623 were significantly higher in patients with PDAC than in healthy donors (HD) and patients with benign pancreatic neoplasms. Chronic pancreatitis (CP), intraductal papillary mucinous neoplasm (IPMN), mucinous cystic neoplasm (MCN), pancreatic neuroendocrine tumor (PNET), serous cystic neoplasm (SCN). Independent Student’s t test, P < 0.001. d Diagnostic AUC value of the circulating exosomal LINC00623 level. AUC1: PDAC (n = 73) vs. HD (n = 42); AUC2: PDAC with normal CA19-9 levels (n = 13) vs. HD (n = 42). e Relative expression of LINC00623 as measured by RT-qPCR in PDAC and matched adjacent normal tissues (n = 40). Independent Student’s t test, P < 0.001. f Representative images of FISH of LINC00623 (red) in 93 pairs of PDAC and adjacent normal tissues. DAPI (blue) was used for nuclear counterstaining. Scale bar = 20 μm. g The expression level of LINC00623 as determined by FISH in PDAC and adjacent normal tissues (n = 93). Independent Student’s t test, P < 0.001. h Kaplan‒Meier survival curve of two groups of patients with PDAC (n = 93): LINC00623(+), patients with high LINC00623 expression; LINC00623(−), patients with low LINC00623 expression. The expression of LINC00623 was determined by FISH. i Univariate Cox regression analyses of the survival of patients with PDAC (n = 93)

Fig. 2

LINC00623 enhanced the proliferation, migration and invasion abilities of PDAC cells in vitro and in vivo. a CCK-8 assays were used to determine the cell proliferation rates. b The results of quantitative analysis of foci numbers are summarized in the bar chart. c The relative migration distance of the indicated cells was determined in the wound healing assay. d The relative numbers of migrated and invaded cells in the indicated groups in the Transwell migration and invasion assays are summarized in the bar chart. Cells transfected with control plasmids were used as controls. a–d The values indicate the mean ± SD of three independent experiments. P values are shown as *P < 0.05, **P < 0.01, or *** P < 0.001. Independent Student’s t test. e The protein expression levels of E-cadherin, N-cadherin and Vimentin in BxPC-3 and PANC-1 cells were determined by Western blotting. GAPDH was used as the loading control. f Representative images of H&E staining and IHC staining of PDO1. PDO1 was transiently transfected with the LINC00623 shRNA or control plasmid. Antibodies against PCNA and Vimentin were used in the IHC assays. g Representative images of subcutaneous tumors formed by the indicated cells. h Tumor weights are expressed as the mean ± SD of six mice (*P < 0.05, independent Student’s t test). i Representative images of IHC staining with anti-PCNA antibody in tumors formed by the indicated cells. j Two in vivo metastasis assays were performed by tail vein injection (left) and splenic injection (right) to evaluate the effect of LINC00623 knockdown on tumor metastasis. Representative images of excised lungs and livers and of H&E staining and IHC staining with an anti-PCNA antibody in metastatic tumors formed by BxPC-3 cells. k The frequency of tumor metastasis in each group is summarized in the bar chart

Fig. 3

LINC00623 binds to NAT10. a GO-CC (cellular component) analysis predicting the potential proteins binding to LINC00623. b Western blotting was performed to evaluate the specific association of NAT10 with biotinylated LINC00623. Lysates of BxPC-3 and PANC-1 cells were harvested for RNA pulldown assays. LINC00623 antisense RNA was used as the negative control. c A RNA-binding protein immunoprecipitation (RIP) assay was performed using an antibody against NAT10. RT-PCR was used to detect LINC00623 enrichment. IgG was used as the isotype control. d Immunofluorescence (IF) staining showed that LINC00623 (red) was colocalized with NAT10 (green) in the nucleus. Scale bar = 10 μm. e Western blotting of NAT10 in samples precipitated by biotinylated full-length LINC00623 (FL) or LINC00623 truncations (Δ1: 1–699 bp; Δ2: 700–1200 bp; Δ3: 1201–1800 bp; Δ4: 1801–2500 bp; Δ5: 2501–3000 bp; Δ6: 3001–3607 bp). FL LINC00623 was used as the positive control. f Flag-RIP assay for LINC00623 showing its fold enrichment in cells transiently transfected with plasmids containing FLAG-tagged full-length and truncated NAT10 constructs (Del1: 1–280 aa; Del2: 281–488 aa; Del3: 558–753 aa; Del4: 753–1052 aa). IgG-RIP was used as the internal control. The values are expressed as the mean ± SD of three independent experiments

Fig. 4

LINC00623 blocks NAT10 ubiquitination and degradation. a The protein levels of NAT10 in BxPC-3 and PANC-1 cells. β-Actin was used as the loading control. b The protein levels of NAT10 in the indicated cells treated with cycloheximide (CHX, 50 μg/mL) for 0, 2, 4, 6, 8, and 10 h. c The protein levels of NAT10 in the indicated cells treated with or without MG132 (10 μM) for 6 h. d BxPC-3 cell lysates were immunoprecipitated (IP) with an anti-NAT10 antibody and immunoblotted with an anti-HA antibody. e IP was performed to detect the association between NAT10 and USP39 in BxPC-3 cells. d–e BxPC-3 cells were transiently transfected with LINC00623 knockdown, LINC00623 overexpression or control plasmids and then treated with MG132 for 4 h before cell lysates were harvested for co-IP with an anti-NAT10 (IP: NAT10) or anti-USP39 (IP: USP39) antibody. f The colocalization of NAT10 (green) and USP39 (red) in BxPC-3 cells was evaluated by IF. Scale bar = 2 μm (left). The percentage of colocalization (Mander’s coefficients) of NAT10 and USP39 is summarized in the bar charts (right). g The ubiquitination levels of NAT10 were determined by Western blotting. BxPC-3 cells were transiently transfected with USP39 silencing (left), USP39 overexpression (right) and control plasmids and then treated with MG132 for 4 h before cell lysates were harvested for co-IP with an anti-NAT10 antibody and immunoblotted with an anti-HA antibody. h The ubiquitination levels of NAT10 in the products of IP with an anti-Flag antibody in lysates from BxPC-3 cells transfected with LINC00623 knockdown or USP39 overexpression and control plasmids

Fig. 5

NAT10 functions as a downstream mediator of LINC00623by remodeling N4-acetylcytidine (ac4C) modification of mRNA. a Representative images of IHC staining with an anti-NAT10 antibody in PDAC, pancreatic intraepithelial neoplasia (PanIN) or matched adjacent normal pancreatic tissues (NP). b The mRNA level of LINC00623 was positively correlated with the protein level of NAT10 in PDAC tissues (n = 93). c Kaplan‒Meier survival curve of two groups of patients with PDAC (n = 93): NAT10 (+), patients with high NAT10 expression; NAT10 (-), patients with low NAT10 expression. The expression of NAT10 was determined by IHC staining. d Schematic diagram of the effects of NAT10 on the stability and translation efficiency of mRNA by catalyzing ac4C modification. e Metagene profile showing the distribution of NAT10 peaks across full-length transcripts containing the 5′UTR, CDS, and 3′UTR. BxPC-3 cell lysates were used for the NAT10 RIP assay. f Metagene profile showing the distribution of ac4C peaks across full-length transcripts containing the 5′UTR, CDS, and 3′UTR. BxPC-3 cell lysates were also used for acRIP. g Venn diagram showing the downstream target genes regulated by NAT10 via ac4C modification in BxPC-3 cells. Group 1: The gene set enriched in NAT10 RIP-seq (RIP-seq); Group 2: The set of target genes enriched in parental cells but not in NAT10-silenced cells according to acRIP-seq (acRIP-seq); Group 3: The genes upregulated or downregulated in NAT10-silenced cells compared with control cells (RNA-seq); Group 4: The mRNA transcripts displaying differences in translation efficiency in NAT10-silenced cells (Ribo-seq). h Functional annotation and pathway enrichment analysis of the predicted downstream target genes of NAT10 according to the Metascape database. i The relative mRNA levels of NAT10, KCNN4, LAMB3 and PHGDH were measured by RT-qPCR. j The protein levels of KCNN4, LAMB3 and PHGDH were determined by Western blotting. k RT-qPCR was used to detect the relative enrichment of KCNN4, LAMB3 and PHGDH mRNAs in NAT10 RIP products. l RT-qPCR was used to detect the relative enrichment of KCNN4, LAMB3 and PHGDH mRNAs in acRIP products. i–l BxPC-3 cells were transfected with NAT10 silencing and control plasmids. Cell lysates were harvested for RIP assays. IgG was used as the isotype control. The values indicate the mean ± SD of three independent experiments. P values are shown as *P < 0.05; **P < 0.01; ***P < 0.001. Independent Student’s t test

Fig. 6

LINC00623 is a potential therapeutic target for PDAC. a Graphical diagram of intratumoral injection of in vivo optimized LINC00623 inhibitor in PDX-bearing mice. b Representative images of xenograft tumors derived from PDX1 or PDX2 in each mouse group. Mice bearing xenograft tumors were treated with antisense oligonucleotide control (ASO-Ctrl) or an in vivo optimized LINC00623 inhibitor (ASO-LINC00623). Xenograft tumor growth was monitored by measuring the tumor volume every six days (c) and weighing the tumors (d). Tumor volume and weight are expressed as the mean ± SD of five mice (*P < 0.05, **P < 0.01; independent Student’s t test). e Representative images of H&E staining, IHC staining and FISH of xenograft tumors. Scale bar = 50 μm. f Schematic diagram of the effect of the LINC00623/NAT10 axis on the tumorigenicity and progression of PDAC