Journal: Journal of Hematology & Oncology

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

doi: 10.1186/s13045-022-01338-9

Figure Lengend Snippet: 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

Article Snippet: The sections were then incubated with specific primary antibodies against PCNA (1:200 dilution, ab15497, Abcam, Cambridge, UK), NAT10 (1:500 dilution, ab194297, Abcam), Vimentin (1:500 dilution, ab92547, Abcam), E-cadherin (1:500 dilution, 20874-1-AP, Proteintech), N-cadherin (1:100 dilution, ab76011, Abcam), LAMB3 (1:500 dilution, 26795-1-AP, Proteintech), PHGDH (1:500 dilution, 14719-1-AP, Proteintech) and KCNN4 (1:200 dilution, 23271-1-AP, Proteintech) at 4 °C overnight.

Techniques: Modification, Immunohistochemistry, Expressing, Control, RNA Sequencing, Functional Assay, Quantitative RT-PCR, Western Blot, Transfection