Fig 2: Low expression of DNAJA4 indicates poor prognosis in NPC patients.A Representative images of IHC staining of DNAJA4 in 212 NPC tissues. The staining of the images was graded as negative, weak, moderate, or strong based on the staining intensity. Scale bars, 100 μm and 20 mm. B Correlations of distant metastasis status with the level of DNAJA4 determined by IHC staining. The p-values were calculated using the chi-square test. C–E Kaplan-Meier curves of relapse-free survival (C), distant metastasis-free survival (D) and overall survival (E) based on DNAJA4 protein level. The p-values were determined using the log-rank test. F–H Forest plots of multivariate Cox regression analyses showing the significance of different clinical prognostic factors for NPC relapse-free survival (F), distant metastasis-free survival (G) and overall survival (H). I The regulatory model of DNAJA4 in NPC. When promoter hypermethylation of DNAJA4 causes its downregulation in NPC cells, the expression of MYH9 is not inhibited, and MYH9 then facilitates the migration and invasion of NPC cells by activating EMT. After exogenous overexpression of DNAJA4, DNAJA4 recruits PSMD2 to promote the protein degradation of MYH9 in a ubiquitination-dependent manner, and finally, the migration and invasion mediated by EMT are blocked.

Fig 3: DNAJA4 hypermethylation causes its downregulation in NPC.A Features of the DNAJA4 gene observed using the UCSC Genome Browser and a schematic diagram of the CpG island sites in the DNAJA4 promoter region. B Methylation levels of DNAJA4 in NPC (n = 24) and normal nasopharynx tissues (n = 24) in the methylation microarray dataset GSE52068; C Methylation levels of DNAJA4 in NPC (n = 25) and normal nasopharynx tissues (n = 25) in the Hong Kong methylation microarray dataset GSE62336. D The methylation rate of DNAJA4 cg16358679 in NPC (n = 6) and normal nasopharynx tissues (n = 6) based on pyrophosphate sequencing. E Relative mRNA levels of DNAJA4 in NPC (n = 15) and normal nasopharynx tissues (n = 30). F Relative protein levels of DNAJA4 in NPC (n = 5) and normal nasopharynx tissues (n = 5). G Relative mRNA levels of DNAJA4 in NPC cell lines and the normal nasopharyngeal epithelial cell line NP69. H Relative protein levels of DNAJA4 in NPC cell lines and the normal nasopharyngeal epithelial cell line NP69. I Relative mRNA levels of DNAJA4 in NPC cell lines and NP69 cells treated with or without the methyltransferase inhibitor DAC. The data in (D, E, G, I) are shown as mean ± SD, and p-values were determined by Student’s t test (*p < 0.05).

Fig 4: DNAJA4 inhibits NPC invasion and metastasis in vivo.SUNE1 cells stably overexpressing DNAJA4 or empty vector were subcutaneously injected into the plantar surface of mice to establish the inguinal lymph node metastasis model. A, B Representative images of foot pad tumours (A) and inguinal lymph nodes (B) in the two groups. C Statistical comparison of lymph node volume between the two groups. D H&E staining of foot pad tumours to analyse the infiltration of cancer cells into mouse muscle and skin. Scale bar, 100 μm. E The infiltration of cancer cells into lymph nodes was evaluated using IHC analysis of pan-cytokeratin. Scale bars, 2 mm and 20 μm. F Statistical analysis of inguinal lymph node metastasis in the two groups. SUNE1 cells stably overexpressing DNAJA4 or empty vector were injected into the tail veins of mice to establish the lung metastatic colonization model. G Representative images of macroscopic tumour nodules formed on the lung surface in the two groups. H H&E staining of the lung tissues to analyse the number and size of metastatic nodules. Scale bars, 5 mm, 2 mm and 20 μm. I Statistical analysis of lung metastatic nodules in the two groups. J Representative images and quantitative analysis of IHC staining of DNAJA4, PSMD2, and MYH9 in transplanted tumour tissues of the two groups. Scale bars, 100 μm and 50 μm. The data in (C, I, J) are shown as the mean ± SD, and p-values were determined by Student’s t test (ns, not significant; *p < 0.05).

Fig 5: Overexpression of DNAJA4 suppresses NPC cell migration, invasion and EMT.A In GSEA of public RNA-seq data (GSE102349), the gene sets related to metastasis and epithelial-mesenchymal transition (EMT) pathways exhibited high enrichment in NPC samples with low DNAJA4 expression. B Wound healing assay of HONE1 and SUNE1 cells transfected with the DNAJA4 overexpression or empty plasmid. C Transwell migration and invasion assays of HONE1 and SUNE1 cells transfected with the DNAJA4 overexpression or empty plasmid. D Representative western blot analysis of the epithelial marker E-cadherin and mesenchymal markers Vimentin, Slug, and Snail in HONE1 and SUNE1 cells transfected with the DNAJA4 overexpression or empty plasmid. E Representative immunofluorescence images of the epithelial marker E-cadherin and mesenchymal marker Vimentin in HONE1 cells transfected with the DNAJA4 overexpression or empty plasmids. Scale bar, 50 μm. The data in (B, C, E) are shown as mean ± SD, and p-values were determined by Student’s t test (*p < 0.05).