NOP2 facilitates EZH2-mediated epithelial-mesenchymal transition by enhancing EZH2 mRNA stability via m5C methylation in lung cancer progression

Abstract

NOP2, a member of the NOL1/NOP2/SUN domain (NSUN) family, is responsible for catalyzing the posttranscriptional modification of RNA through 5-methylcytosine (m5C). Dysregulation of m5C modification has been linked to the pathogenesis of various malignant tumors. Herein, we investigated the expression of NOP2 in lung adenocarcinoma (LUAD) tissues and cells, and found that it was significantly upregulated. Moreover, lentivirus-mediated overexpression of NOP2 in vitro resulted in enhanced migration and invasion capabilities of lung cancer cells, while in vivo experiments demonstrated its ability to promote the growth and metastasis of xenograft tumors. In contrast, knockdown of NOP2 effectively inhibited the growth and metastasis of lung cancer cells. RNA-sequencing was conducted to ascertain the downstream targets of NOP2, and the findings revealed a significant upregulation in EZH2 mRNA expression upon overexpression of NOP2. Subsequent validation experiments demonstrated that NOP2 exerted an m5C-dependent influence on the stability of EZH2 mRNA. Additionally, our investigations revealed a co-regulatory relationship between NOP2 and the m5C reader protein ALYREF in modulating the stability of EZH2 mRNA. Notably, the NOP2/EZH2 axis facilitated the malignant phenotype of lung cancer cells by inducing epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Mechanistically, ChIP analysis proved that EZH2 counteracted the impact of NOP2 on the occupancy capacity of EZH2 and H3K27me3 in the promoter regions of E-cadherin, a gene crucial for regulating EMT. In a word, our research highlights the significant role of NOP2 in LUAD and offers novel mechanistic insights into the NOP2/ALYREF/EZH2 axis, which holds promise as a potential target for lung cancer therapy.

Fig. 1. NOP2 is upregulated in lung cancer tissues and predicts poor prognosis.

A TIMER 2.0 database was used for pan‐cancer analysis of NOP2. B, C Differential analysis of NOP2 expression in LUAD and normal tissues. D, E Kaplan–Meier analysis and receiver operating characteristic curve of NOP2. F The heat map of the relationship between NOP2 expression and different clinicopathologic features of LUAD. G, H Forest plots of cox regression analysis of the relationship between NOP2 expression and different clinicopathologic features of LUAD. I Representative IHC staining images and J IHC scores of NOP2 expression in paired LUAD tissues and adjacent non-tumor tissues. Scale bar = 50 μm. K Western blot for NOP2 expression in several specimens from LUAD patients. L, M RT-qPCR and western blot were used to compare the difference in NOP2 expression in lung cancer cells and normal human lung epithelial cells. Statistical methods: independent samples t-test (J), one-way ANOVA (L). Data are presented as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. NOP2 promotes migration and invasion of lung cancer cells.

A, B RT-qPCR was performed to verify the overexpression and knockdown efficiency of NOP2. C, D Western blot was performed to verify the overexpression and knockdown efficiency of NOP2. E–H The migration ability of lung cancer cells was determined by wound-healing assays. Scale bar = 100μm. I–L The invasion ability of lung cancer cells was shown by transwell assays. Scale bar = 100μm. Statistical methods: independent samples t-test (A, E, F, I, J), one-way ANOVA (B, G, H, K, L). Data are shown as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. NOP2 does not affect the proliferation, cell cycle and apoptosis of lung cancer cells.

A–D The effect of NOP2 on the proliferation of lung cancer cells was detected in CCK‐8 proliferation assay. E–H The effect of NOP2 on the proliferation of H1650 and H1299 cells was detected in EdU (Scale bar = 100μm) and colony-formation assays. I–L Cell cycle and apoptosis of H358 and A549 cells were detected by flow cytometry. Statistical methods: independent samples t-test (A, B, F, G, I, J), one-way ANOVA (C, D, F, H, K, L). Data are presented as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. EZH2 is a downstream gene of NOP2 in lung cancer cells.

A, D Volcano plot and heat map of RNA-sequencing showing the genes whose expression level was positively or negatively correlated with the expression level of NOP2. B, C GO and KEGG analysis for differential genes obtained by RNA-sequencing. E RT-qPCR was performed to detect mRNA levels of five differential genes associated with migration and invasion. F STRING database analysis of the protein interaction relationship. The types of connection represented by the different colored edges between the nodes are indicated in the figure. EZH2 expression upon NOP2 knockdown or overexpression were determined by G, H RT-qPCR and I, J western blot. K ENCORI database analysis of the correlation between NOP2 and EZH2 expression in LUAD. L Differential analysis of EZH2 expression in LUAD and normal tissues. M Kaplan–Meier analysis of EZH2. N The heat map of the relationship between EZH2 expression and different clinicopathologic features of LUAD. Statistical methods: independent samples t-test (E, G), one-way ANOVA (H). Data are shown as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. “Writer” NOP2 and “Reader” ALYREF promote the stability of EZH2 mRNA via m5C.

A, I The results of RIP and RT-qPCR in H1650 and H1299 cells. IgG was used as a negative control to preclude nonspecific binding. B The results of methylated-RIP for H1650 and H1299 cells. The relative m5 C enrichment of EZH2 mRNA for each group was normalized to the Input. C EZH2 5’UTR containing either wild-type or mutant (C-to-A mutation) m5C sites was cloned into luciferase reporter vector. D Relative luciferase activity of the wild-type and mutant form of EZH2 5’UTR reporter vectors in H1299 cells transfected with sh-control or sh-NOP2, respectively. E, F The relative expression of EZH2 mRNA was detected after treating H1650 and H1299 cells with 5 μg/mL actinomycin D for indicated times. J, K Do the same. G H1299 cells in the control group and NOP2 overexpression group were treated with 100 μg/mL CHX for the indicated times, and protein expression of EZH2 was analyzed by western blot analysis. H H1299 cells transfected with sh-control or sh-NOP2 were treated with 200 ng/mL puromycin for the indicated time and the whole cell lysates were detected by western blot. L The expression level of EZH2 protein in A549 and H1299 cells of each group. M, N The results of the transwell assays. Scale bar = 100μm. Statistical methods: independent samples t-test (A, B, D, E, F, I, J, K), one-way ANOVA (N). Data are presented as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. NOP2 promotes EMT via regulating EZH2 in lung cancer cells.

A The morphology of A549 and H1299 cells was observed using a microscope. Scale bar = 50 μm. B, C Protein expression levels in lung cancer cells were detected by western blot. D The effect of NOP2 on the expression of different molecules in H1650 and H1299 cells was detected by immunofluorescence. Scale bar = 50 μm. E, F The expression levels of the relevant proteins in H358 and H1650 cells were detected by western blot.

Fig. 7. EZH2 deficiency can counterbalance the positive effect of NOP2 on lung cancer cells in vitro.

A RT-qPCR was performed to detect the expression level of E-cadherin mRNA in each group of H1299 cells. B, C Binding of EZH2 and H3K27me3 antibodies to the E-cadherin promoter was detected by ChIP assay in H1299 cells. D–F Set up opposite NOP2 and EZH2 expression groups and do the same experiments in H1650 cells. Transwell (G–J) and wound-healing assays (K–N) were performed to detect the migratory and invasive abilities of lung cancer cells. Scale bar = 100 μm. Statistical methods: one-way ANOVA (A–F, H, J, L, N). Data are presented as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 8. EZH2 deficiency represses the positive effect of NOP2 on lung cancer progression in vivo.

A–C Tumor growth and weight were monitored in mice. D, E IHC staining of tumor sections and western blot results of tumor proteins. Scale bar = 50 μm. F Images of lungs in an in vivo mouse metastatic tumor model and H&E staining images of lung sections. Scale bar = 100 μm. G Lung metastases in all mice and the number of nodules were counted. H Schematic summarizing the NOP2/EZH2/EMT axis and its role in modulating lung cancer progression. Statistical methods: one-way ANOVA (B, C, G). Data are presented as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.