The RNA-binding protein PCBP1 represses lung adenocarcinoma progression by stabilizing DKK1 mRNA and subsequently downregulating β-catenin

Abstract

Background: PolyC-RNA-binding protein 1 (PCBP1) functions as a tumour suppressor and RNA regulator that is downregulated in human cancers. Here, we aimed to reveal the biological function of PCBP1 in lung adenocarcinoma (LUAD).

Methods: First, PCBP1 was identified as an important biomarker that maintains LUAD through The Cancer Genome Atlas (TCGA) project screening and confirmed by immunohistochemistry and qPCR. Via colony formation, CCK8, IncuCyte cell proliferation, wound healing and Transwell assays, we confirmed that PCBP1 was closely related to the proliferation and migration of LUAD cells. The downstream gene DKK1 was discovered by RNA sequencing of PCBP1 knockdown cells. The underlying mechanisms were further investigated using western blot, qPCR, RIP, RNA pulldown and mRNA stability assays.

Results: We demonstrate that PCBP1 is downregulated in LUAD tumour tissues. The reduction in PCBP1 promotes the proliferation, migration and invasion of LUAD in vitro and in vivo. Mechanistically, the RNA-binding protein PCBP1 represses LUAD by stabilizing DKK1 mRNA. Subsequently, decreased expression of the DKK1 protein relieves the inhibitory effect on the Wnt/β-catenin signalling pathway. Taken together, these results show that PCBP1 acts as a tumour suppressor gene, inhibiting the tumorigenesis of LUAD.

Conclusions: We found that PCBP1 inhibits LUAD development by upregulating DKK1 to inactivate the Wnt/β-catenin pathway. Our findings highlight the potential of PCBP1 as a promising therapeutic target.

Keywords: DKK1; LUAD; PCBP1; Wnt signalling pathway.

Fig. 1

Identification of the prognostic and potential biological value of PCBP1. A Heatmap of differentially expressed genes between cancer and adjacent tissues in LUAD patients and the early death group and long-term survival group. B Kaplan–Meier survival curves of overall survival between high PCBP1 expression and low PCBP1 expression patients from TCGA and GSE19188. C PCBP1 expression between patients with distant metastasis and patients without metastasis. D PCBP1 expression in LUAD patients with different TNM stages. E IHC staining of human LUAD tissues and paired adjacent normal tissues; scale bars, 100 μm. F, G Comparison of PCBP1 detected by IHC staining (F) and qPCR (G) between tumour and peritumoral tissues and patients with different tumour sizes, N staging and tumour stages. H Kaplan–Meier analysis of overall survival was stratified by protein expression levels of PCBP1. I Kaplan–Meier analysis of overall survival was stratified by mRNA expression levels of PCBP1. *p < 0.05; **p < 0.01; ns, not significant

Fig. 2

The effect of PCBP1 on the biological behaviour of LUAD in vitro. A shPCBP1 A549 cell colony formation ability. B shPCBP1 H358 cell colony formation ability. C PCBP1-OE A549 cell colony formation ability. D–F The proliferation rates of shPCBP1 A549 (D), shPCBP1 H358 (E), and PCBP1-OE A549 (F) cells were measured by IncuCyte ZOOM™. G–I Estimating the cell migration ability in shPCBP1 A549 (G), shPCBP1 H358 (H), and PCBP1-OE A549 (I) cells was performed by the IncuCyte™ Wound Healing assay. *p < 0.05; **p < 0.01; ns, not significant

Fig. 3

The expression of PCBP1 with respect to metastasis. A Volcano plot of DEGs in RNA-seq. B GO analysis showing the top gene functions that were mostly different among the PCBP1 high and low expression groups based on RNA-seq. C Heatmap showing the EMT-related DEGs identified by RNA-seq. D KEGG pathway enrichment analysis of DEGs. E The heatmap shows differential genes in WNT signalling pathways in A549 RNA‐seq data. F GSEA validated the enhanced activity of “Epithelial-Mesenchymal Transition”. G Validation of 22 selected genes by qPCR in PCBP1 knockdown and overexpression cells

Fig. 4

PCBP1 mediates DKK1 mRNA stability. A Graph based on the above results showing that DKK1 was the key gene. B qPCR analysis of the levels of DKK1 in PCBP1 knockdown and overexpression cells. C Western blot analysis of the levels of DKK1 in PCBP1 knockdown and overexpression cells. D The interaction of PCBP1 with DKK1 was examined by RIP-qPCR in A549 cells. E The interaction of PCBP1 with DKK1 was assessed by RNA pull-down assays followed by western blot analysis and silver staining. F mRNA expression of PCBP1-overexpressing cells and control cells treated with actinomycin D was examined by qPCR. *p < 0.05; **p < 0.01; ns, not significant

Fig. 5

PCBP1/DKK1/β-catenin regulates migration and EMT in LUAD cells. A Western blotting analysis of the expression of β-catenin, phosphor-β-catenin, Vimentin, Claudin-1, PCBP1 and Tubulin. B shPCBP1 A549 cell migration was estimated by Transwell assays. C The knockdown efficiency of DKK1 in A549 cells was determined by RT–qPCR analyses. D Western blot assays were used to examine the expression levels of DKK1, β-catenin, phospho-β-catenin, Vimentin, Claudin-1, PCBP1 and Tubulin in A549 control, A549 PCBP1-OE, A549 PCBP1-OE + siDKK1#1, and A549 PCBP1-OE + siDKK1#2 cells. (E) Cell migration was determined by Transwell assay in each group. *p < 0.05; **p < 0.01; ns, not significant

Fig. 6

PCBP1 inhibits tumour growth and metastasis in LUAD in vivo. A Body weight of the mouse A549 cell lung metastasis model. B Representative macroscopic lung images upon necropsy of mice with postimplant shPCBP1 and control A549 cells and HE images. C Quantification of lung metastases in mice bearing either shPCBP1 or control tumours. D Body weight of the mouse H358 cell lung metastasis model. E Representative macroscopic lung images upon necropsy of mice with postimplant shPCBP1 and control H358 cells and HE images. F The number of nodules per lung was quantified by HE staining. G, H A549 and H358 cells stably transfected with control and shPCBP1 were injected subcutaneously into nude mice. Tumour growth curves were plotted. I A549 cells stably transfected with control and PCBP1 were injected subcutaneously into nude mice. Tumour growth curves were plotted. (J) The weights of the excised tumours were measured. *p < 0.05; **p < 0.01; ns, not significant

Fig. 7

High levels of PCBP1 and DKK1 expression in LUAD predict good clinical outcome. A Representative images of DKK1 IHC staining; scale bars, 100 μm. The expression of DKK1 in peritumor and tumour tissues evaluated by immunohistochemistry. B Representative images of β-catenin IHC staining; scale bars, 100 μm. The expression of β-catenin in peritumor and tumour tissues evaluated by immunohistochemistry. C Kaplan–Meier analysis of overall survival was stratified by the protein expression levels of DKK1 and β-catenin. D Comparison of DKK1 and β-catenin protein expression detected by IHC between patients with different tumour sizes, N stages and tumour stages. E Representative images of PCBP1, DKK1 and β-catenin IHC staining; scale bars, 100 μm. F The correlation between PCBP1, DKK1and β-catenin at the protein level. G Correlation analysis of PCBP1 and DKK1 in TCGA and GSE19188. H Graphical summary of the PCBP1-DKK1- β-catenin axis regulating the proliferation and migration of lung cancer cells. *p < 0.05; **p < 0.01; ns, not significant