Prp19 Is an Independent Prognostic Marker and Promotes Neuroblastoma Metastasis by Regulating the Hippo-YAP Signaling Pathway

Abstract

Pre-mRNA processing factor 19 (Prp19) was previously reported to be involved in tumor progression. However, Prp19 expression and its functions remain elusive in neuroblastoma. Here, we aim to identify the functions and mechanisms of Prp19 in neuroblastoma. Neuroblastic tumor tissue microarrays and two independent validation data sets indicate that Prp19 is associated with high-risk markers and bone marrow metastasis and serves as a prognostic marker for worse clinical outcomes with neuroblastoma. Gain- and loss-of-expression assays reveal that Prp19 promotes invasion, migration, and epithelial-mesenchymal transition (EMT) of neuroblastoma cells in vitro. Bioinformatics analysis of RNA-seq data shows that the expressions of YAP and its downstream genes are significantly inhibited after downregulation of Prp19. Prp19 and YAP expression in metastatic lymph nodes is higher than in situ neuroblastoma tissue. Further experiments show that Prp19 regulates YAP expression and consequently affects cell invasion, migration, and EMT in neuroblastoma by pre-mRNA splicing of YAP. In conclusion, our findings provide the first evidence that Prp19 is a potential therapeutic target and prognostic biomarker for patients with neuroblastoma.

Keywords: Prp19; RNA splicing; YAP; metastasis; neuroblastoma.

Figure 1

Expression and roles of Prp19 in neuroblastoma. (A) Representative images of different levels (0–4) of IHC staining of Prp19 and the proportions of five levels. Scale bar: 100 μm. (B) Prp19 expression between NB/GNB-N and GNB-I in TMA. The data were analyzed by Mann-Whitney U-test (n = 62). (C,D) Proteins of 7 NB/GNB-N and 7 GNB-I tumor tissue proteins extracted for immunoblotting to detect Prp19 expression, and Image J was used to quantify protein bands. The data in (D) were analyzed by Mann-Whitney U-test (n = 14). *p < 0.05 and ***p < 0.001.

Figure 2

Prp19's prognostic value in TMA cohort and data sets. (A,D) Kaplan-Meier analysis of OS and EFS for the TMA cohort based on Prp19 expression with the log-rank test P-value indicated (n = 50). (B,E) Kaplan-Meier analysis of OS and EFS for the SEQC data set based on Prp19 expression with the log-rank test p value indicated (n = 498). (C,F) Kaplan-Meier analysis of OS and EFS for the NRC data set based on Prp19 expression with the log-rank test p-value indicated (n = 275).

Figure 3

Effects of Prp19 on cell biological behavior of neuroblastoma cells. (A) WB detected the expression levels of Prp19 in neuroblastoma cell lines SK-N-BE (2), SK-N-AS, SH-SY5Y, IMR-32, and LAN1, and two cell lines [SK-N-BE (2) and SK-N-AS] with higher expression of Prp19 were selected for subsequent experiments. (B) Prp19 was knocked down in SK-N-BE (2) and SK-N-AS using siRNA. The first siRNA was selected for subsequent experiments according to knockdown efficiency detected by WB. (C,D) Transwell array tested the invasive ability of SK-N-BE (2) and SK-N-AS between siPrp19 and siNC or between overexpression of Prp19 and control, and the number of cells crossing the bottom of the chamber were analyzed by GraphPad Prism 5. Scale bar: 200 μm p = 0.0022, respectively. (E,F) The cell migration ability was estimated using a wound-healing assay. The images were captured at indicated time after wounding (magnification: 50×; scale bar: 250 μm) p = 0.0022, respectively. (G,H) WB tested the expression of Prp19, E-cad, and MMP9 in Prp19 downregulation or overexpression cells. *p < 0.05 and **p < 0.01.

Figure 4

Bioinformatics analysis of RNA-seq data. SK-N-BE (2) cells were transfected with siPrp19 for 48 h, and then subjected to RNA sequencing. (A) Volcano plot showing distribution of differential expression genes Prp19 downregulation (|Fold of changes| >1.5, FDR <0.05). (B) Disease ontology (DO) analysis enriched the affected diseases after Prp19 knockdown, which were mainly concentrated in nervous system cancer. (C) KEGG enriched the affected pathways after Prp19 knockdown, indicating that the Hippo signaling pathway was suppressed. (D) PCR analyzed the YAP mRNA level after Prp19 knockdown in SK-N-BE (2) and SK-N-AS. (E) RNA-seq analyzed the mRNA level of YAP and its downstream genes after Prp19 knockdown. It indicated that YAP and its downstream genes decreased following Prp19 downregulation. (F) The protein level of YAP, cyclin D1, and CTGF after downregulation or overexpression of Prp19. *p < 0.05.

Figure 5

Effects of YAP on cell biological behavior of neuroblastoma cells. (A) Transwell array tested the invasive ability of SK-N-BE (2) and SK-N-AS between siYAP and siNC, and the number of cells crossing the bottom of the chamber were analyzed by GraphPad Prism 5. Scale bar: 100 μm p = 0.0079, respectively. (B) The cell migration ability was estimated using the wound-healing assay after YAP downregulation. The images were captured at the time shown on the graph after wounding (magnification: 50×; scale bar: 250 μm) p = 0.0022, respectively. (C) WB tested the expression of YAP, E-cad, and MMP9 after downregulation of YAP. (D) WB tested the expression of YAP, CTGF, cyclin D1, FGF1, and Prp19 after downregulation of YAP. *p < 0.05 and **p < 0.01.

Figure 6

Expression of Prp19 and YAP are higher in metastatic lymph nodes than primary sites. (A) The expression of Prp19 in metastatic lymph nodes of patients 1 and 2 is higher than that in primary tumor. (B) The expression of Prp19 in metastatic lymph nodes of patients 1 and 2 is higher than that in primary tumor. The average intDen in each group was analyzed by Mann-Whitney U-test (n = 6 per group). *p < 0.05 and **p < 0.01.

Figure 7

Prp19 is required for efficient intron removal of YAP. (A) A schematic diagram of exons and introns of the YAP gene and the primer sets designed for PCR as well as the RT-PCR shown in (B,C). (B) PCR analysis using primer sets shown in (A) to compare splicing efficiencies of YAP introns splicing in Prp19 siRNA-transfected and Prp19 overexpression cells. (C) qPCR analysis using primer sets shown in (A) to compare splicing efficiencies of YAP introns splicing in Prp19 siRNA-transfected and Prp19 overexpression cells. *p < 0.05.