PCLAF promotes neuroblastoma G1/S cell cycle progression via the E2F1/PTTG1 axis

Abstract

PCLAF (PCNA clamp-associated factor), also known as PAF15/ KIAA0101, is overexpressed in most human cancers and is a predominant regulator of tumor progression. However, its biological function in neuroblastoma remains unclear. PCLAF is extremely overexpressed in neuroblastoma and is associated with poor prognosis. Through the analysis of various data sets, we found that the high expression of PCLAF is positively correlated with increased stage and high risk of neuroblastoma. Most importantly, knocking down PCLAF could restrict the proliferation of neuroblastoma cells in vitro and in vitro. By analyzing RNA-seq data, we found that the enrichment of cell cycle-related pathway genes was most significant among the differentially expressed downregulated genes after reducing the expression of PCLAF. In addition, PCLAF accelerated the G1/S transition of the neuroblastoma cell cycle by activating the E2F1/PTTG1 signaling pathway. In this study, we reveal the mechanism by which PCLAF facilitates cell cycle progression and recommend that the PCLAF/E2F1/PTTG1 axis is a therapeutic target in neuroblastoma.

Fig. 1. Expression and roles of PCLAF in neuroblastoma.

(A) Representative pictures of distinct levels (0–4) of IHC staining of PCLAF and the proportions of five levels; (B, C) 10NB, 8GNB and 8GN tumor tissue proteins extracted for Western Blot to discover PCLAF expression, and Image J was used to quantify protein bands. The data in (C) were analyzed by Mann-Whitney U-test (n = 28); (D) Kaplan-Meier analysis of OS in TMA of 70 neuroblastoma samples based on PCLAF expression with the log-rank test P value indicated; (E–H) Kaplan-Meier analysis of OS and EFS for the SEQC data set (n = 498) and the NRC data set (n = 275), based on PCLAF expression with the log-rank test p value indicated; (I–K) PCLAF expression levels in stage (St) 1-4 S tumors was show in box plot. (*p < 0.05, ***p < 0.001and ****p < 0.0001, and bar graphs represent the mean ± SEM).

Fig. 2. PCLAF is highly expressed and promotes the proliferation of neuroblastoma cells.

(A) Western blotting examined the protein expression of PCLAF in 5 neuroblastoma cell lines; (B, C) Interference efficiency of 2 small interfering RNAs with different sequences on PCLAF in SK-N-BE (2) and SH-SY5Y cell lines, detected by qRT-PCR and Western blotting; (D) The in vitro proliferation function of PCLAF was evaluated by CCK-8; (E) SK-N-BE(2) and SH-SY5Y cell lines with PCLAF siRNA performed imaging analysis after 48 h; (F) The in vitro proliferation function of PCLAF was evaluated by flow cytometry EdU labeling detection; (G) Western blotting was used to detect cell proliferation-related proteins; (H) PCLAF knockdown led to cell apoptosis in a flow cytometric apoptosis assay; (I) Apoptosis related markers were detected by western blot in neuroblastoma cells. (*p < 0.05, **p < 0.01, ***p < 0.001and ****p < 0.0001, and bar graphs represent the mean ± SEM).

Fig. 3. PCLAF triggers pathways related to cell cycle regulation in RNA-seq data.

(A, B) Heatmap of Gene Ontology (GO) enriched terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched terms colored by p-values; (C) The volcanic map was drawn according to the gene distribution. The abscissa is log2FoldChange, and the ordinate is -log10(p value); (D) Cluster heat map of 6 samples. Red represents high expressed genes and green represents low expressed genes; (E) The GO enrichment analysis results of differentially expressed genes; (F) The KEGG enrichment analysis results of differentially expressed genes. Select the top 20 pathways with the smallest p value for display; (G, H) Flow cytometry was used to analyze the cell cycle of neuroblastoma cell lines. (*p < 0.05, **p < 0.01, ***p < 0.001and ****p < 0.0001, and bar graphs represent the mean ± SEM).

Fig. 4. PCLAF can regulate PTTG1 to promote G1/S cell cycle transition.

(A, B) RNA-seq analyzed the mRNA level of the cell cycle signaling pathway after PCLAF knockdown; (C) Western blotting examined the protein expression of PCLAF in 5 neuroblastoma cell lines; (D) The expression of PTTG1 and PCLAF in clinical tissues was measured by western blot; (E) Spearman correlation analysis of the relationship between PCLAF and PTTG1 in identical clinical tissues (r = 0.774, P < 0.01); (F, G) qRT-PCR and Western blot were used to detect the changes in PTTG1 mRNA and protein levels after interference PCLAF in SK-N-BE(2) and SH-SY5Y cell lines; (H, I) Interference efficiency of 3 small interfering RNAs with different sequences on PTTG1 in SK-N-BE (2) and SH-SY5Y cell lines, detected by qRT-PCR and Western blotting; (J, K) Flow cytometry was used to analyze the cell cycle of neuroblastoma cell lines. (*p < 0.05, **p < 0.01, ***p < 0.001and ****p < 0.0001, and bar graphs represent the mean ± SEM).

Fig. 5. PCLAF regulates PTTG1 by the transcription factor E2F1.

(A) Family analysis of transcription factors based on the RNA sequencing data; (B) Spearman analysis was used to detect the correlations between PCLAF and E2F1 and PTTG1 in the neuroblastoma database; (C, D) Prediction of the sequence logo of E2F1 and the binding sites of E2F1 on the PTTG1 promoter region were predicted by JASPAR; (E) Graphical representation of enrichment for E2F1 on the regulatory regions of PTTG1 in HeLa, K562, and LM2 cells. Red boxes show regions with E2F1 enrichment; (F) The mRNA level of PTTG1 after decreasing E2F1 by siE2F1 in SK-N-BE (2) and SH-SY5Y cell lines; (G) The protein levels of PTTG1 after decreasing E2F1 by siE2F1 in SK-N-BE (2) and SH-SY5Y cell lines; (H) The mRNA level of E2F1 after decreasing PCLAF by siPCLAF in SK-N-BE (2) and SH-SY5Y cell lines.; (I) The protein levels of E2F1 after decreasing PCLAF by siPCLAF in SK-N-BE (2) and SH-SY5Y cell lines. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, and bar graphs represent the mean ± SEM).

Fig. 6. PCLAF knockdown restrained the growth of neuroblastoma in vivo.

(A) The volume of tumors obtained from xenografts was diminished in the PCLAF-knockdown group by shRNA from the SK-N-BE (2) cell line; (B, C) The volume and weight of tumors formed by PCLAF knockdown cells were dramatically smaller than that of the control; (D) The expression of PCLAF, PTTG1 and CyclinD1 were examined by western blot in tumors obtained from xenografts; (E) H&E staining of subcutaneous tumors in BALB/c nude mice; (F, G) Immunohistochemical staining of subcutaneous tumors on target of PCLAF, PTTG1 andCyclinD1 separately. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, and bar graphs represent the mean ± SEM).