Loss of histone H4 lysine 20 trimethylation in osteosarcoma is associated with aberrant expression ofhistone methyltransferase SUV420H2

Abstract

Epigenetic modifications of histones have crucial roles in various types of cancers. The aberrant trimethylation of histone H4 at lysine 20 (H4K20) has been implicated in carcinogenesis. At present, the status of trimethylation at H4k20 (H4K20me3) in osteosarcoma (OS), the predominant bone cancer in humans, is unknown. In the present study, a genome-wide decrease was observed in H4K20me3 levels in OS tissues and cell lines. Reduced levels of lysine methyltransferase 5C (SUV420H2), the histone methyltranferase responsible for modification of H4K20me3, was also observed in OS cells with the associated loss of H4K20me3. Furthermore, a total of 507 SUV420H2-regulated genes were identified through RNA-seq and a number of candidate genes were further validated. Bioinformatic analysis revealed an association between SUV420H2 and multiple signaling pathway, including the mitogen-activated protein kinase, P53, transforming growth factor and the ErbB pathways. These results demonstrated that there are aberrant levels of H4K20me3 and SUV420H2 in OS, and highlighted H4K20me3 as a candidate biomarker for the early detection of OS.

Keywords: epigenetics; histone H4 lysine 20; histone methyltransferase; lysine methyltransferase 5C; osteosarcoma.

Figure 1.

Modifications to H4K20me3 in OS. (A) Representative samples of H4K20me3 immunostaining in tissue samples (400× magnification) from patients with OS, ranging from low (+1) to high (+3). H4K20me3 positive spots exhibited brown staining with a nuclear localization. (B) Levels of H4K20me3 were significantly lower in the OS samples than in normal bone tissue. (C) H4K20me3 levels were reduced in HOS, U2OS and MG-63 osteosarcoma cell lines compared to hFOB1.19 cells. Histone H4 and β-actin served as loading controls. Data are presented as the mean ± standard deviation of two independent experiments; *P<0.05 OS cell lines vs. hFOB1.19. ****P<0.0001. H4, histone H4; H4k20me3, trimethylation at histone H4 lysine 20; OS, osteosarcoma.

Figure 2.

Reduced expression levels of SUV420H2 are found in OS cell lines. (A) Reverse transcription-quantitative PCR of SUV420H1 (left) and SUV420H2 (right) in OS cell lines, compared with the normal control hFOB1.19 cell line. mRNA expression levels were normalized to GAPDH. Data are presented as the mean ± SEM of three independent experiments; *P<0.05 OS cell lines vs. hFOB1.19. (B) Expression of SUV420H2 protein in OS cell lines was analyzed by WB analysis using anti-SUV420H2 antibodies and β-actin served as a loading control. OS, osteosarcoma; SUV420H1, lysine methyltransferase 5B; SUV420H2, lysine methyltransferase 5C.

Figure 3.

Kaplan-Meier overall survival for patients with osteosarcoma in the (A) GSE21257 and (B) GSE42352 database based on SUV420H2 expression levels (low vs high expression level groups). SUV420H2, lysine methyltransferase 5C.

Figure 4.

Downstream genes of SUV420H2 were identified using RNA-sequencing. (A) Reverse transcription-quantitative PCR was used to examine the knockdown effect of SUV420H2 expression in HOS cells transfected with siRNA specific to SUV420H2 (si SUV420H2#1 and si SUV420H2#2) compared to control siRNA (siEGFP and siNC). Data are presented as the mean ± SEM of three independent experiments. (B) The knockdown efficiency of SUV420H2 in protein level was confirmed using western blotting. Levels of H4K20me3 were also investigated. Total histone H4 and β-actin were loaded as controls. (C) Heatmap of up and downregulated genes following depletion of SUV420H2. RNA sequence analyses were conducted on HOS cells treated with SUV420H2-specific siRNA (si SUV420H2#1) or EGFP control siRNA for 96 h. *P<0.05 vs. siEGFP. EGFP, enhanced green fluorescent protein; H4K20me3, trimethylation at histone H4 lysine20; si, small interfering RNA; SUV420H2, lysine methyltransferase 5C.

Figure 5.

GO and KEGG analyses of RNA-seq data. (A) Analysis of GO term enrichment of the biological process category of genes with increased (left) or decreased (right) expression after SUV420H2 depletion. The top 10 GO terms based on the P-value are plotted. (B) KEGG pathway enrichment analysis of up (left) or downregulated (right) genes identified in SUV420H2 deleted HOS cells. cGMP-PKG, cyclic GMP-dependent protein kinase; ErbB2, erb-b2 receptor protein kinase 2; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; SUV420H2, lysine methyltransferase 5C; TGF, transforming growth factor.

Figure 6.

Validation of RNA-sequencing results using RT-qPCR. Expression of selected SUV420H2-associated genes, including (A) upregulated and (B) downregulated genes upon SUV420H2 depletion, was validated in control (siEGFP) and SUV420H2-depleted (siSUV420H2#1 and siSUV420H2#2) HOS cells using RT-qPCR analysis. The relative mRNA expression levels (normalized to GAPDH) in siSUV420H2#1 and siSUV420H2#2 samples were compared to that in the control siEGFP sample. Data are presented as the mean ± SEM of three experimental repeats; *P<0.05, siSUV420H2 vs. siEGFP. RT-qPCR, reverse transcription-quantitative PCR; si, small interfering RNA; LAPTM5, lysosomal protein transmembrane 5; FOS, Fos proto-oncogene; PTX3, pentraxin 3; VEPH1, ventricular zone expressed PH domain containing 1; SEMA3C, semaphorin 3C; CSF2, colony stimulating factor 2; AMPD3, adenosine monophosphate deaminase 3; GDF15, growth differentiation factor 15; KDM6B, lysine demethylase 6B; PIK3IP1, phosphoinositide-3-kinase interacting protein 1; CNN2, calponin 2; BMF, Bcl2 modifying factor; TTYH3, tweety family member 3; SGK1, serum/glucocorticoid regulated kinase 1; EGLN2, egl-9 family hypoxia inducible factor 2; SMOC1, SPARC related modular calcium binding 1; JARID2, jumonji and AT-rich interaction domain containing 2; TOM1, target of myb1 membrane trafficking protein; BCORL1, BCL6 corepressor like 1; PLK1, polo-like kinase 1.