Transcriptomics-Based Investigation of Molecular Mechanisms Underlying Apoptosis Induced by ZnO Nanoparticles in Human Diffuse Large B-Cell Lymphoma

Figures & data

Figure 1 Evaluation of the cytotoxicity of ZnO NPs against DLBCL cells and normal cells. The viability of DLBCL cells (A) and PBMCs (B) following treatment with different concentrations of ZnO NPs for 120 hours was determined using MTT assay. The type of cellular death caused by ZnO NPs (30 μg/mL) in DLBCL cells (C) was identified using annexin V-FITC and propidium iodide (PI) with flow cytometer. The data of flow cytometer were presented using column graph (D). ZnO NPs-induced production of ROS was measured in DLBCL cells using DCFH-DA (E). All experiments were performed in triplicate. Asterisk symbol was used to define p values; p ≤ 0.05 is *p ≤ 0.01 is **p ≤ 0.001 is*** and p ≤ 0.0001 is****.

Abbreviation: PBMCs, peripheral blood mononuclear cells.

Figure 2 ZnO NPs induced significant changes in the transcriptomic profile of DLBCL cells. Volcano plot demonstrates differentially expressed genes (DEGs: FC≥3; p≤0.008; corrected p≤0.05) in the ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells (A). Hierarchical cluster analysis with heatmap presentation were done for the DEGs (B). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Table 1 Biological Processes and Pathways Enrichment Analysis of the Differentially Expressed Genes (DEGs: FC≥3; P≤0.008; Corrected P≤0.05) in the ZnO NPs-Treated DLBCL Cells Compared with the Untreated DLBCL Cells

GO or Pathway Accession No	GO or Pathway Term	Reg	Corr. p value	Count in Selection	Count in Total
GO:0097501	Stress response to metal ion	Up	4.94×10^19	10	16
GO:0061687	Detoxification of inorganic compound	Up	4.94×10^19	10	16
GO:0006882	Cellular zinc ion homeostasis	Up	3.51×10^15	10	35
GO:0071294	Cellular response to zinc ion	Up	8.02×10^14	8	12
GO:0071241	Cellular response to inorganic substance	Up	1.83×10^12	14	219
GO:0071248	Cellular response to metal ion	Up	6.38×10^11	11	93
R-HSA-5661231	Metallothioneins bind metals	Up	1.36×10^9	8	16
GO:0010035	Response to inorganic substance	Up	3.36×10^7	13	360
GO:0006873	Cellular ion homeostasis	Up	2.84×10^6	14	684
GO:0045926	Negative regulation of growth	Up	3.38×10^6	10	210
GO:0006950	Response to stress	Up	4.34×10^6	33	4085
GO:0048878	Chemical homeostasis	Up	1.75×10^5	17	1213
R-HSA-3371568	Attenuation phase	Up	3.91×10^5	7	47
R-HSA-3371571	HSF1-dependent transactivation	Up	1.28×10^4	7	59
GO:0070887	Cellular response to chemical stimulus	Up	5.28×10^4	23	2220
GO:0042221	Response to chemical	Up	0.008	28	3716
GO:0034599	Cellular response to oxidative stress	Up	0.02	6	302
GO:0044815	DNA packaging complex	Down	1.74×10^12	17	102
GO:0006305	DNA alkylation	Down	1.66×10^11	14	68
GO:0006306	DNA methylation	Down	1.66×10^11	14	68
GO:0006335	DNA replication-dependent nucleosome assembly	Down	6.86×10^10	10	31
GO:0031497	Chromatin assembly	Down	1.02×10^7	14	134
REAC:R-HSA-157118	Signaling by NOTCH	Down	1.68×10^7	18	232
REAC:R-HSA-68875	Mitotic Prophase	Down	3.16×10^7	11	135
GO:0045814	Negative regulation of gene expression by epigenetic	Down	6.69×10^7	12	106
GO:0070076	Histone lysine demethylation	Down	1.15×10^6	8	34
REAC:R-HSA-195721	Signaling by WNT	Down	1.49×10^6	15	327
GO:0016577	Histone demethylation	Down	1.83×10^6	8	36
REAC:R-HSA-171306	Packaging Of Telomere Ends	Down	3.19×10^6	7	52
GO:0032451	Demethylase activity	Down	5.02×10^6	8	41
REAC:R-HSA-195258	RHO GTPase Effectors	Down	5.75×10^6	14	319
REAC:R-HSA-68886	M Phase of cell cycle	Down	4.27×10^5	14	385
REAC:R-HSA-194315	Signaling by Rho GTPases	Down	0.0002	14	449
REAC:R-HSA-69278	Cell Cycle, Mitotic	Down	0.001	14	527
GO:0000723	Telomere maintenance	Down	0.005	7	75
GO:0045892	Negative regulation of transcription	Down	0.02	24	983
GO:1902679	Negative regulation of RNA biosynthetic process	Down	0.03	24	1016
GO:0030097	Hemopoiesis	Down	0.05	18	905

Abbreviations: GO, gene ontology; Reg, expression regulation of the genes that enriched GO or pathway terms; Corr p value, corrected p value that was calculated on the basis of Benjamini–Hochberg false discovery rate (FDR); Count in selection, number of DEGs that were assigned to a GO or pathway term; Count in total, number of all genes that have been known to function in a GO or pathway term.

Figure 3 Genes identified with over-expression in the ZnO NPs-treated DLBCL cells compared with the untreated DLBCL cells. Hierarchical cluster analysis with heatmap presentation was conducted on the over-expressed genes (FC≥3; p≤0.008; corrected p≤0.05) that significantly over-represented the biological processes and pathways: cellular response to zinc ions (A), response to chemicals (B) and negative regulator of growth (C). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 4 Genes that were under-expressed in the ZnO NPs-treated DLBCL cells compared with the untreated DLBCL cells. Hierarchical cluster analysis with heatmap presentation was conducted on the under-expressed genes (FC≥3; p≤0.008; corrected p≤0.05) that significantly over-represented the biological processes and pathways: negative regulation of transcription (A), chromatin assembly (B), telomere maintenance (C) and NOTCH signaling (D). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 5 Expression profile of oxidative stress responsive gene. Hierarchical cluster analysis with heatmap presentation was done on oxidative stress responsive gene (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs treated DLBCL cells versus untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 6 Expression profile of apoptotic gene. Hierarchical cluster analysis with heatmap presentation were constructed for anti-apoptotic genes (A) and pro-apoptotic genes (B) (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 7 Expression profile of oncogenes. Hierarchical cluster analysis with heatmap presentation were constructed for oncogenes (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 8 Expression profile of B-cell receptor signaling genes. Hierarchical cluster analysis with heatmap presentation were constructed for the genes (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 9 Expression profile of nuclear factor kappa B signaling genes. Hierarchical cluster analysis with heatmap presentation were constructed for the genes (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).