Transcriptomic screening of novel targets of sericin in human hepatocellular carcinoma cells

Abstract

Sericin, a natural protein derived from Bombyx mori, is known to ameliorate liver tissue damage; however, its molecular mechanism remains unclear. Herein, we aimed to identify the possible novel targets of sericin in hepatocytes and related cellular pathways. RNA sequencing analysis indicated that a low dose of sericin resulted in 18 differentially expressed genes (DEGs) being upregulated and 68 DEGs being downregulated, while 61 DEGs were upregulated and 265 DEGs were downregulated in response to a high dose of sericin (FDR ≤ 0.05, fold change > 1.50). Functional analysis revealed that a low dose of sericin regulated pathways associated with the complement and coagulation cascade, metallothionine, and histone demethylate (HDMs), whereas a high dose of sericin was associated with pathways involved in lipid metabolism, mitogen-activated protein kinase (MAPK) signaling and autophagy. The gene network analysis highlighted twelve genes, A2M, SERPINA5, MT2A, MT1G, MT1E, ARID5B, POU2F1, APOB, TRAF6, HSPA8, FGFR1, and OGT, as novel targets of sericin. Network analysis of transcription factor activity revealed that sericin affects NFE2L2, TFAP2C, STAT1, GATA3, CREB1 and CEBPA. Additionally, the protective effects of sericin depended on the counterregulation of APOB, POU2F1, OGT, TRAF6, and HSPA5. These findings suggest that sericin exerts hepatoprotective effects through diverse pathways at different doses, providing novel potential targets for the treatment of liver diseases.

Keywords: Hepatoprotection; Sericin; Transcriptome profiling.

Figure 1

Cytotoxic effect of sericin on HepG2 cells.

Figure 2

Genes that are differentially expressed after sericin administration (blue and red represent downregulated and upregulated genes, respectively). Volcano plots illustrating the magnitude and significance of DEGs between the (A) 0.125 mg/mL sericin-treated group vs. untreated group, (B) 1 mg/mL sericin-treated group vs. untreated group, and (C) 0.125 mg/mL sericin-treated group vs. 1 mg/mL sericin-treated group.

Figure 3

Venn diagram showing the shared DEGs identified by pairwise analyses.

Figure 4

Pathway enrichment analysis of DEGs between the 0.125 mg/mL treatment group and the untreated group. (A) KEGG pathway enrichment. (B) Reactome pathway enrichment: the y-axis indicates the pathway name. The x-axis indicates the enrichment factor in each pathway. The bubble size indicates the number of genes. The color bar indicates the corrected p-value; orange represents a higher value, and light orange represents a lower value. The UpSet plot below indicates the interactions of the sets. The X-axis indicates the intersections of enriched terms. The main plot shows a heatmap of genes at corresponding intersections, colored depending on the up- or downregulation value. The UpSet plot below indicates the interactions of the sets. The X-axis indicates the UpSet plot of intersections of enriched terms. The main plot shows a heatmap of genes at corresponding intersections, colored by up- or downregulation.

Figure 5

Pathway enrichment analysis of DEGs between the 1 mg/mL treatment group and the untreated group. (A) KEGG pathway enrichment. (B) Reactome pathway enrichment: the y-axis indicates the pathway name. The x-axis indicates the enrichment factor in each pathway. The bubble size indicates the number of genes. The color bar indicates the corrected p-value; orange represents a higher value, and light orange represents a lower value. The UpSet plot below indicates the interactions of the sets. The X-axis indicates the intersections of enriched terms. The main plot shows a heatmap of genes at corresponding intersections, colored depending on the up- or downregulation values.

Figure 6

Pathway enrichment analysis of common DEGs between 0.125 mg/mL and 1 mg/mL sericin vs. untreated sericin. (A) KEGG pathway enrichment analysis of the common DEGs between the 0.125 mg/mL and 1 mg/mL groups. (B) KEGG and (C) Reactome pathway enrichment analysis for the contrast DEGs in the same comparisons. The y-axis indicates the pathway name. The x-axis indicates the enrichment factor in each pathway. The bubble size indicates the number of genes. The color bar indicates the corrected p-value; orange represents a higher value, and light orange represents a lower value. The UpSet plot below indicates the interactions of the sets. The X-axis indicates the intersections of enriched terms. The main plot shows a heatmap of genes at corresponding intersections, colored depending on the up- or downregulation values.

Figure 7

The critical protein complex affected by sericin treatment according to the PPI network. (A) Formation of fibrin clots. (B) Metallothionines bind metals. (C) Autophagy. (D) Selective autophagy. (E) Macroautophagy. Red indicates downregulation, and green indicates upregulation.

Figure 8

Box plot visualizing the changes in transcription factor activity upon sericin administration. (A) Transcription factor activity at 0.125 mg/mL. (B) Transcription factor activity at 1 mg/mL. The X-axis indicates the transcription factor pathway. The y-axis indicates the transcription factor score. The purple box indicates deactivation. The pink box indicates activation.

Figure 9

Cell viability in acetaminophen-induced hepatotoxicity treated with sericin. Data are expressed as means ± SEM. Statistical analysis was performed using a paired t-test. The asterisk (*) indicates a statistically significant difference (p < 0.05) between the sericin-treated groups (0.125 and 1 mg/mL) and the untreated control group.

Figure 10

Expression of genes associated with sericin-mediated hepatoprotection in response to hepatotoxicity induced by acetaminophen.

Figure 11

RT‒PCR illustrating the changes in the expression of the selected genes in HepG2 cells treated with 0.125 mg/mL sericin vs. untreated and 1 mg/mL sericin vs. untreated. The PCR products were normalized to GAPDH and are expressed as the fold change. The data are expressed as the mean ± SEM.