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. 2024 Aug 7;14(1):18356.
doi: 10.1038/s41598-024-68069-4.

6-Gingerol modulates miRNAs and PODXL gene expression via methyltransferase enzymes in NB4 cells: an in silico and in vitro study

Ali Afgar  1 , Mahdiyeh Ramezani Zadeh Kermani  2 , Athareh Pabarja  3 , Amir Reza Afgar  1 , Batoul Kavyani  4 , Hossein Arezoomand  5 , Saeed Zanganeh  6 , Mohammad Javad Sanaei  7 , Mahla Sattarzadeh Bardsiri  8   9 , Reza Vahidi  10
Affiliations

6-Gingerol modulates miRNAs and PODXL gene expression via methyltransferase enzymes in NB4 cells: an in silico and in vitro study

Ali Afgar et al. Sci Rep. .

Abstract

This investigation delves into the influence of predicted microRNAs on DNA methyltransferases (DNMTs) and the PODXL gene within the NB4 cell line, aiming to elucidate their roles in the pathogenesis of acute myeloid leukemia (AML). A comprehensive methodological framework was adopted to explore the therapeutic implications of 6-gingerol on DNMTs. This encompassed a suite of bioinformatics tools for protein structure prediction, docking, molecular dynamics, and ADMET profiling, alongside empirical assessments of miRNA and PODXL expression levels. Such a multifaceted strategy facilitated an in-depth understanding of 6-gingerol's potential efficacy in DNMT modulation. The findings indicate a nuanced interplay where 6-gingerol administration modulated miRNA expression levels, decreasing in DNMT1 and DNMT3A expression in NB4 cells. This alteration indirectly influenced PODXL expression, contributing to the manifestation of oncogenic phenotypes. The overexpression of DNMT1 and DNMT3A in NB4 cells may contribute to AML, which appears modulable via microRNAs such as miR-193a and miR-200c. Post-treatment with 6-gingerol, DNMT1 and DNMT3A expression alterations were observed, culminating in the upregulation of miR-193a and miR-200c. This cascade effect led to the dysregulation of tumor suppressor genes in cancer cells, including downregulation of PODXL, and the emergence of cancerous traits. These insights underscore the therapeutic promise of 6-gingerol in targeting DNMTs and microRNAs within the AML context.

Keywords: PODXL gene; AML; Docking; Methyltransferase; MiRNA; Molecular dynamic simulation; NB4 cell line.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Secondary structure plot of DNMT1 (A), DNMT3A (B), and DNMT3B (C). The SOPMA server predicted the secondary structure of three proteins: DNMT1, DNMT3A, and DNMT3B. The results showed that DNMT1 had 46.50% random coil, 28.90% alpha helix, 18.87% extended strand, and 5.73% beta-turn. DNMT3A had 46.88% random coil, 30.48% alpha helix, 16.55% extended strand, and 6.10% beta-turn. DNMT3B had 53.25% random coil, 26.36% alpha helix, 14.94% extended strand, and 5.45% beta-turn.
Figure 2
Figure 2
3D structures of DNMT1 (A), DNMT3A (B), DNMT3B (C), and 6-gingerol (D). The structures were visualized with Pymol software Version 2.5.8. The four proposed shapes represent the three-dimensional structures of the obtained molecules before docking and molecular dynamics simulations.
Figure 3
Figure 3
3D Docking gingerol with DNMT1 (A), DNMT3A (B), and DNMT3B (C) by HDOCK Server (The structures were visualized with Pymol software Version 2.5.8). As shown in the figure above, gingerol is correctly positioned in the binding pockets or cavities of the target proteins.
Figure 4
Figure 4
2D interactions of docking 6-gingerol with DNMT1 (A), DNMT3A (B), and DNMT3B (C) by Ligplot+. The results indicate that 6-gingerol, the active ingredient, can form multiple chemical bonds with the enzymes, including hydrogen and hydrophobic bonds. This demonstrates the potential effect of 6-gingerol on the enzymatic activity.
Figure 5
Figure 5
Convergence assessment of Molecular Dynamics Simulations of 6-gingerol-DNMTs. The RMSD of backbone atoms was calculated using the g_rms function of GROMACS for 40 ns. The plot in (A) showed that DNMT3A reached a stable conformation with less deviation faster than DNMT1, indicating better docking with 6-gingerol. The Rg of the protein–ligand complexes was calculated using the gmx_gyrate function of GROMACS for 40 ns. The plot in (B) showed that DNMT1 and DNMT3A had mean Rg deviations of 3.695 nm and 1.766 nm, respectively, suggesting that DNMT3A had a more compact structure than DNMT1. The RMSFs of the residues were calculated using the gmx_rmsf module of GROMACS for 40 ns. The plot in (C) showed that some residues of DNMT1 had high peaks, indicating high dynamics and flexibility, while some residues of DNMT3A had low peaks, indicating low fluctuations and stability. The number of H-bonds between the protein and the ligand was recorded using the gmx_hbond tool of GROMACS for 40 ns. The plot in (D) showed that 6-gingerol formed 0_4 and 0_5 H-bonds with DNMT1 and DNMT3A, respectively, and that DNMT3A had a higher average number of H-bonds than DNMT1, implying a better interaction with 6-gingerol. The SASA values of the complexes were computed using the gmx_sasa function of GROMACS for 40 ns. The plot in (E) showed that DNMT1 and DNMT3B had average SASA values of 571.168 nm2 and 128.233 nm2, respectively, while DNMT3A had the lowest SASA value, indicating that 6-gingerol had a stronger bond with DNMT3A than with water molecules.
Figure 6
Figure 6
Cell viability and flow cytometry analysis of 6-gingerol effect on NB4 cells. As presented in (A), the 6-gingerol treatment induced a dose-dependent inhibition of cell viability, and the IC50 value was determined as 183 µM. Consistent with the cytotoxic effect, the apoptotic effect was observed after 24-h exposure to IC50 concentration of the 6-gingerol (B,D). In other words, unlike the control group (C), treatment of cells with 183 µM of 6-gingerol decreased the number of viable cells and increased (P value < 0.05) the number of apoptotic cells (Annexin V + cells).
Figure 7
Figure 7
Morphologic characterization of NB4 cells before (A) and after treatment with 100 (B), 150 (C), and 200 µM (D) of 6-gingerol (DAPI staining, × 100 magnification). As shown, 6-gingerol reduced the number of viable cells in a dose-dependent manner, with the most significant reduction observed at 200 µM. Furthermore, DAPI staining revealed that 6-gingerol also caused nuclear changes, such as chromatin condensation/fragmentation (red arrow) and nuclear blebbing (white arrow).
Figure 8
Figure 8
(A) Upon comparative analysis, NB4 cells subjected to 6-gingerol exhibited a significant upregulation of miR-193a and miR-200c, as evidenced by P values less than 0.05. Conversely, in comparison to the control group of untreated NB4 cells, the expression levels of miR-548 and miR-148a-5p remained statistically unchanged. Furthermore (B), the study observed a discernible decrease in the expression of DNMT1, DNMT3A, and PODXL in the 6-gingerol-treated NB4 cells relative to their untreated counterparts, with P value < 0.05 for DNMT1 and PODXL, and (P value < 0.01) for DNMT3A. The expression of DNMT3B, however, did not demonstrate a significant difference.
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