Huashi Jiedu Decoction Enhances 5-Fluorouracil Efficacy in Gastric Cancer via miRNA-21-3p/p53 Pathway

Abstract

Purpose: To explore the mechanism of Huashi Jiedu Decoction (HJD) synergizing with 5-fluorouracil (5-Fu) in gastric cancer (GC) therapy.

Methods: MicroRNAs (miRNAs) and genes involved in HJD-mediated GC treatment were identified using ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry, network pharmacology, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and molecular docking. The effects of HJD on 5-Fu sensitivity in BGC-823 cells were evaluated with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and Western blotting. Synergistic effects in vector-transfected and miRNA-21-3p knockdown cells were assessed by colony formation, wound healing, transwell assays, and flow cytometry (FCM). An in vivo study evaluated the impact of HJD on 5-Fu sensitivity, measuring miRNA-21-3p, tumor protein p53 (p53), N-cadherin, vimentin, and E-cadherin in tumors, along with tumor volume and weight.

Results: miRNA-21-3p and p53 were key targets in HJD's therapeutic effect on GC. RT-qPCR showed that HJD combined with 5-Fu reduced miRNA-21-3p and upregulated p53 in vector cells and increased p53 mRNA (p < 0.01) and protein (p < 0.05) compared to 5-Fu alone. These effects were abolished in miRNA-21-3p knockdown cells. The combination reduced colony formation by 48.92% (p < 0.01), suppressed transwell migration by 28.5% (p < 0.01), and inhibited wound healing by 81.9% compared to 5-Fu monotherapy (p < 0.001), with no effects in knockdown cells. FCM showed a 15.1% increase in G₀/G₁ phase arrest (p < 0.05). In vivo, the combination significantly reduced tumor volume (p < 0.05) and weight by 18.7%, with concomitant miRNA-21-3p downregulation (p < 0.0001), EMT marker suppression (N-cadherin, vimentin), and tumor suppressor activation (p53, E-cadherin) versus 5-Fu alone.

Conclusion: HJD enhances 5-Fu's effects on GC by regulating the miRNA-21-3p/p53 pathway and modulating cadherin expression, supporting its potential as an adjunctive treatment in GC.

Keywords: 5-fluorouracil; gastric cancer; huashi jiedu decoction; miRNAs; p53.

Graphical abstract

Figure 1

General experimental progress.

Figure 2

Total ion chromatogram of HJD obtained by UHPLC-Q-Orbitrap HRMS analysis in (A) positive ion mode and (B) negative ion mode. Peaks are annotated as follows: (a) 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4Hchromen-4-one, (b) eicosapentaenoic acid methyl ester, (c) deoxycholic acid, (d) berberine, (e) quercetin, (f) kaempferol, (g) estrone, (h) 7-hydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one, (i) sinomenine, (j) gibberellic acid, (k) arachidonic acid, (l) artemisinin, (m) hispidulin, (n) linolenic acid ethyl ester.

Figure 3

miRNA-21 is overexpressed in GC and associated with gastric carcinogenesis and metastasis. (A) Volcano plot of differentially expressed miRNAs in GC vs normal tissues (n=450: 40 normal; 410 GC). (B) Heatmap of the top 18 upregulated and downregulated miRNAs (n=450: 40 normal; 410 GC). (C) miRNA-21 expression in normal gastric tissues, primary GC, and metastatic GC tissues (n=433: 41 normal; 180 primary GC; 212 metastatic GC). *p < 0.05, ****p < 0.001.

Figure 4

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggest miRNA-21 is associated with GC metastatic behaviors, cell cycle progression, and p53 pathway activity. (A) GO and KEGG enrichment analysis of miRNA-21-high-expressing differentially expressed genes (DEGs) in GC. (B) GO and KEGG enrichment analysis of miRNA-21-low-expressing DEGs in GC.

Figure 5

Identification of DEGs in GC using integrated data from the UCSC XENA and Genotype-Tissue Expression (GETx) databases, and prediction of potential therapeutic targets for HJD treatment. (A) Volcano plot of 6,508 DEGs in GC (n=500: 83 normal, 417 GC cases). (B) Heatmap displaying the top 25 upregulated and bottom 25 downregulated DEGs. (C) Venn diagram illustrating the intersection of HJD target genes with GC-associated DEGs, revealing 162 potential therapeutic targets. (D) Decoction-compound-target network for HJD: drug (hexagon), 14 active compounds (rhombus), and 162 targets (circle).

Figure 6

MiRNA-21-3p and p53 are proposed as potential key regulatory factors mediating the therapeutic effects of HJD in GC. (A) Network analysis of HJD target genes associated with miRNA-21-3p and miRNA-21-5p in GC treatment. Edge thickness correlates with interaction strength (thicker edges: stronger associations). (B) Molecular docking model illustrating the binding interface between miRNA-21-3p and p53.

Figure 7

The cytotoxicity of HJD or 5-Fu on BGC-823 cells, the synergistic effect of HJD in combination with 5-Fu, and the preliminary validation of the miRNA-21-3p/p53 pathway. (A) Inhibitory effects of 5-Fu or HJD on BGC-823 cell growth (IC50: 7.74 mg/L for 5-Fu and 3.02 mg/mL for HJD, n = 6 per group). (B) Synergistic effects of HJD and 5-Fu on BGC-823 cell viability (MTT assay at 24 h and 48 h, n = 6 per group). (C and D) RT-qPCR analysis of miRNA-21-3p and p53 expression in BGC-823 cells treated with fixed 5-Fu and increasing HJD concentrations (n = 6 per group, ANOVA). (E) p53 mRNA levels (RT-qPCR) in BGC-823 cells with negative transfection or miRNA-21-3p knockdown across four groups: control, 5-Fu, HJD, and combination (n = 6 per group, ANOVA). (F) p53 mRNA expression (RT-qPCR) in control groups (negative transfection vs miRNA-21-3p knockdown, n = 6 per group, t-test). (G) p53 mRNA expression (RT-qPCR) in synergistic groups (negative transfection vs miRNA-21-3p knockdown, n = 6 per group, t-test). (H–J) p53 protein expression (Western blot) in BGC-823 cells with negative transfection or miRNA-21-3p knockdown (n = 6 per group, ANOVA). (K) p53 protein levels (Western blot) in control groups (negative transfection vs miRNA-21-3p knockdown, n = 6 per group, t-test). (L) p53 protein levels (Western blot) in synergistic groups (negative transfection vs miRNA-21-3p knockdown, n = 6 per group, t-test). Data: mean ± SEM. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, N.S. indicates no statistical significance.

Figure 8

Continued.

Figure 8

HJD synergizes with 5-Fu via miRNA-21-3p to suppress proliferation, migration, and G₀/G₁ phase arrest in BGC-823 cells. (A and B) Vector BGC-823 cells and miRNA-21-3p down from four groups (control, 5-Fu, HJD, and synergistic) were subjected to cell cloning assay (n = 6 per group). Quantification in panel B. (C, E and F) Transwell migration assay was conducted on the aforementioned groups. Quantification E and F (n = 6 per group). (D, G and H) Wound healing assay was conducted on the aforementioned groups. Quantification in G and H (n = 6 per group). (I–K) FCM assay was conducted on the aforementioned groups. Quantification in J and K (n = 5 per group). Data are presented as the means ±SEM. Values were tested by ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. indicates no statistical significance.

Figure 9

Continued.

Figure 9

HJD, in combination with 5-Fu, inhibited GC proliferation and metastasis in vivo by modulating miRNA-21-3p downregulation and p53 upregulation. (A) The difference of mice tumor size in the four groups (control, 5-Fu, HJD, and synergistic) (n = 5 per group). (B) The tumor weight in the relevant groups at the end of the experiment (n = 5 per group). (C) The tumor volume of the relevant groups (n = 5 per group). (D–F) Relative expressions of miRNA-21-3p, p53, and E-cadherin, N-cadherin, and Vimentin in the tumor tissues of four groups were detected by RT-QPCR (n = 5 per group). (G–I) Protein expressions of p53, E-cadherin, and N-cadherin in the GC tissues of four groups were detected by WB (n = 5 per group). (J) Representative immunofluorescence images of E-cadherin, N-cadherin, and Vimentin in the GC tissues of mice (n = 5 per group). (K) Fluorescence intensity of E-cadherin, N-cadherin, and Vimentin in the GC tissues of mice of the aforementioned groups (n = 5 per group). The values were tested by ANOVA. *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 10

HJD increases the sensitivity of GC to 5-Fu, and the miRNA-21-3p/p53 pathway is involved.