Edodes Cultured Extract Regulates Immune Stress During Puberty and Modulates MicroRNAs Involved in Mammary Gland Development and Breast Cancer Suppression

Abstract

Background: Immune stressors, such as lipopolysaccharides (LPS), profoundly affect microbiota balance, leading to gut dysbiosis. This imbalance disrupts the metabolic phenotype and structural integrity of the gut, increasing intestinal permeability. During puberty, a critical surge in estrogen levels is crucial for mammary gland development. However, inflammation originating from the gut in this period may interfere with this development, potentially heightening breast cancer risk later. The long-term effects of pubertal inflammation on mammary development and breast cancer risk are underexplored. Such episodes can dysregulate cytokine levels and microRNA expression, altering mammary cell gene expression, and predisposing them to tumorigenesis.

Methods: This study hypothesizes that prebiotics, specifically Lentinula edodes Cultured Extract (AHCC), can counteract LPS's adverse effects. Using BALB/c mice, an acute LPS dose was administered at puberty, and breast cancer predisposition was assessed at 13 weeks. Cytokine and tumor-related microRNA levels, tumor development, and cancer stem cells were explored through immunoassays and qRT-PCR.

Results: Results show that LPS induces lasting effects on cytokine and microRNA expression in mammary glands and tumors. AHCC modulates cytokine expression, including IL-1β, IL-17A/F, and IL-23, and mitigates LPS-induced IL-6 in mammary glands. It also regulates microRNA expression linked to tumor progression and suppression, particularly counteracting the upregulation of oncogenic miR-21, miR-92, and miR-155. Although AHCC slightly alters some tumor-suppressive microRNAs, these changes are modest, highlighting a complex regulatory role that warrants further study.

Conclusion: These findings underscore the potential of dietary interventions like AHCC to mitigate pubertal LPS-induced inflammation on mammary gland development and tumor formation, suggesting a preventive strategy against breast cancer.

Keywords: LPS; breast cancer; mammary glands development; microRNAs; microbiome and dysbiosis; prebiotics; pro‐inflammatory cytokines; tumor development.

FIGURE 1

Gut‐immune interactions, mammary gland development, and experimental design. (A) The interplay between gut and microbiota and mammary gland development early in life and potential breast cancer later in life. An activated immune system produces pro‐inflammatory factors, such as TNFα, IL‐1β, and Cox‐2. Given that the gut immune system is a part of mucosal‐associated lymphoid tissue (MALT), exposure to inflammatory agents or immune stressors during critical periods, such as puberty, has the potential to disrupt the normal development of mammary glands and alter their gene expression. This can ultimately contribute to the acquisition of a CSC phenotype and the development of breast cancer. (B) Study design showing the experimental timeline, groups, dietary intervention, LPS injection, and 4T1 cells injection.

FIGURE 2

Effects of AHCC and LPS on tumor characteristics. (A) represent the tumor volume, weight, and CSCs. (B) Indicate the number and size of CSCS in the treatment groups. (C) Concentrations of IL‐1β, IL‐6, IL‐10, IL‐23, TGF‐β1, TGF‐β2, and TGF‐β3 in the tumor samples. (D) Concentrations of IL‐1β, IL‐6, IL‐10, IL‐17A, IL‐17F, IL‐23, TGF‐β1, and TGF‐β3 in the mammary gland samples. The mice were randomly assigned to receive either AHCC (2 g/kg BW/d) in their drinking water or plain drinking water for 2 weeks, 1 week before, and 1 week after the pubertal LPS injection. Two‐way ANOVA and Tukey's post hoc tests were used to compare groups. All values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

Effects of AHCC and LPS on microRNA Expression in the Tumors. (A) The enrichment gene ontology plot indicates the role of microRNAs in the important pathways affecting tumor development. The left side of the plot is the heatmap, showing the log2 expression of the selected microRNAs. (B) The relative expression of candidate microRNAs, including let‐7a, let‐7c, miR‐21, miR‐34a, miR‐92, miR‐125, miR‐140, miR‐155, miR‐181c, miR‐200c, and miR‐425 in the tumor of adult mice 4 weeks after 4T1 cells injection. The mice were randomly assigned to receive either AHCC (2 g/kg BW/d) in their drinking water or plain drinking water for 2 weeks, 1 week before, and 1 week after the pubertal LPS injection. Two‐way ANOVA and Tukey's post hoc tests were used to compare groups. All values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.000.

FIGURE 4

Effects of AHCC and LPS on microRNA expression in the adjacent mammary glands. (A) The enrichment ontology plot indicating the role of microRNAs in the important pathways affecting tumor development. The left side of the plot is the heatmap, showing the log2 expression of the selected microRNAs. (B) The relative expression of candidate microRNAs, including miR‐34a, miR‐125, miR‐145, miR‐181, miR‐184, miR‐188, miR‐200c, miR‐205, miR‐223, and miR‐425, in the mammary glands of adult mice 4 weeks after 4T1 cells injection. The mice were randomly assigned to receive either AHCC (2 g/kg BW/d) in their drinking water or plain drinking water for 2 weeks, 1 week before, and 1 week after the pubertal LPS injection. Two‐way ANOVA and Tukey's post hoc tests were used to compare groups. All values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001.