The Role of lncRNAs in the Protective Action of Tamoxifen on the Ovaries of Tumor-Bearing Rats Receiving Cyclophosphamide

Abstract

Infertility due to ovarian toxicity is a common side effect of cancer treatment in premenopausal women. Tamoxifen (TAM) is a selective estrogen receptor modulator that prevented radiation- and chemotherapy-induced ovarian failure in preclinical studies. In the current study, we examined the potential regulatory role of long noncoding RNAs (lncRNAs) in the mechanism of action of TAM in the ovaries of tumor-bearing rats receiving cyclophosphamide (CPA) as cancer therapy. We identified 166 lncRNAs, among which 49 were demonstrated to be differentially expressed (DELs) in the ovaries of rats receiving TAM and CPA compared to those receiving only CPA. A total of 24 DELs were upregulated and 25 downregulated by tamoxifen. The identified DELs shared the characteristics of noncoding RNAs described in other reproductive tissues. Eleven of the identified DELs displayed divergent modes of action, regulating target transcripts via both cis- and trans-acting pathways. Functional enrichment analysis revealed that, among target genes ascribed to the identified DELs, the majority were involved in apoptosis, cell adhesion, immune response, and ovarian aging. The presented data suggest that the molecular mechanisms behind tamoxifen's protective effects in the ovaries may involve lncRNA-dependent regulation of critical signaling pathways related to inhibition of follicular transition and ovarian aging, along with the suppression of apoptosis and regulation of cell adhesion. Employing a tumor-bearing animal model undergoing chemotherapy, which accurately reflects the conditions of mammary cancer, reinforces the obtained results. Given that tamoxifen remains a key player in the management and prevention of breast cancer, understanding its ovarian-specific actions in cancer patients is crucial and requires detailed functional studies to clarify the underlying molecular mechanisms.

Keywords: cancer treatment; cyclophosphamide; fertility preservation; long noncoding RNA (lncRNA); ovarian reserve; tamoxifen.

Figure 1

Characterization of the identified differentially expressed lncRNAs (DELs). (A) Graphical representation of the first (PC1) and second (PC2) principal components (PCs) affecting the lncRNA expression pattern, (B) distance matrix of differentially expressed lncRNAs (DELs; P-adjusted < 0.05 and log2FC ≥ |1.0|), and (C) genomic localization of the identified lncRNAs. (D–F)The comparison of genomic features (mean ± SEM) of the identified lncRNAs and mRNAs. The lncRNAs and mRNAs were compared in terms of average transcript length (D), exon length (E), and exon number (F). nt: nucleotides.

Figure 2

MA (A) and Volcano (B) plot presenting differentially expressed genes (DEGs; P-adjusted < 0.05 and log2FC ≥ |1.0|) and lncRNAs (DELs; P-adjusted < 0.05 and log2FC ≥ |1.0|) identified in the ovaries of tumor-bearing rats on the basis of the expression comparison performed between the CPA + TAM group and the CPA group. Red circles depict upregulated DEGs/DELs and green circles represent downregulated DEGs/DELs. Gray circles represent DEGs or DELs with no significant changes in expression.

Figure 3

The visualization of the expression patterns of trans-regulated DEGs and DELs in the ovaries of tumor-bearing rats. (A) Pearson correlation between DELs and their potential DEG targets; (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of target DEGs affected by DELs; (C) Statistical significance of the correlations presented in panel A; (D) Gene Ontology (GO) analysis of DEGs affected by DELs. The figure presents only GO subcategories containing the highest number of target DEGs.

Figure 4

Identification (A) and functional enrichment analysis (B) of target DEGs potentially regulated by 11 DELs exhibiting divergent regulation of target gene expression (both in cis- and trans-).

Figure 5

Real-Time PCR validation of the selected differentially expressed lncRNAs identified (RNA-Seq) in the ovaries of tumor-bearing rats (CPA + TAM vs. CPA). The asterisk depicts statistically significant differences (p < 0.05) in CPA+TAM group compared to CPA group.

Figure 6

Interaction network of target DEGs potentially regulated by lncRNAs (DELs) identified in the ovaries of rats treated with cyclophosphamide (CPA) plus tamoxifen vs. rats treated with CPA alone. The network was generated by STRING (confidence score: 0.4) using DEGs (P-adjusted < 0.05 and log2 fold change ≥ 1.0) related to apoptosis and cell adhesion. Enrichment p-value: 1.0 × 10⁻¹⁶.

Figure 7

Experimental design of the study performed on rats. CPA was injected intraperitoneally, n = 25/group (modified from Nynca et al., 2023 [23]).