Identification of circRNA-miRNA-mRNA network in luminal breast cancers by integrated analysis of microarray datasets

Abstract

Introduction: Circular RNAs (circRNAs) regulatory network is important in human cancer. We, therefore, mapped the regulatory networks driven by circRNA in luminal-subtype breast cancer. Methods: Breast cancer-related microarray datasets from GEO database were analyzed for the differentially expressed circRNAs, miRNAs, and mRNAs. The potential downstream RNAs were collected using Circular RNA Interactome or Targetscan database. Protein-protein interaction (PPI) analysis was performed for the filtered genes to identify hub genes. The functions were annotated by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. CircRNA-miRNA-mRNA networks were mapped using Cytoscape software. Hsa_circ_0086735-miR-1296-5p-STAT1 axis was used for verification. The expression levels of hsa_circ_0086735, miR-1296-5p, and STAT1 mRNA were confirmed by qRT-PCR in luminal-subtype tissues and cell lines. The interactions among them were verified by Luciferase reporter assay and RNA pull-down assay. Cell proliferation and apoptosis were assayed. Overall and distant metastasis-free survival was analyzed. Results: A total of 70 genes were finally targeted and enriched in multi-process and multi-pathway. Networks containing 96 circRNA-miRNA-mRNA axes were constructed. Hsa_circ_0086735 and STAT1 mRNA was upregulated in luminal breast cancer, while miR-1296-5p was downregulated. Hsa_circ_0086735-miR-1296-5p-STAT1 axis promotes breast cancer progression and contributes to tamoxifen resistance. High hsa_circ_0086735 was associated with poor overall and distant metastasis-free survival. Discussion: This study identified the hsa_circ_0086735-miR-1296-5p-STAT1 as an important regulatory axis in luminal-subtype breast cancer, aiding to determine potential therapeutic targets.

Keywords: circRNA; hsa_circ_0086735-miR-1296-5p-STAT1; luminal breast cancer; network; tamoxifen resistance.

FIGURE 1

Flowchart of the construction of circRNA-miRNA-mRNA network.

FIGURE 2

The differentially expressed circRNAs in tamoxifen-resistant and luminal-subtype breast cancer. (A) Volcano map of up/downregulated circRNAs in GSE182471 analyzed by GEOR2. The red dots indicated upregulated circRNAs and the blue dots indicated downregulated circRNAs. (B) Venn diagrams of the shared differentially expressed circRNAs among GSE182471, GSE111504, GSE159980, and 101,410. (C) Heatmap of eight up/downregulated circRNAs according to the expression in GSE182471.

FIGURE 3

The differentially expressed miRNAs (DE-miRNAs) in luminal-subtype breast cancer. (A) Volcano map of the shared miRNAs from GSE32022, GSE110204, and GSE121172. The orange dots indicated upregulated micRNAs and the blue dots indicated downregulated miRNAs. (B) and (C) Venn diagrams of the overlapped miRNAs among DE-miRNAs and the circRNA-targeting miRNAs.

FIGURE 4

Bioinformatics of the differentially expressed mRNAs. (A) Volcano map of differentially expressed mRNAs in GSE48390 analyzed by GEOR2. (B) Protein-protein interaction (PPI) networks of the shared mRNA between differentially expressed mRNAs and miRNA-targeting mRNAs. (C) The enriched GO terms by the shared mRNAs. (D) The enriched KEGG pathways of the shared mRNAs. (E) The top 20 pathways enriched by the shared mRNAs.

FIGURE 5

Networks. (A) and (B) Differentially expressed circRNA-miRNA-mRNA networks. The diamond represented circRNAs, the rectangles miRNAs, and ellipses mRNAs. Red and yellow color represent up- and downregulation in luminal-B tumor tissues, respectively. The black border indicated the pathway derived from the top 10 hub genes. (C) Network of the main circRNA-miRNA-mRNA-pathway.

FIGURE 6

Expression of hsa_circ_0086735 in luminal-subtype breast cancer. Luminal-subtype breast cancer tissues expressed higher hsa_circ_0086735 (A), lower miR-1296-5p (B), and higher STAT1 mRNA (C) compared to those in adjacent normal tissues. Expression of hsa_circ_0086735 (D), miR-1296-5p (E), and STAT1 mRNA (F) in luminal-subtype cell lines compared to those in MCF10-A. ***p < 0.001.

FIGURE 7

Hsa_circ_0086735 sponged to miR-1296-5p, and miR-1296-5p targeted to STAT1. (A) Predicted complementary binding sites of hsa_circ_0086735 against miR-1296-5p. (B) The expression level of hsa_circ_0086735 was reversely related to that of miR-1296-5p. (C) Luciferase reporter assays confirm the binding between hsa_circ_0086735 and miR-1296-5p. The pmirGL3 reporter vectors were cloned with the wild-type hsa_circ_0086735 with the potential miR-1296-5p binding sites to construct WT-circ plasmids or mutant of these sites to construct MUT-circ plasmids, and then transfected into MCF-7 cells with miR-1296-5p mimic (mm-miR) or inhibitor (in-miR) or corresponding negative controls (mm-NC or in-NC). (D) In vitro pull-down of hsa_circ_0086735 with biotin-labeled miR-1296-5p (biotin-miR) or negative control (biotin-NC) in ZR-75-1 cells. (E) Predicted complementary binding sites of miR-1296-5p against STAT1. (F) Luciferase reporter assays confirm the binding between miR-1296-5p and STAT1. The pmirGL3 reporter vectors were cloned with the wild-type STAT1 with the potential miR-1296-5p binding sites to construct WT-STAT1 plasmids or mutant of these sites to construct MUT-STATA1 plasmids, and then transfected into ZR-75-1 cells with miR-1296-5p mimic (mm-miR) or inhibitor (in-miR) or corresponding negative controls (mm-NC or in-NC). (G) In vitro pull-down of STAT1 mRNA with biotin-labeled miR-1296-5p (biotin-miR) or negative control (biotin-NC) in MCF7 cells. (H) The expression level of miR-1296-5p was reversely related to that of STAT1. ***p < 0.001.

FIGURE 8

The effect of hsa_circ_0086735-miR-1296-5p-STAT1 axis on proliferation and apoptosis of luminal-subtype cell lines. (A) and (B) The transfection efficiency was confirmed by qRT-PCR analysis. CCK8 was performed on MCF7 (C) and ZR-75-1 (D) cells. Apoptosis assay by AnnexinV-FITC/PI staining for MCF7 (E) and ZR-75-1 (F) cells. (G) Overall survival of 87 breast cancer patients whose tumors had high or low hsa_circ_0086735 expression. ***p < 0.001, compared with si-NC group. ^†† p < 0.01, ^††† p < 0.001, compared with si-circ+in-NC group. ^&& p < 0.01, ^&&& p < 0.001, compared with si-circ+in-miR + ctr-STAT1 group. si-NC: negative siRNA control for hsa_circ_0086735; si-circ: siRNA for hsa_circ_0086735; in-NC: inhibitor negative control; in-miR: miR-1296-5p inhibitor; ctr-STAT1: negative siRNA control for STAT1; si-STAT1: siRNA for STAT1.

FIGURE 9

The effect of hsa_circ_0086735-miR-1296-5p-STAT1 axis on the proliferation of tamoxifen-resistant cells. (A) Hsa_circ_0086735 was upregulated in tamoxifen-resistant cells. (B) The transfection efficiency was confirmed by qRT-PCR analysis. (C) The IC50 values of different cell lines. (D) The cell proliferation by CCK-8 assay. (E) Apoptosis assay by AnnexinV-FITC/PI staining. (F) Distant metastasis-free survival of 87 breast cancer patients whose tumors had high or low hsa_circ_0086735 expression. ***p < 0.001, compared with si-NC group. ^†† p < 0.01, ^††† p < 0.001, compared with si-circ+in-NC group. ^&& p < 0.01, ^&&& p < 0.001, compared with si-circ+in-miR + ctr-STAT1 group. si-NC: negative siRNA control for hsa_circ_0086735; si-circ: siRNA for hsa_circ_0086735; in-NC: inhibitor negative control; in-miR: miR-1296-5p inhibitor; ctr-STAT1: negative siRNA control for STAT1; si-STAT1: siRNA for STAT1.