The UALCAN (http://ualcan.path.uab.edu) is a comprehensive and interactive web resource for analyzing genomics data from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/), which was used to determine the expression of miR-3609 in breast cancer. It allows researchers to analyze the relative expression of potential genes of interest in tumors and normal samples, and in various tumor subgroups (12).

The cBioPortal (https://www.cbioportal.org) is an open-access resource for interactive exploration of multidimensional cancer genomics datasets (13). Genetic alterations of miR-3609 was obtained from cBioPortal, based on TCGA database.

The Kaplan Meier Plotter database (https://kmplot.com/analysis) is able to assess the effect of 54 k genes (mRNAs, miRNAs and proteins) on the survival of patients with 21 different types of cancer. Sources for the databases include Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/) and TCGA (14). The overall survival of patients with breast cancer was analyzed using the Kaplan-Meier Plotter database.

LinkedOmics (http://www.linkedomics.org/login.php) is a publicly available portal that contains multi-omics and clinical data from 32 types of cancer, and 11,158 patients from TCGA project (15). It also includes mass spectrometry-based proteomics data generated by the Clinical Proteomics Tumor Analysis Consortium for TCGA breast, colorectal and ovarian tumors.

The differentially expressed genes associated with miR-3609 were screened from TCGA BRCA cohort (n=755), using the LinkFinder module in the LinkedOmics database. Spearman's correlation coefficient analysis was performed to assess the correlation between the miR-3609 expression and genes differentially expressed in breast cancer.

Gene Ontology (GO; http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.kegg.jp/) pathway enrichment analyses of the differentially expressed genes were performed using the LinkInterpreter module, the results of which were signed and ranked using the gene set enrichment analysis (GSEA) tool in the LinkedOmics.

STRING (https://string-db.org) is a public database that predicts protein-protein interactions (PPIs), and aims to achieve a comprehensive and objective global network and present them with a unique set of computational predictions (16).

To establish a PPI network, the present study screened out co-expressed genes with interaction scores >0.4. Cytoscape software (version 3.8.0; https://cytoscape.org) which is an open source software platform for visualizing molecular interaction networks and biological pathways and integrating these networks with annotations, gene expression profiles and other state data, was used to visualize the PPI network, and hub genes were identified using cytoHubba plug-in (version 0.1; https://cytoscape.org) and the degree topological algorithm.

The TNBC cell line, MDA-MB-231 was purchased from the American Type Culture Collection and maintained in DMEM (Hyclone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G and 100 µg/ml streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), at 37˚C in a humidified 5% CO₂ atmosphere.

miR-3609 mimics (40, 80 and 160 nM; 5'-CAAA GUGAUGAGUAAUACUGGCUG-3' and 5'-GCCAGUA UUACUCAUCACUUUGUU-3'), nonspecific miRNA mimics (5'-UUCUCCGAACGUGUCACGUTT-3' and 5'-ACGUGACACGUUCGGAGAATT-3') were synthesized by Shanghai GeneChem Co., Ltd., and transfected into MDA-MB-231 cells using Lipofectamine^® RNAiMax reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. After transfection at 37˚C for 24 h, miR-3609 expression in MDA-MB-231 cells transfected with 80 nM miR-3609 mimics increased the most (Fig. S1), MDA-MB-231 cells transfected with 80 nM miR-3609 mimics were used for subsequent analyses. Non-specific miRNA mimics, synthesized by Shanghai GeneChem Co., Ltd., were used as the negative controls. Transfected cells were used for subsequent experimentation 48 or 72 h post-transfection.

MDA-MB-231 cells were transfected with either miR-3609 mimics or a non-specific miRNA mimic (both at 80 nM), using Lipofectamine RNAiMax reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Transfected MDA-MB-231 cells were seeded into 96-well plates at a density of 2x10³ cells/well and incubated for 24, 48, 72, 96 or 120 h at 37˚C in DMEM (Hyclone; Cytiva). Cell viability was assessed via the Cell Counting Kit-8 (CCK-8) assay (Suzhou Yuheng Biotechnology Co., Ltd.) for 2 h.

Following transfection with either miR-3609 mimics or a non-specific miRNA mimic for 48 h, MDA-MB-231 cells were collected after centrifugation with 172.2 x g for 5 min at room temperature, and washed twice with pre-cooled PBS. Subsequently, 1.0x10⁴ cells were fixed with 500 µl of 70% cold ethanol overnight at 4˚C, followed by addition of 500 µl of propidium iodide (PI) solution (11.6 µg/ml PI, 2 mg/ml RNaseA in PBS; Beijing Solarbio Science & Technology Co., Ltd.), and the mixture was incubated at room temperature for 30-60 min, in the dark. DNA content analysis was performed using the FACS Calibur instrument (BD FACSCantoII™) and CellQuest software (modfit 5.1; BD Biosciences). The experiments were performed in triplicate.

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). Data are presented as the mean ± SD of at least three independent experiments. Unpaired Student's t-test was used to compare differences between two groups (Figs. 1A, and 4C and D). One-way ANOVA followed by Tukey's post hoc test was used to compare differences between multiple groups (Fig. S1). P<0.05 was considered to indicate a statistically significant difference.

The present study determined the expression of miR-3609 between normal and primary breast tumor samples in TCGA database (Fig. 1A). The results revealed no significant differences between the two groups. The genetic alterations of miR-3609 in 1,108 breast invasive carcinoma samples in TCGA database were detected, using cBioPortal. The results demonstrated that 15 cases had miR-3609 alterations (1.4%) (Fig. 1B and C).

After investigating the overall survival of miR-3609 in breast cancer using the Kaplan-Meier Plotter database, the results demonstrated no significant differences in overall survival for miR-3609 among all patients with breast cancer from TCGA database. However, high miR-3609 expression was associated with better overall survival in patients with TNBC (Fig. 2A and B).

The present study analyzed the co-expressed genes of miR-3609 in 755 patients with breast cancer, using the LinkedOmics database. The volcano plot depicts genes positively and negatively correlated with miR-3609 (Fig. 3A). The top 50 significant gene sets positively or negatively correlated with miR-3609 are depicted in the heat maps, respectively (Fig. 3B and C).

GO analysis demonstrated that differentially expressed genes associated with miR-3609 were mainly located in ‘spliceosomal complex’, ‘ribosome’, ‘condensed chromosome’ and ‘exoribonuclease complex’, and these cellular components may have participated in ‘RNA splicing’, ‘rRNA metabolic process’, ‘spinal cord development’, ‘G₀-G₁ transition’ or other biological processes. They acted as structural constituents of the ribosome and mRNA binding (Fig. 3D-F). KEGG pathway analysis demonstrated that the functions of the associated genes were primarily enriched in ribosome and spliceosome (Fig. 3G).

To confirm G₀-G₁ transition for biological processes of miR-3609, the cell cycle assay was performed. The results demonstrated that the proportion of MDA-MB-231 cells transfected with miR-3609 mimics in the G₀/G₁ phase was significantly higher (P=0.032), and that in the G₂/M phase was significantly lower (P=0.018) compared with the control group (Fig. 4A-C), suggesting that miR-3609 induces cell cycle arrest of TNBC cells in the G₀/G₁ phase.

The CCK-8 assay was performed to assess the viability of MDA-MB-231 cells. The results demonstrated that the viability of MDA-MB-231 cells transfected with miR-3609 mimics was significantly lower compared with MDA-MB-231 cells transfected with non-specific miRNA mimic (P=0.0285), suggesting that high miR-3609 expression decreases the viability of MDA-MB-231 cells (Fig. 4D).

The 422 significantly co-expressed genes were selected to construct aPPI network using the STRING database (P<0.001), and Cytoscape (cytoHubba plug-in) was used to identify the hub genes, along with the degree topological algorithm. Based on the degree score, the genes with the highest scores [interleukin enhancer binding factor 3 (ILF3), ELAV like RNA binding protein 1 (ELAVL1), heterogeneous nuclear ribonucleoprotein (HNRNP)L, HNRNPK, HNRNPH1 and polypyrimidine tract binding protein 1 (PTBP1)] were identified as potential hub genes (Fig. 5A and B). The gene of ILF3 encodes a double-stranded RNA (dsRNA)-binding protein that complexes with other proteins, dsRNAs, small noncoding RNAs and mRNAs to regulate gene expression and stabilize mRNAs (17). The protein encoded by ELAVL1 is a member of the ELAVL family of RNA-binding proteins that contain several RNA recognition motifs, and selectively bind AU-rich elements found in the 3' untranslated regions of mRNAs (18). Heterogeneous nuclear RNAs which include mRNA precursors and mature mRNAs are associated with specific proteins to form heterogenous ribonucleoprotein (hnRNP) complexes (19). HNRNPL is stably associated with hnRNP complexes, and is likely to play a major role in the formation, packaging, processing, and function of mRNA, along with other hnRNP proteins (20). HNRNPH1 encodes a member of a subfamily of ubiquitously expressed hnRNPs (19). Moreover, both HNRNPK and PTBP1 belong to the subfamily of ubiquitously expressed hnRNPs (21,22).

The overall survival of the hub genes in breast cancer was assessed using the Kaplan-Meier Plotter database. The results demonstrated that low HNRNPL expression was associated with a longer overall survival time (P<0.05; Fig. 6).