MicroRNA-671-3p inhibits the development of breast cancer: A study based on in vitro experiments, in-house quantitative polymerase chain reaction and bioinformatics analysis

Abstract

MicroRNAs (miRNAs or miRs) are highly conserved small noncoding RNA molecules involved in gene regulation. An increasing number of studies have demonstrated that miRNAs act as oncogenes or antioncogenes in various types of cancer, including breast cancer (BC). However, the exact role of miR‑671‑3p in BC has not yet been reported. In the present study, in vitro experiments were implemented to explore the effects of miR‑671‑3p on the proliferation and apoptosis of BC cells, and reverse transcription‑quantitative polymerase chain reaction was conducted using in‑house clinical BC samples to address the expression level and clinical value of miR‑671‑3p in BC. Simultaneously, miR‑671‑3p target genes were collected, and subsequent bioinformatics analyses were executed to probe the potential signaling pathway through which miR‑671‑3p influenced the occurrence and progression of BC. According to the results, the expression level of miR‑671‑3p was lower in BC tissues compared with that in adjacent non‑tumorous tissues (P=0.048), and the area under the curve was 0.697 (95% confidence interval=0.538‑0.856), with a sensitivity and specificity of 0.818 and 0.579, respectively. Forced miR‑671‑3p expression in the BC cell line MDA‑MB‑231 evidently arrested cell proliferation and induced cell apoptosis. Furthermore, in silico enrichment analyses suggested that miR‑671‑3p may be involved in the initiation and progression of BC through the targeting of genes associated with the Wnt signaling pathway. In conclusion, the present study findings suggested that miR‑671‑3p may function as a tumor suppressor in BC by influencing the Wnt signaling cascade, which provides a prospective molecular target for the therapy of BC.

Figure 1

Workflow of the present study. miR, microRNA; BC, breast cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; GEO, Gene Expression Omnibus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction.

Figure 2

miR-671-3p mimic inhibited the proliferation and viability of MDA-MB-231 cells. (A) Transfection efficiency of miR-671-3p. Effect of miR-671-3p mimic on (B) cell proliferation determined by MTS Assay, and (C) cell viability determined by CellTiter-Blue Cell Viability Assay. ^*P<0.05, ^**P<0.01 and ***P<0.001 vs. negative control at 0 h. miR, microRNA.

Figure 3

miR-671-3p mimic accelerated the apoptosis of MDA-MB-231 cells. (A) Effect of miR-671-3p mimic on cell apoptosis in terms of the caspase-3/7 activity, as determined by an Apo-ONE Homogeneous Caspase-3/7 assay. (B) Effect of miR-671-3p mimic on cell apoptosis, determined by fluorescence microscopy following Hoechst 33342/PI double-fluorescent chromatin staining. (C) Viable and apoptotic cells were observed under a microscope following Hoechst 33342/PI staining (magnification, x200) at 96 h. Viable cells exhibited Hoechst 33342-positive and PI-negative staining; early apoptotic cells exhibited Hoechst 33342-positive and PI-negative staining, with blue fragmentation in the cells; late apoptotic cells exhibited Hoechst 33342-positive and PI-positive staining, with red fragmentation in the cells; and necrotic cells exhibited PI-positive staining with debris signals. ^*P<0.05 and ^**P<0.01 vs. negative control at 0 h. miR, microRNA; PI, propidium iodide.

Figure 4

Expression level and clinical value of miR-671-3p in BC based on in-house reverse transcription-quantitative polymerase chain reaction. (A) Expression levels of miR-671-3p in ANT and BC tissues. (B) Receiver operating characteristic curve and (C) prognostic value of miR-671-3p in BC. miR, microRNA; BC, breast cancer; ANT, adjacent non-tumorous tissues; AUC, area under the curve.

Figure 5

Expression level of miR-671-3p in BC based on data obtained from the GEO and ArrayExpress databases. (A) Forest plot of the 11 datasets evaluating miR-671-3p expression in BC (random-effects model). SMD <0 indicates that miR-671-3p is downregulated in BC. (B) Sensitivity analysis evaluating the stability of the meta-analysis. (C) Funnel plot assessing the publication bias in the 11 datasets. miR, microRNA; BC, breast cancer; GEO, Gene Expression Omnibus; SMD, standard mean difference; 95% CI, 95% confidence interval; s.e., standard error.

Figure 6

Top 5 enriched GO annotations of the putative target genes of miR-671-3p. The top 5 enriched (A) biological processes, (B) molecular functions, and (C) cellular components are shown. miR, microRNA; GO, Gene Ontology.

Figure 7

Top 10 enriched KEGG pathways of the putative target genes of miR-671-3p. miR, microRNA; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 8

PPI and miRNA-gene regulatory networks. (A) PPI network of 13 miR-671-3p target genes involved in the Wnt signaling pathway. (B) miRNA-gene regulatory network based on 13 miR-671-3p-gene pairs. miR, microRNA; PPI, protein-protein interaction.

Figure 9

Heatmap of 10 differently expressed targets of miR-671-3p in BC and normal breast tissues based on data obtained from TCGA. miR, microRNA; BC, breast cancer; TCGA, The Cancer Genome Atlas.

Figure 10

Expression levels of 10 differently expressed targets of miR-671-3p in BC and normal breast tissues based on data obtained from TCGA. (A) WNT7B, (B) PLCB3, (C) CCND1, (D) VANGL1 and (E) FRAT2 were upregulated in BC. (F) CCND2, (G) SFPR1, (H) PRICKLE2, (I) FZD5 and (J) FZD4 were downregulated in BC. miR, microRNA; BC, breast cancer; TCGA, The Cancer Genome Atlas.