J Breast Cancer.  2019 Jun;22(2):219-236. 10.4048/jbc.2019.22.e19.

miR-148b-3p, miR-190b, and miR-429 Regulate Cell Progression and Act as Potential Biomarkers for Breast Cancer

Affiliations
  • 1Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Yunnan, China. shengmm@aliyun.com
  • 2Department of Mammary Gland and Thyroid Disease, First People's Hospital of Yunnan Province, Yunnan, China.

Abstract

PURPOSE
Breast cancer is the most frequently diagnosed malignancy in women worldwide. MicroRNAs (miRNAs) are thought to serve as potential biomarkers in various cancers, including breast cancer.
METHODS
We evaluated the miRNA expression profiles in 1,083 breast cancer samples and 104 normal breast tissues from The Cancer Genome Atlas database. We used the edgeR package of R software to analyze the differentially expressed miRNAs in normal and cancer tissues, and screened survival-related miRNAs by Kaplan-Meier analysis. A receiver operating characteristic curve was generated to evaluate the accuracy of these miRNAs as molecular markers for breast cancer diagnosis. Furthermore, the functional role of these miRNAs was verified using cell experiments. Targets of candidate miRNAs were predicted using 9 online databases, and Gene Ontology (GO) functional annotation and pathway analyses were conducted using Database for Annotation, Visualization and Integrated Discovery online tool.
RESULTS
A total of 68 miRNAs showed significantly different expression patterns between the groups (p < 0.001), and 13 of these miRNAs were significantly associated with poor survival (p < 0.05). Three miRNAs with high specificity and sensitivity, namely, miR-148b-3p, miR-190b, and miR-429, were selected. In vitro experiments showed that the overexpression of these 3 miRNAs significantly promoted the proliferation and migration of MDA-MB-468 and T47D cells and reduced the apoptosis of T47D cells. GO and pathway enrichment analyses revealed that the targets of these dysregulated miRNAs were involved in many critical cancer-related biological processes and pathways.
CONCLUSION
The miR-148b-3p, miR-190b, and miR-429 may serve as potential diagnostic and prognostic markers for breast cancer. This study demonstrated the roles of these 3 miRNAs in the initiation and progression of breast cancer.

Keyword

Biological marker; Biological phenomenon; Breast neoplasm; MicroRNA

MeSH Terms

Apoptosis
Biological Phenomena
Biological Processes
Biomarkers*
Breast Neoplasms*
Breast*
Diagnosis
Female
Gene Ontology
Genome
Humans
In Vitro Techniques
Kaplan-Meier Estimate
MicroRNAs
ROC Curve
Sensitivity and Specificity
Biomarkers
MicroRNAs

Figure

  • Figure 1 Identification of the 3 miRNAs. (A) Overall workflow of the study. (B) Kaplan-Meier survival curves showing different overall survival in groups of patients with low and high miRNAs expression. (C) ROC curves analysis for miR-148b-3p, miR-190b, and miR-429 differentiating tumor specimens from normal specimens. miRNA = microRNA; HR = hazard ratio; CI = confidence interval; AUC = area under the curve; ROC = receiver operating characteristic.

  • Figure 2 The expression of miR-148b-3p, miR-190b, and miR-429 was enhanced in breast cancer tissues and cell lines. (A) Expression levels of the 3 miRNAs in TCGA. (B) Expression levels of the 3 miRNAs were examined in breast cancer samples. (C) Expression levels of the 3 miRNAs were examined in 7 cell lines. TCGA = The Cancer Genome Atlas; miRNA = microRNA. *p < 0.05, †p < 0.01, ‡p < 0.001 vs. MCF-10A using 1-way analysis of variance.

  • Figure 3 Effect of miR-148b-3p, miR-190b and miR-429 on breast cancer cell proliferation. The proliferative rate of T47D (A) and MDA-MB-468 (B) transfected with miRNA-control, miRNA mimics or miRNA inhibitors. The data were representative of 3 technical repeats with the mean ± standard deviation. miRNA = microRNA; EdU = 5-Ethynyl-2′-deoxyuridine. *p < 0.05, †p < 0.001 vs. control using 1-way analysis of variance.

  • Figure 4 Effect of miR-148b-3p, miR-190b and miR-429 on breast cancer cell apoptosis. A. The influence of miRNAs on T47D cells apoptosis was evaluated by flow cytometry. B. The PARP and cleaved PARP protein levels were measured after transfection with miRNA inhibitors in T47D cells. The data were representative of 3 technical repeats with the mean ± standard deviation. PARP = poly (ADP-ribose) polymerase; miRNA = microRNA; GAPDH = glyceraldehyde 3-phosphate dehydrogenase. *p < 0.05, †p < 0.01 vs. control using 1-way analysis of variance.

  • Figure 5 Effect of miR-148b-3p, miR-190b and miR-429 on breast cancer cell migration. The migration capacity of MDA-MB-468 cell transfected with miRNA mimics (A) or miRNA inhibitors (B) was detected by using wound-healing assay. (C) The capacity of MDA-MB-231 cell migration was decreased after treatment with miR-190b mimic based on the result of wound-healing assay. D. The ability of cell migration in MDA-MB-231 cells transfected with miR-190b mimic or inhibitor based on transwell. The data were representative of 3 technical repeats with the mean ± standard deviation. miRNA = microRNA. *p < 0.05, †p < 0.01, and ‡p < 0.001 vs. control using 1-way analysis of variance.

  • Figure 6 Interaction network of lncRNA, mRNA and pathways in breast cancer tumors. Each hexagon indicates miRNA. Each ellipse indicates mRNA. Each rhombus indicates pathways. lncRNA = long non-coding RNA; mRNA =messenger RNA; miRNA = microRNA.


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