MicroRNA Expression Profiling on Paired Primary and Lymph Node Metastatic Breast Cancer Revealed Distinct microRNA Profile Associated With LNM

Abstract

Breast cancer (BC) is the foremost cause of cancer-related deaths in women. BC patients are oftentimes presented with lymph node metastasis (LNM), which increases their risk of recurrence. Compelling data have recently implicated microRNAs in promoting BC metastasis. Therefore, the identification of microRNA (miRNA)-based molecular signature associated with LNM could provide an opportunity for a more personalized treatment for BC patients with high risk of LNM. In current study, we performed comprehensive miRNA profiling in matched primary breast and LNM and identified 40 miRNAs, which were differentially expressed in LNM compared to primary tumors. The expression of 14 miRNAs (Up: hsa-miR-155-5p, hsa-miR-150-5p, hsa-miR-146a-5p, hsa-miR-142-5p and down: hsa-miR-200a-3p, hsa-miR-200b-3p, hsa-miR-200c-3p, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-214-3p, hsa-miR-141-3p, hsa-miR-127-3p, hsa-miR-125a-5p, and hsa-let-7c-5p) was subsequently validated in a second cohort of 32 breast and 32 matched LNM tumor tissues. Mechanistically, forced expression of hsa-miR-205-5p, or hsa-miR-214-3p epigenetically inhibited MDA-MB-231 cell proliferation, colony formation, and cell migration. Global gene expression profiling on MDA-MB-231 cells overexpressing hsa-miR-205-5p, or hsa-miR-214-3p in combination with in silico target prediction and ingenuity pathway analyses identified multiple bona fide targets for hsa-miR-205-5p, hsa-miR-214-3p affecting cellular proliferation and migration. Interestingly, interrogation of the expression levels of hsa-miR-205 and hsa-miR-214 in the METABRIC breast cancer dataset revealed significantly poor overall survival in patients with downregulated expression of miR-205 [HR = 0.75 (0.61-0.91)], p = 0.003 and hsa-miR-214 [HR = 0.74 (0.59-0.93) p = 0.008]. Our data unraveled the miRNA-transcriptional landscape associated with LNM and provide novel insight on the role of several miRNAs in promoting BC LNM, and suggest their potential utilization in the clinical management of BC patients.

Keywords: breast cancer; hsa-miR-205-5p; hsa-miR-214-3p; lymph node metastasis; miRNA signature.

Figure 1

Hierarchical clustering depicting the expression of differentially expressed miRNAs in lymph node metastatic (LNM) and matched primary breast cancer (BC). (A) Hierarchical clustering of 12 LNM and 12 matched primary BC tissue based on differentially expressed miRNA levels. Each column represents a sample and each row represents a miRNA. Expression level of each miRNA in a single sample is depicted according to the color scale. (B) Principal component analysis (PCA) for the miRNA transcriptome of 12 LNM and their matched BC tissue.

Figure 2

Validation of the expression of 14 miRNAs identified from microarray data in a second cohort of 32 LNM and 32 matched primary BC tissue Taqman low density array (TLDA) qRT-PCR. P-values were calculated using two-tailed t-test and are indicated on each plot. Data are presented as “delta CT” using dot plots.

Figure 3

MiR-205-5p and miR-214-3p inhibits MDA-MB-231 BC cell proliferation, colony formation and migration. (A) Cell viability of MDA-MB-231 cells transfected with hsa-miR-205, hsa-miR-214-3p, or scrambled-control measured on day 5 using alamarblue assay. Data are presented as mean ± S.E.M., n = 6. (B) Clonogenic assay showing the colony forming capability of MDA-MB-231 cells transfected with hsa-miR-205, hsa-miR-214-3p, or scrambled-control. Plates were stained with Diff-Quik stain set on day 10. Wells are representative of two independent experiments for each condition. (C,D) Migration capability of MDA-MB-231 cells transfected with hsa-miR-205, hsa-miR-214-3p compared to scrambled-control. The two-tailed t-test was used to compare different treatment groups. **p < 0.01; ***p < 0.001.

Figure 4

Identification of miR-205-5p and miR-214-3p bona fide gene targets in BC cells. (A) Hierarchical clustering on differentially expressed transcripts in MDA-MB-231 cells transfected with hsa-miR-205, hsa-miR-214 compared to scramble control. Each column represents one replica and each row represents a transcript. Expression level of each gene in a single sample is depicted according to the color scale. (C,D) Venn diagram depicting the overlap between the predicted gene targets for hsa-miR-205-5p or hsa-miR-214-3p (based on TargetScan algorithm) and the downregulated genes in MDA-MB-231 cells overexpressing the respective miRNA. (B) The expression levels of selected genes targets for hsa-miR-205-5p or hsa-miR-214-3p were validated using qRT-PCR in MDA-MB-231 cells overexpressing the respective miRNA. Data are presented as mean ± S.E., n = 6. The two-tailed t-test was used to compare different treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Multiple signaling networks are regulated by hsa-miR-205-5p based on ingenuity pathway analysis (IPA). (A) Illustration of the top inhibited mechanistic networks (FOXO1, AREG and MYC) based on the identified hsa-miR-205-5p gene targets and IPA analysis. (B) Functional networks illustrating the involvement of the indicated hsa-miR-205-5p gene targets in regulating cell proliferation, cell spreading, and organization of cytoskeleton functional based on IPA analysis.

Figure 6

Regulated of the WT1 network, cell survival, invasion, and chemotaxis by hsa-miR-214. (A) Illustration of the WT1 mechanistic network based on the identified hsa-miR-214-3p gene targets and IPA analysis. (B) Functional networks illustrating the involvement of the indicated hsa-miR-214-3p gene targets in regulating cell proliferation, cell spreading, and organization of cytoskeleton based on IPA analysis.

Figure 7

Reduced expression of hsa-miR-205-3p or hsa-214-3p is associated with poor prognosis in BC patients. Kaplan-Meier curves illustrating the duration of overall survival (OS) of 1,262 BC patients stratified into high vs. low based on hsa-miR-205 (HR = 0.75, p = 0.003, A) or hsa-miR-214 (HR = 0.74, p = 0. 008, B) expression from the METABRIC BC dataset.