J Breast Cancer.  2019 Mar;22(1):52-66. 10.4048/jbc.2019.22.e4.

Screening of Recurrence Related MicroRNA in Ductal Carcinoma In Situ and Functional Study of MicroRNA-654-5p

• Li S
• Pu T
• Xiao L
• Gao H
• Li L
• Ye F
• Liu Y
• Bu H

• KMID: 2441851
• DOI: http://doi.org/10.4048/jbc.2019.22.e4

Abstract

PURPOSE
Ductal carcinoma in situ (DCIS) contributes to 20%-30% of newly diagnosed cases of breast cancer in China. Although the breast cancer-specific mortality of DCIS is extremely low, a small proportion of DCIS patients still show relapse or metastasis, leading to poor prognosis. Little is known about the molecular mechanism for DCIS metastasis, partly due to the limited number of poor prognosis patients. This study analyzed the clinicopathological features and screened key microRNAs (miRNAs) contributing to local or distant recurrence.
METHODS
The clinicopathological features of DCIS were evaluated and survival analysis were performed to clarify risk factors associated with poor prognosis. Using miRNA arrays and real-time quantitative polymerase chain reaction (RT-qPCR) on DCIS formalin-fixed and paraffin-embedded samples with or without microinvasion with different clinical outcomes, potential DCIS metastasis-related miRNAs were screened out and further validated. The influence of one identified miRNA, miRNA-654-5p, on DCIS progression was analyzed.
RESULTS
Poor prognosis was significantly associated with larger tumor size and higher lymph node metastasis rate (both p < 0.05). Both were independent prognostic factors for DCIS. According to RT-qPCR results, distinct miRNA expression profiles were identified between DCIS and DCIS with microinvasion (DCIS-Mi) patients. In the DCIS panel, miRNA-654-5p was significantly upregulated in the patients with poor prognosis. In vitro, miRNA-654-5p promoted MDA-MB-231 cell mobility in healing tests and metastasis in the Transwell study.
CONCLUSION
The panel of high-risk miRNAs in DCIS and DCIS-Mi differs markedly. miRNA-654-5p is significantly upregulated DCIS patients having poor prognosis and may be essential for local and distant recurrence in DCIS.

Keyword

Disease-free survival; Epithelial-mesenchymal transition; Noninfiltrating intraductal carcinoma; MicroRNAs

MeSH Terms

Breast
Breast Neoplasms
Carcinoma, Ductal*
Carcinoma, Intraductal, Noninfiltrating*
China
Disease-Free Survival
Epithelial-Mesenchymal Transition
Humans
In Vitro Techniques
Lymph Nodes
Mass Screening*
MicroRNAs*
Mortality
Neoplasm Metastasis
Polymerase Chain Reaction
Prognosis
Recurrence*
Risk Factors
MicroRNAs

Figure

• 

  Figure 1 Flow chart of our study design. miRNA profiling was performed using a microRNA array analysis of three good prognoses and three metastatic cases from different patients. The selected metastasis related miRNAs (n = 5) in DCIS and DCIS-Mi in 2 independent sets of good and metastatic formalin-fixed and paraffin-embedded samples (4 metastatic and 12 nonmetastatic DCIS patients; 5 metastatic and 17 nonmetastatic DCIS-Mi patients) using qPCR. miRNA = MicroRNA; DCIS = ductal carcinoma in situ; DCIS-Mi = ductal carcinoma in situ with microinvasion; qPCR = quantitative polymerase chain reaction.

• 

  Figure 2 A total of 77 differentially expressed miRNAs in metastatic versus good prognosis patients. (A) 77 miRNAs in three pairs of poor and good prognosis patients with or without microinvasion by miRNA microarray analysis (the red part represents the upregulated miRNAs, while the green part represents the downregulated miRNAs). (B) The 77 miRNAs had a greater than 1.5-fold upregulated or downregulated fold-change value. (C) Volcano plot; red pots represented the miRNA consistently overexpressed or under expressed more than 1.5-fold at p < 0.05. (D) The better the repeatability, the closer the plots were to the diagonal line in the scatter plot. miRNA = MicroRNA.

• 

  Figure 3 The screening of the distinct prognostic miRNAs by quantitative polymerase chain reaction. (A, B) The 66 miRNAs had a greater than 1.5-fold upregulated or downregulated fold-change value in DCIS and DCIS-Mi, respectively. (C, D) Five differentially expressed miRNAs were screened in DCIS and DCIS-Mi. miRNA = MicroRNA; DCIS = ductal carcinoma in situ; DCIS-Mi = ductal carcinoma in situ with microinvasion.

• 

  Figure 4 The expression of miRNA-654-5p in different prognosis. (A) The expression of miRNA-654-5p was verified by quantitative polymerase chain reaction in different prognosis and consistently high in poor prognosis. (B) According to the variation of relative distance in cluster analysis dendrogram, the expression of miRNA-654-5p in different prognosis can be classified alone.

• 

  Figure 5 High expression of miRNA-654-5p promoted motility in breast cancer cells. (A) The basic expression of miRNA-654-5p in 3 different breast cancer cell lines. (B, C) The efficiency after transfection of miRNA-654-5p mimics in MDA-MB-231 cells. (D) Wound healing indicated that the miRNA-654-5p mimic group migrated significantly faster than the control group (×40). (E, F) The decreased area in the treatment group was apparently more than that in the negative control group after 24 hours (p < 0.05). (G, H) The number of cells migrated to the lower wells in the treatment group were more than the negative group (×40, ×100, p < 0.05). cq = cycle quantification; NC = negative control.

• 

  Figure 6 Upregulated miRNA-654-5p changed the expression of the protein related to the metastasis verified by quantitative polymerase chain reaction and Western blotting. (A) For the expression in the negative control and the standard, the expression of the gene involved in migration was upregulated, especially the expression of snail (p < 0.05). (B) Cellular lysates from the experimental group cells were used to measure epithelial-mesenchymal transition-related protein expression by western blot. (C) F-actin staining (left panels) of MDA-MB-231 cells transfected with synthetic miRNA-654-5p for 4 days. DAPI staining was used to detect the nuclei. The merged panels showed different cellular morphology. DAPI = 4′,6-diamidino-2-phenylindole; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; NC = negative control.

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