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. 2017 Oct 6;8(65):108712-108725.
doi: 10.18632/oncotarget.21561. eCollection 2017 Dec 12.

Downregulation of miR-221-3p and upregulation of its target gene PARP1 are prognostic biomarkers for triple negative breast cancer patients and associated with poor prognosis

Ling Deng  1 , Qianqian Lei  1   2 , Yu Wang  1 , Zhu Wang  1 , Guiqin Xie  1 , Xiaorong Zhong  1 , Yanping Wang  1 , Nianyong Chen  3 , Yan Qiu  4 , Tianjie Pu  4 , Hong Bu  4 , Hong Zheng  1   3
Affiliations

Downregulation of miR-221-3p and upregulation of its target gene PARP1 are prognostic biomarkers for triple negative breast cancer patients and associated with poor prognosis

Ling Deng et al. Oncotarget. .

Abstract

The purpose of this study was to identify microRNAs (miRNAs) closely associated with the prognosis of triple-negative breast cancer (TNBC) and their possible targets. This study recruited 125 early-stage TNBC patients, including 40 cases in the experimental group (20 cases with poor prognoses vs. 20 cases with good prognoses) and 85 cases in the validation group (27 cases with poor prognoses vs. 58 cases with good prognoses). In the experimental group, miRNA microarray showed 34 differentially expressed miRNAs in patients with different prognoses. We selected 5 miRNAs for validation. The differential expression of miR-221-3p was further verified in the experimental and validation groups using real-time polymerase chain reaction (PCR). High miR-221-3p expression was associated with better 5-year disease-free survival (DFS) (HR = 0.480; 95% CI, 0.263-0.879; p = 0.017) of TNBC patients. High expression of its target gene PARP1 predicted poorer 5-year DFS (HR = 2.236, 95% CI, 1.209-4.136, p = 0.010). MiR-221-3p down-regulated PARP1 by targeting its 3'-untranslated region. In conclusion, low miR-221-3p expression may contribute to the poor outcome of TNBC patients through regulating PARP1. MiR-221-3p likely plays a role as a PARP1 inhibitor by directly regulating PARP1 expression, thereby affecting the prognoses of TNBC patients.

Keywords: PARP1; miR-221-3p; microRNA; prognosis; triple negative breast cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST No potential conflicts of interest are disclosed.

Figures

Figure 1
Figure 1. Validation of differentially expressed miRNAs by real-time PCR
The relative expression levels of miR-203 (A), miR-140-5p (B), and miR-221-3p (C) are shown for 40 specimens of the experimental group. (D) Relative expression of miR-221-3p in the validation group. (E) Relative expression of miR-221-3p in 12 paired cancer and normal tissues in the control group. (F) Relative expression of miR-222-3p in the control group. (G) Relative expression of miR-221-3p and miR-222-3p in the cancer tissues of the control group. Long horizontal marks represent the mean expression of miRNAs in patients with different prognoses, and short horizontal marks represent the standard error of the mean miRNA expression. * p < 0.05; # p > 0.05; A, B, C, E, F, G, t test; D, non-parametric test.
Figure 2
Figure 2. Association between miR-221-3p expression and 5-year disease-free survival (DFS) of TNBC patients
High miR-221-3p expression was associated with better 5-yearDFS in overall patients (A), patients following anthracyclines treatment (B), or patients with lymph node positivity (C). But no association was observed in patients with lymph node negativity (D).
Figure 3
Figure 3. PARP1 expression by IHC staining
PARP1expression was evaluated by IHC staining and QS-score: (A) high PARP1 expression; and (B) low PARP1 expression (×100).
Figure 4
Figure 4. High PARP1 expression was associated with poorer 5-year DFS of TNBC patients
High PARP1 expression was associated with poorer 5-year DFS in overall patients (A) or patients following anthracyclines treatment (B).
Figure 5
Figure 5. Negative regulation of PARP1 expression by miR-221-3p
MiR-221-3p mimic with different doses were transfected into MDA-MB-231 cell line (A-C), and relative miR-221-3pexpression to untreated control was increased in cells with different doses (*p < 0.05, t test, A). Relative PARP1 mRNA (*p < 0.05, t test, B) and protein (C) expression to untreated control were decreased in transfected dose with 50 nM or 100 nM, but there was no difference between 50 nM transfected cells and 100 nM transfected cells. Then 50 nM of miR-221-3p mimics was chosen to continue the experiments in the MDA-MB-231 cell line (D-E), which showed PARP1 mRNA (*p <0.05, t test, D) and protein levels (E) were markedly down-regulated. MiR-124 mimic served as the positive control. Transfection with the miR-221-3p inhibitor increased the PARP1 mRNA (*p< 0.05, t-test, F) and protein (G) levels in three TNBC cell lines. (H) Sequences of miR-221-3p, PARP1-3’UTR, and mutant PARP1-3’UTR in the dual luciferase experiment. (I) Cotransfection with the luciferase constructs containing the wild-type PARP1 3’-UTR and miR-221-3p precursor resulted in 48% decline in the luciferase activity compared with the control cells (* p< 0.05).
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