J Breast Cancer.  2018 Jun;21(2):142-149. 10.4048/jbc.2018.21.2.142.

Combined Let-7a and H19 Signature: A Prognostic Index of Progression-Free Survival in Primary Breast Cancer Patients

• Guo Q
• Wen R
• Shao B
• Li Y
• Jin X
• Deng H
• Wu J
• Su F
• Yu F

• KMID: 2413932
• DOI: http://doi.org/10.4048/jbc.2018.21.2.142

Abstract

PURPOSE
The long non-coding RNA H19, a conservatively imprinted gene, acts as a molecular sponge for the let-7 family, which has been identified as a set of tumor suppressors. However, the combined prognostic value of H19 and let-7a signature in breast cancer patients remains unclear.
METHODS
In this research we assessed the prognostic value of the combined H19 and let-7a signature in breast cancer patients by retrospectively reviewing that data of 79 patients who underwent neoadjuvant chemotherapy; we also investigated the expression and function of H19 in breast cancer cell lines in vitro. Survival data were calculated using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate survival analyses were conducted using the Cox proportional hazards regression method. As determined using X-tile, the optimal cutoff value for the risk score to assess progression-free survival (PFS) based on the combined signature was -0.1.
RESULTS
Patients with an overall positive treatment response had higher let-7a and lower H19 levels. In addition, let-7a expression was negatively correlated with H19 expression. Patients with a risk score of >-0.1 had shorter overall survival and PFS. In vitro data showed that chemoresistant cell lines exhibit higher H19 and lower let-7a levels and knockdown H19 restores paclitaxel sensitivity.
CONCLUSION
Our results suggest that the combined let-7a and H19 signature is a novel prognostic factor for breast cancer patients treated with neoadjuvant chemotherapy.

Keyword

Breast neoplasms; H19 long non-coding RNA; Hsa-let-7a-1 microRNA; Neoadjuvant therapy; Prognosis

MeSH Terms

Breast Neoplasms*
Breast*
Cell Line
Disease-Free Survival*
Drug Therapy
Humans
In Vitro Techniques
Methods
Neoadjuvant Therapy
Paclitaxel
Porifera
Prognosis
Retrospective Studies
RNA, Long Noncoding
Paclitaxel
RNA, Long Noncoding

Figure

• 

  Figure 1 H19 and let-7a expression levels in 79 primary breast cancer patients as measured using quantitative real-time polymerase chain reaction. (A) let-7a is expressed at higher levels in patients with a complete response (CR)/partial response (PR) than in patients with stable disease (SD)/progressive disease (PD). (B) H19 is expressed at higher levels in patients with SD/PD than in patients with CR/PR. (C) The expression of let-7a is negatively associated with that of H19 (r=–0.566, p<0.001). The fold difference in expression was normalized against the sample with the lowest expression levels.GAPDH=glyceraldehyde-3-phosphate dehydrogenase; U6=U6 small nuclear RNA. *p<0.001.

• 

  Figure 2 Drug-resistant breast cancer cell lines (MDA-MB-231 paclitaxel-resistant cell line [MDA-MB-231p]) express high levels of H19, and knockdown of H19 expression (as measured using quantitative real-time polymerase chain reaction [qRT-PCR]) restores the chemosensitivity of these cells. (A) Based on the qRT-PCR results, the average expression level of H19 in the paclitaxel cell line was 13.04-fold (p=0.002) higher than the average H19 expression level in the control MDA-MB-231 cell line. (B) Based on the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT) assay, the half maximal inhibitory concentration (IC50) values of paclitaxel in MDA-MB-231 cells and paclitaxel-resistant cells were 53.83±4.16 nM and 372.20±59.81 nM, respectively. After knockdown of H19 in MDA-MB-231p cells, the IC50 values were 74.44±8.40 nM (p=0.008) and 65.45±6.44 nM (p=0.007), respectively. (C) Flow cytometry of cells stained with Annexin V-fluorescein isothiocyanate was used to investigate drug-induced apoptosis. After 24 hours of paclitaxel treatment, the apoptosis rate of MDA-MB-231p cells was only 3.5%±0.9%; however, the two sets of small interfering RNA (siRNA)-transfected cells exhibited apoptosis rates of 13.8%±1.5% (p=0.004) and 18.2%±1.6% (p=0.001).GAPDH=glyceraldehyde-3-phosphate dehydrogenase; NC=negative control; PI=propidium iodide. *p<0.01; †p<0.01 vs. MDA-MB-231p and NC; ‡p<0.01 vs. mock and siRNA-NC.

• 

  Figure 3 Progression-free survival (PFS) and overall survival (OS) curves of 79 primary breast cancer patients with different risk score values. (A) The median OS was 16 months (range, 4–29 months) in the risk score ≥–0.1 group and 20 months (range, 7–32 months) in the risk score <–0.1 group (hazard ratio [HR], 0.126; 95% confidence interval [CI], 0.020−0.806; p=0.001). (B) The median PFS was 18 months (range, 7–32 months) in the risk score ≥–0.1 group and 14 months (range, 4–29 months) in the risk score <–0.1 group (HR, 0.221; 95% CI, 0.094−0.518; p=0.001). (C) Comparisons of the areas under the receiver operating characteristic curve for survival status according to the risk score and the let-7a and H19 levels. The areas were 0.790, 0.683, and 0.667, respectively.lnc=long non-coding; PI=propidium iodide.

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