The Effect of MicroRNA-124 Overexpression on Anti-Tumor Drug Sensitivity

<div><p>MicroRNAs play critical roles in regulating various physiological processes, including growth and development. Previous studies have shown that microRNA-124 (miR-124) participates not only in regulation of early neurogenesis but also in suppression of tumorigenesis. In the present study, we found that overexpression of miR-124 was associated with reduced DNA repair capacity in cultured cancer cells and increased sensitivity of cells to DNA-damaging anti-tumor drugs, specifically those that cause the formation of DNA strand-breaks (SBs). We then examined which DNA repair–related genes, particularly the genes of SB repair, were regulated by miR-124. Two SB repair–related genes, encoding ATM interactor (ATMIN) and poly (ADP-ribose) polymerase 1 (PARP1), were strongly affected by miR-124 overexpression, by binding of miR-124 to the 3¢-untranslated region of their mRNAs. As a result, the capacity of cells to repair DNA SBs, such as those resulting from homologous recombination, was significantly reduced upon miR-124 overexpression. A particularly important therapeutic implication of this finding is that overexpression of miR-124 enhanced cell sensitivity to multiple DNA-damaging agents via ATMIN- and PARP1-mediated mechanisms. The translational relevance of this role of miR-124 in anti-tumor drug sensitivity is suggested by the finding that increased miR-124 expression correlates with better breast cancer prognosis, specifically in patients receiving chemotherapy. These findings suggest that miR-124 could potentially be used as a therapeutic agent to improve the efficacy of chemotherapy with DNA-damaging agents.</p></div

MiR-124 overexpression increases sensitivity to specific anti-tumor drugs/treatments.

<p>(A) Survival of breast cancer cells (MDA-MB-231) stably transfected with the empty lentivirus vector (vector control, VC) or the miR-124-overexpressing construct (miR-124) after the indicated treatments. (B) Survival of osteosarcoma cells (U-2 OS) stably transfected with the empty lentivirus vector (vector control, VC) or the miR-124-overexpressing construct (miR-124) after the indicated treatments. *Significant difference (<i>P</i> < 0.05) between the miR-124 and VC-transfected cells.</p

MiR-124 overexpression reduces DNA SB repair capacity.

<p>(A) HR repair capacity in miR-124-overexpressing cells and cells transfected with the antisense microRNA control (anti-miR-CON) or antisense miR-124 inhibitor (anti-miR-124). (B) Comet assay showing that miR-124 overexpression (miR-124) reduces SB repair relative to the vector control (VC) after treatment with CPT. (C) Immunostaining of DNA SBs (detected by the presence of γ-H2AX) in cells after IR treatment. Red: anti-γ-H2AX; blue: nuclei (left panel). The γ-H2AX signal was quantified and compared with the cells transfected with VC or miR-124 (right panel). All of the experiments shown were conducted in U-2 OS cells, and similar results were obtained in MDA-MB-231 cells. *<i>P</i> < 0.05 in individual comparisons.</p

Higher miR-124 expression correlates with better prognosis for breast cancer patients receiving chemotherapy.

<p>Overall survival of breast cancer patients who received (left panel) or did not receive chemotherapy (right panel) stratified by expression of miR-124. Log-rank test <i>P</i> values are shown. Data were generated using the Kaplan Meier plotter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128472#pone.0128472.ref016" target="_blank">16</a>].</p

ATMIN and PARP1 mRNAs are targets of miR-124.

<p>(A) Relative luciferase activity in U-2 OS cells carrying the luciferase reporter (pGL4) linked to the sense-oriented (normally orientated) 3′-UTR of putative miR-124 targets <i>RAD17</i>, <i>UBE2B</i>, <i>PARP1</i>, <i>FANCF</i>, and <i>ATMIN</i> or the empty reporter vector control (VC). *Significantly different (<i>P</i> < 0.05) from the VC group. (B) Two predicted miR-124 binding sequences in <i>ATMIN-3</i>′-UTR (upper panel, ATMIN-3′-UTR) were mutated (upper panel, ATMIN-3′-UTR MT1 and ATMIN-3′-UTR MT2). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the sense-oriented (normally oriented) (forward) <i>ATMIN</i>-3′-UTR (miR-124-F), reverse-oriented <i>ATMIN</i>-3′-UTR (miR-124-R), ATMIN-3′-UTR MT1 (miR-124-MT1), ATMIN-3′-UTR MT2 (miR-124-MT2), or the mutant carrying mutations at both of the two predicted sites (miR-124-MT1&MT2). (C) One predicted miR-124 binding sequence in <i>PARP1</i>-3′-UTR (upper panel, PARP1-3'-UTR) was mutated (upper panel, PARP1-3'-UTR MT). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the [sense-oriented (normally oriented) (forward) PARP1-3'-UTR (miR-124-F), reverse-oriented PARP1-3'-UTR (miR-124-R, or PARP1-3'-UTR MT (miR-124-MT). (D) MiR-124 regulates expression ATMIN and PARP1. Western blot of ATMIN and PARP1 in U-2 OS cells transiently transfected with the VC or miR-124-expressing vector (miR-124; upper panel), or with a 2′-O-methyl-modified antisense inhibitor of miR-124 (Anti-miR-124; lower panel) or 2′-O-methyl negative control (Anti-miR) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128472#pone.0128472.ref048" target="_blank">48</a>]. Tubulin was included as a protein loading control, and the numbers indicate the expression relative to expression in the VC or Anti-miR control, measured by densitometry. *<i>P</i> < 0.05 relative to VC (A) or to miR-124-R (B and C).</p

Introduction of ATMIN and PARP1 reverses the DNA repair defect induced by miR-124 overexpression.

<p>(A) HR repair capacity in cells transfected with the indicated combinations of vector control (VC) or the constructs overexpressing miR-124, ATMIN, or PARP1. *Significantly different (<i>P</i> < 0.05) from VC. ^#Significantly different (<i>P</i> < 0.05) from the miR-124-overexpressing group. (B) Comet assay to detect DNA SBs in cells overexpressing VC, miR-124, miR-124 + ATMIN, or miR-124 + PARP1 after treatment with CPT. (C) Effects of ATMIN and PARP1 overexpression on miR-124 overexpression–enhanced sensitivity to CPT (left panel) or ETO (right panel). *Significantly different (<i>P</i> < 0.05) from VC.</p

Expression of miR-124 mRNA in breast cancer cell lines and U-2 OS (osteosarcoma) cells.

<p>MiR-124 expression level was determined by quantitative PCR and expressed as the fold change relative to RNU6B. Expression in the immortalized breast epithelial cell line, H184B5F5/M10, was used as the reference.</p

Multi-center study on patient selection for and the oncologic safety of intraoperative radiotherapy (IORT) with the Xoft Axxent® eBx® System for the management of early stage breast cancer in Taiwan

<div><p>Background</p><p>In this multi-center study, we report the patient selection criteria for and preliminary oncologic outcomes associated with intraoperative radiotherapy (IORT) delivered by the Xoft Axxent® eBx® system for early-stage breast cancer in Taiwan.</p><p>Methods</p><p>Patients with early breast cancer in Taiwan received breast conserving surgery and received IORT with Xoft Axxent® eBx® System during 2013–2015 was search from database of Taiwan IORT study cooperative group (T-IORTSCG). Patients’ clinicopathologic characteristics and early post-operative results were collected and reported.</p><p>Results</p><p>During the study period, 26 hospitals in Taiwan performed a total of 261 Xoft IORT procedures for breast cancer. The mean age of them was 52.9 ± 9.8 years (37–72), and tumor size was 1.5 ± 0.8 cm (0.1–4.2 cm) for invasive cancer and 1.2 ± 0.8 cm (range, 0.2–3.0 cm) for ductal carcinoma in situ (DCIS) lesions. Lymph node metastasis was found in 6 (2.3%) patients. The patients received IORT in Taiwan differed markedly from those used in the ELIOT and TARGIT-A studies. Specifically, patients selected for IORT in Taiwan tended to be younger, their tumors tended to be larger and the prevalence of lymph node metastasis tended to be lower. Among these 261 patients, 8 (3.1%) patients required whole breast radiotherapy. During a mean follow up of 15.6 months, locoregional recurrence was observed in 2 (0.8%) patients.</p><p>Conclusion</p><p>In real world experience, patients received IORT differed quite significantly with criteria formulated by trials. The preliminary results of IORT in Taiwan showed it is well acceptable by patients and clinicians.</p></div

Comparison of patients selection criteria of Xoft IORT in Taiwan with ELIOT and TARGIT-A trials.

<p>Comparison of patients selection criteria of Xoft IORT in Taiwan with ELIOT and TARGIT-A trials.</p

The development and application of Xoft IORT system in Taiwan.

<p>(a) The development and application of Xoft IORT system in Taiwan from 2013–2015. The T-IORTSCG comprises members from major IORT centers in Taiwan, and included 5 centers in 2013, 18 in 2014, and 26 in 2015. The number of IORT performed per year and the cumulative number of IORT performed in the past 3 years were provided. (b) Illustration of pre- and post-operative breast appearance of patients received conventional radiotherapy. (c) Illustration of pre- and post-operative breast appearance of patients received intra-operative radiotherapy.</p