Human ovarian cancer cell line SKOV-3 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (50 U/ml; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

All molecular analyses were carried out on primary biopsies. Tissues were formalin-fixed and embedded in paraffin. Pathologists confirmed the existence of at least 80% tumor cells in the clinical specimens.

The Instituto Nacional de Cancerologia (Mexico) provided the ovarian tumor and normal tissue collection. The Instituto Nacional de Cancerologia (Mexico) ethics committee approved the protocols concerning the use of human tissues. A signed informed form consent was obtained from each participant or a representative prior to release for research use.

Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from patients with ovarian cancer. Total RNA was isolated using the RNeasy FFPE kit (Qiagen, Valencia, CA, USA) according to the manufacture's protocol. Briefly, 5–10 FFPE sections of 10 µm were incubated two times in xylene for 1 h at 63°C for deparaffinization. Then, total RNA was extracted according to the manufacturer's protocol. In addition, total RNA from SKOV-3 cells was isolated using the TRIzol protocol (Ambion, Austin, TX, USA), and concentration and purity were evaluated by spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA) followed by 1% agarose-formaldehyde gel electrophoresis.

Quantitative real-time RT-PCR (qRT-PCR) analysis for miRNA expression was performed using the TaqMan MicroRNA Assay kits (Assay ID 001178; Thermo Fisher Scientific, Inc.). Total RNA (100 ng) was reverse transcribed using a looped-RT specific primer targeting the let-7d-3p mature sequence CUAUACGACCUG CUGCCUUUCU, dNTPs (100 mM; New England Biolabs, Ipswich, MA, USA), reverse transcriptase MultiScribe (50 U/µl; Thermo Fisher Scientific, Inc.), 10X buffer, RNase inhibitor (20 U/µl; Promega, Madison, WI, USA) and 4.16 µl RNase-free water. Retrotranscription reaction (1:15) was mixed with master mix TaqMan (Universal PCR Master Mix, No AmpErase UNG, 2X; Thermo Fisher Scientific, Inc.), and the corresponding specific TaqMan PCR probe. PCR reaction was performed in a GeneAmp System 9700 (Applied Biosystems, Foster City, CA, USA) as follows: 95°C for 10 min, and 40 cycles at 95°C for 15 sec and 60°C for 1 min. Tests were normalized using RNU44 as control.

Let-7d-3p (90 nM) inhibitor (MH10785; Thermo Fisher Scientific, Inc.) and scramble (30 nM) sequence (AM17110, Thermo Fisher Scientific, Inc.) were used as negative control (https://www.thermofisher.com/order/genome-database/details/mirna/MC10785). Both, the inhibitor and scramble sequences were individually transfected into SKOV-3 cells using siPORT amine transfection agent (Ambion). Briefly, antagomiR let-7d-3p and scramble were added to wells containing 1×10⁷ SKOV-3 cells and incubated for 48 h. Then, total RNA was extracted using Trizol and efficacy of antagomiR treatment in endogenous let-7d-3p downregulation was evaluated by qRT-PCR using specific stem-looped RT oligonucleotide and TaqMan probe (ID: 001178; Thermo Fisher Scientific, Inc.) as implemented in the TaqMan MicroRNA Assays protocol.

SKOV-3 cells (1×10⁵) treated with let-7d-3p antagomiR (90 nM), or scramble sequence (30 nM) were seeded in triplicate in a 6-well plate and grown to 80% confluence. Twenty-four hours post-transfection a vertical wound was traced in the cell monolayer. After 12 and 24 h, cells were fixed with 4% paraformaldehyde and the scratched area was quantified. For migration assays, SKOV-3 cells (1×10⁵) were transfected with let-7d-3p antagomiR or scramble sequence, and then transferred to 0.5 ml serum-free medium and placed in the upper Transwell chambers (Corning Inc., Corning, NY, USA), whereas the lower chamber was loaded with 0.8 ml medium containing 10% FBS. The total number of cells that migrated into the lower chamber was manually counted after 24 h incubation at 37°C. Experiments were performed three times by triplicate and results are expressed as mean ± SD. P<0.05 was considered as statistically significant.

For cell proliferation studies, the MTT reagent [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to SKOV-3 cells (1×10⁵) and incubated for 3.5 h at 37°C. Then, dissolution buffer (99% isopropanol, 0.3% HCl, 0.7% NP-40) was added to cells and incubated for 15 min. Absorbance was recorded at 24 and 48 h using a spectrophotometer (570–630 nm). Data were analyzed using the BioStat software.

SKOV-3 cells (2×10⁵) were seeded by triplicate in a 6-well plate and treated for 48 h as follows: i) siPORT transfection agent (mock); ii) scramble sequence (negative control, 30 nM); and iii) let-7d-3p antagomiR (90 nM). Then, carboplatin (50 µM) was added to let-7d-3p inhibitor-transfected cells or alone in non-transfected cells and incubated for 24 h. Then, cells were harvested, washed twice with PBS 1X, resuspended in 100 µl buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂), and processed following the manufacturer's instructions (Annexin V-FLUOS staining kit; Roche Diagnostics, Basel, Switzerland). Briefly, cells were stained with 2 µl Annexin V-FITC and 2 µl propidium iodide (PI) mixed with 100 µl incubation buffer for 15 min, washed with 500 µl binding buffer and resuspended in 300 µl PBS 1X. Apoptosis events were analyzed on the FACSCalibur flow cytometer (BDIS; Becton-Dickinson, Franklin Lakes, NJ, USA). Annexin V and PI emissions were detected in the FL-1 and FL-2 channels, respectively. For each sample, data from 20,000 cells were acquired in list mode on logarithmic scales. Data were analyzed using the Summit V4.3 software and results were represented as the total percentage of apoptotic cells as the sum of both early and late phases of apoptosis (Annexin V-FITC-positive). Assays were performed by triplicate and data was expressed as mean ± SD. P<0.05 was considered as statistically significant.

miRNA target genes were predicted using TargetScan and PicTar software. Only gene targets predicted by the two algorithms were included in further analysis. Cellular pathways and processes potentially affected by let-7c-3p were predicted using DAVID 6.7 software.

Experiments were performed three times by triplicate and results are represented as mean ± SD. One-way analysis of variance (ANOVA) followed by Tukey's test were used to compare the differences between means. A P<0.05 was considered as statistically significant.

Ovarian tumor samples were collected between January 2010 and September 2012 from 34 patients diagnosed with EOC who underwent carboplatin/paclitaxel neoadjuvant treatment followed by debulking surgery at Instituto Nacional de Cancerologia. Tumor tissues were histologically analyzed by a pathologist to confirm at least 80% of tumor cells and then processed for total RNA isolation for downstream analysis. An overview of the major clinical and pathological features of tumors and patients included in the present study is provided in Table I. The average age of the patients at surgery was 52.1 years (range 35–74). Disease stage and tumor grade were classified according to the International Federation of Gynecology and Obstetrics (FIGO) and the World Health Organization (WHO) criteria, respectively. The majority of patients were diagnosed with stage III (44.1%) and IV (44.1%) disease, whereas 11.7% were at stage I. Tumor grade 1, 2 and 3 were found in 4, 3, and 17 women, respectively. The most common histology was serous papillary high grade (67.6%), and the remaining was endometrioid (17.6%), mucinous (9.0%), serous papillary low grade (3.0%), and clear cell (3.0%). At the time of diagnosis 16 (47%) patients showed no metastasis whereas 18 (53.0%) had distal metastasis. The median follow-up time was 34 months, ranging from 0.6 to 71 months. After neoadjuvant chemotherapy 17 patients (50%) were classified as complete responders and 2 (6%) as stable, whereas 12 (35.2%) had a partial response. Response was monitored by measuring the levels of cancer antigen 125 (CA125 or mucin 16) and CT scan. CA125 is a well-accepted protein marker found on the surface of ovarian cancer cells and diverse types of cancer. Serial measurements of CA125 were routinely used to monitor tumor response and survival during chemotherapy.

In order to study the expression of members of the let-7 family in primary EOC tumors and normal ovarian tissues, we used stem-loop qRT-PCR as implemented in MicroRNA assay protocol (Thermo Fisher Scientific, Inc.). After comparative 2^−ΔΔCt analyses we found that let-7a-3p and let-7d-3p were significantly (P<0.05) upregulated in ovarian tumors (n=40) in comparison to normal ovarian tissues (n=18) (Fig. 1A and B). In contrast, no significant change in the expression of let-7f between the groups was found (Fig. 1C).

A high percentage of patients diagnosed with locally advanced ovarian cancer have an unfavorable response to conventional treatment. This situation is aggravated as no useful molecular predictors for therapy response are currently used in clinical practice (14). We aimed to determine whether let-7 miRNA expression is associated with response to neoadjuvant therapy. We analyzed by qRT-PCR the expression of let-7 in ovarian cancer patients that had achieved a positive response and no response to carboplatin/paclitaxel regimen. Notably, data showed that the differential expression of let-7d-3p in ovarian tumors was able to discriminate between the patients that showed response to therapy and the non-responder group (P<0.036) (Fig. 2). In contrast, no significant differences in the expression of let-7a-3p and let-7f between the responder and no-responder groups were found.

Expression analysis of let-7 members allowed us to evidence that let-7d-3p was significantly upregulated in ovarian tumors in comparison to normal ovarian tissues. To study the biological relevance of let-7d-3p we first confirmed its upregulation in SKOV-3 ovarian cancer cells relative to ovarian normal tissues (Fig. 3A). Then, knockdown of let-7d-3p expression was performed using a specific antagomiR. Data showed that transfection of increasing concentrations (30, 60 and 90 nM) of let-7d-3p inhibitor significantly downregulated the endogenous let-7d-3p expression in a dose-dependent manner (Fig. 3B). We next investigated whether the forced inhibition of let-7d-3p had effects on cell proliferation in vitro. Data from the MTT assays showed that the growth rate of SKOV-3 cells transfected with let-7d-3p inhibitor (90 nM) was significantly (P<0.05) decreased up to 80% in comparison with non-transfected control cells after 96 h (Fig. 3C). Then, we performed scratch/wound-healing assays to evaluate the contribution of let-7d-3p inhibition in cell migration. Unexpectedly, data indicated no changes in the restoration of monolayers of cells transfected with antagomiR let-7d-3p (90 nM) in comparison to non-treated and scramble transfected control cells at 24 h (Fig. 3D and E). Similar results in SKOV-3 cell migration were obtained using Transwell chamber assays at 24 h (data not shown).

Standard treatment options for ovarian cancer patients include the use of platinum salt-based therapy. However, the effectiveness of this regimen is poor and additional therapeutic strategies are needed to improve clinical outcome. Therefore, we investigated whether the inhibition of let-7d-3p may induce apoptosis resulting in the potential sensitization of cancer cells to platinum chemotherapy. The number of apoptotic cancer cells in cultures treated with the let-7d-3p inhibitor was assessed using Annexin V-FITC assays. Our results showed that the percentage of apoptotic cells was significantly increased (P<0.05) from 9.8% in the non-treated control cells to 29.6% in cells treated with let-7d-3p inhibitor for 48 h (Fig. 4A and B). As expected, carboplatin monotherapy used as a control of cell death resulted in a marked increase in apoptotic cells (64.5%) in comparison to mock and scramble transfected control cells. Notably, a significant increase (P<0.05) in cell death up to 93.8% was found in let-7d-3p-deficient cells treated with carboplatin in comparison to the controls indicating a synergistic effect in apoptosis exerted by antagomiR therapy (Fig. 4A and B).

To evaluate the potential chemosensitizing effect of let-7d-3p, we next analyzed the cell viability effects of its inhibition in combination with carboplatin cytotoxic therapy. Data showed that while treatment with the let-7d-3p inhibitor (90 nM) alone slightly affected ovarian cancer cell viability, a combination of let-7d-3p plus carboplatin (5 mM) resulted in a marked increase in cell cytotoxicity (Fig. 4C). These data suggested that let-7d-3p sensitizes SKOV-3 cells to carboplatin therapy, at least in part, by cell death induction.

Prediction of let-7d-3p targets based on GO categories identified several genes involved in key cellular processes and signaling pathways related to tumor development, progression and drug-resistance including ABC transporters, ErbB, RAS and HIF-1 pathways (Fig. 5A and B). For example, activation of ErbB signaling contributes to chemoradiotherapy resistance phenotypes in ovarian, breast and cervical cancer, suggesting that let-7d-3p overexpression could be associated with a complete response to therapy in ovarian cancer through similar modulation of ErbB signaling (16–18). However, additional experimental data is needed to confirm this hypothesis.