mir-660-p53-mir-486 Network: A New Key Regulatory Pathway in Lung Tumorigenesis.

Lung cancer is the most frequent cause of cancer-related death worldwide, with limited therapeutic options and rapid development of drug resistance. MicroRNAs, a class of small non-coding RNAs that control different physiological processes, have been associated with cancer development, as either oncomiRNAs or tumor-suppressor miRNAs. In the present study we investigated the interaction between mir-486-5p and mir-660-5p, two independent tumor-suppressor miRNAs, to assess their possible role and synergistic effect in lung cancer treatment. Our data show that mir-660-5p over-expression in A549 lung cancer cells induced a remarkable increase in mir-486-5p expression level and activity, detected as a reduction of its target gene, p85. mir-486-5p expression was confirmed by microRNA in situ hybridization. mir-660-5p modulated mir-486-5p through the silencing of Mouse Double Minute 2 (MDM2), one of its direct target, and then through p53 stimulation. This regulatory pathway was effective in A549, but not in H1299; therefore, only in the context of a functional p53 protein. Our findings support the conclusion that mir-486-5p is positively regulated by mir-660-5p in lung cancer cell lines, through the mir-660-MDM2-p53 pathway, making mir-660-5p even more interesting for its potential successful use in lung cancer therapy.

c-Myc shuttled by tumour-derived extracellular vesicles promotes lung bronchial cell proliferation through miR-19b and miR-92a.

Lung cancer causes approximately one fifth of all cancer deaths. Tumour cells actively communicate with the surrounding microenvironment to support malignant progression. Extracellular vesicles (EVs) play a pivotal role in intercellular communication and modulate recipient cells by delivering their contents, including proteins and nucleic acids such as microRNAs (miRNAs). We isolated EVs from the conditioned medium (CM) of human lung cancer cell lines and plasma of lung cancer patients and cancer-free smokers using an ultracentrifugation method. A significant increase in bronchial HBEC-KRASV12high cell proliferation, confirmed by cell cycle analysis, was observed after treatment with cancer-derived EVs. Lung cancer-derived EVs induced transcription of the pri-miR-92a gene, resulting in the overexpression of mature miR-19b and miR-92a in recipient bronchial cells. Modulation of these two miRNAs using miRNA mimics or inhibitors confirmed their ability to promote proliferation. In silico analysis and experimental validation showed that miR-19b and miR-92a impaired the TGF-beta (TGFB) pathway and identified TGFBRI and TGFBRII as target genes involved in EV-mediated bronchial cell proliferation. Interestingly, the oncoprotein c-Myc, a well-known miR-17-92 cluster activator, was detected only in the EVs derived from lung cancer patients and cell lines and was able to modulate the proliferation of HBEC-KRASV12high recipient cells. These data support the role of c-Myc shuttling in lung cancer-derived EVs in inducing the upregulation of onco-miR-19b and miR-92a expression with concomitant impairment of the TGFB signalling pathway in recipient cells.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 is a Novel Post-Transcriptional Regulator of Ewing Sarcoma Malignancy.

Purpose: Large-scale sequencing studies have indicated that besides genomic alterations, the posttranscriptional regulation of gene expression or epigenetic mechanisms largely influences the clinical behavior of Ewing sarcoma. We investigated the significance of the RNA-binding protein IGF2BP3 in the regulation of Ewing sarcoma aggressiveness.Experimental Design: Explorative study was performed in 14 patients with localized Ewing sarcoma using RNA sequencing. Next, 128 patients with localized Ewing sarcoma were divided into two cohorts. In the training set, 29 Ewing sarcoma samples were analyzed using Affymetrix GeneChip arrays. In the validation set, 99 Ewing sarcoma samples were examined using qRT-PCR. Patient-derived cell lines and experimental models were used for functional studies.Results:Univariate and multivariate analyses indicated IGF2BP3 as a potent indicator of poor prognosis. Furthermore, ABCF1 mRNA was identified as a novel partner of IGF2BP3. Functional studies indicated IGF2BP3 as an oncogenic driver and ABCF1 mRNA as a sponge that by binding IGF2BP3, partly repressed its functions. The combined evaluation of IGF2BP3 and ABCF1 could identify different patient outcomes-high IGF2BP3 and low ABCF1 levels indicated poor survival (25%), whereas low IGF2BP3 and high ABCF1 levels indicated favorable survival (85.5%). The bromodomain and extraterminal domain inhibitor (BETi) JQ1 decreased IGF2BP3 expression, modified the expression of its validated targets and inhibited the capability of Ewing sarcoma cells to grow under anchorage-independent conditions.Conclusions: The combined assessment of IGF2BP3 and ABCF1 predicts recurrence in Ewing sarcoma patients. Thus, for patients with high expression of IGF2BP3 and poor probability of survival, the use of BETis should be clinically evaluated. Clin Cancer Res; 24(15); 3704-16. ©2018 AACR.

Detection of microRNAs Using Chip-Based QuantStudio 3D Digital PCR.

Digital PCR (dPCR) is an innovative approach for detection and quantification of nucleic acid that offers an alternative method to conventional real-time quantitative PCR for absolute quantification. dPCR is a highly precise and sensitive technique that does not require a standard reference, making it a suitable method for the detection of microRNAs. The potential of these small noncoding RNA as biomarkers is on the rise, especially due to their presence in body fluids, making them easily accessible. Nevertheless, the problem of lack of consensus regarding an optimal method for miRNAs normalization compromises their use. Here, we describe an innovative method for the absolute quantification of miRNAs across different types of biological samples using a chip-based platform, the QuantStudio 3D digital PCR.