The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker.

BackgroundThe microRNA 125b is a double-faced gene expression regulator described both as a tumor suppressor gene (in solid tumors) and an oncogene (in hematologic malignancies). In human breast cancer, it is one of the most down-regulated miRNAs and is able to modulate ERBB2/3 expression. Here, we investigated its targets in breast cancer cell lines after miRNA-mimic transfection. We examined the interactions of the validated targets with ERBB2 oncogene and the correlation of miR-125b expression with clinical variables.MethodsMiR-125b possible targets were identified after transfecting a miRNA-mimic in MCF7 cell line and analyzing gene expression modifications with Agilent microarrays and Sylamer bioinformatic tool. Erythropoietin (EPO) and its receptor (EPOR) were validated as targets of miR-125b by luciferase assay and their expression was assessed by RT-qPCR in 42 breast cancers and 13 normal samples. The molecular talk between EPOR and ERBB2 transcripts, through miR-125b, was explored transfecting MDA-MD-453 and MDA-MB-157 with ERBB2 RNA and using RT-qPCR.ResultsWe identified a panel of genes down-regulated after miR-125b transfection and putative targets of miR-125b. Among them, we validated erythropoietin (EPO) and its receptor (EPOR) - frequently overexpressed in breast cancer - as true targets of miR-125b. Moreover, we explored possible correlations with clinical variables and we found a down-regulation of miR-125b in metastatic breast cancers and a significant positive correlation between EPOR and ERBB2/HER2 levels, that are both targets of miR-125b and function as competing endogenous RNAs (ceRNAs).ConclusionsTaken together our results show a mechanism for EPO/EPOR and ERBB2 co-regulation in breast cancer and confirm the importance of miR-125b in controlling clinically-relevant cancer features.

BackgroundThe anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (moAbs) cetuximab or panitumumab are administered to colorectal cancer (CRC) patients who harbor wild-type RAS proto-oncogenes. However, a percentage of patients do not respond to this treatment. In addition to mutations in the RAS genes, mutations in other genes, such as BRAF, PI3KCA, or PTEN, could be involved in the resistance to anti-EGFR moAb therapy.MethodsIn order to develop a comprehensive approach for the detection of mutations and to eventually identify other genes responsible for resistance to anti-EGFR moAbs, we investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab. Among these, 37 samples were responsive and 28 were resistant.ResultsWe confirmed that mutations in EGFR-pathway genes (KRAS, NRAS, BRAF, PI3KCA) were relevant for conferring resistance to therapy and could predict response (p = 0.001). After exclusion of KRAS, NRAS, BRAF and PI3KCA combined mutations could still significantly associate to resistant phenotype (p = 0.045, by Fisher exact test). In addition, mutations in FBXW7 and SMAD4 were prevalent in cases that were non-responsive to anti-EGFR moAb. After we combined the mutations of all genes (excluding KRAS), the ability to predict response to therapy improved significantly (p = 0.002, by Fisher exact test).ConclusionsThe combination of mutations at KRAS and at the five gene panel demonstrates the usefulness and feasibility of multigene sequencing to assess response to anti-EGFR moAbs. The application of parallel sequencing technology in clinical practice, in addition to its innate ability to simultaneously examine the genetic status of several cancer genes, proved to be more accurate and sensitive than the presently in use traditional approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1752-5) contains supplementary material, which is available to authorized users.

Taken together, these results indicate that naturally occurring cytokinesis failure in primary cardiomyocytes is due to an aberrant mitotic microtubule apparatus resulting in inefficient anchorage of the actomyosin ring to the plasma cell membrane. Thus, cardiomyocyte binucleation and division can be discriminated by the analysis of RhoA as well as IQGAP3 localization.

microRNA miR-221 is frequently over-expressed in a variety of human neoplasms. Aim of this study was to identify new miR-221 gene targets to improve our understanding on the molecular tumor-promoting mechanisms affected by miR-221. Gene expression profiling of miR-221-transfected-SNU-398 cells was analyzed by the Sylamer algorithm to verify the enrichment of miR-221 targets among down-modulated genes. This analysis revealed that enforced expression of miR-221 in SNU-398 cells caused the down-regulation of 602 mRNAs carrying sequences homologous to miR-221 seed sequence within their 3′UTRs. Pathways analysis performed on these genes revealed their prominent involvement in cell proliferation and apoptosis. Activation of E2F, MYC, NFkB, and β-catenin pathways was experimentally proven. Some of the new miR-221 target genes, including RB1, WEE1 (cell cycle inhibitors), APAF1 (pro-apoptotic), ANXA1, CTCF (transcriptional repressor), were individually validated as miR-221 targets in SNU-398, HepG2, and HEK293 cell lines. By identifying a large set of miR-221 gene targets, this study improves our knowledge about miR-221 molecular mechanisms involved in tumorigenesis. The modulation of mRNA level of 602 genes confirms the ability of miR-221 to promote cancer by affecting multiple oncogenic pathways.