Low Expression of miR-18a as a Characteristic of Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) occurs in both adults and children but the response to chemotherapy and survival is significantly worse in the adults. We aimed to study whether the expression of immune system-associated miRNAs would differ between adult and pediatric patients with ALL at the time of diagnosis. MATERIALS AND METHODS: Inflammation-associated miRNA analysis was performed in 19 adults and 79 pediatric patients with ALL and involved miR-10, miR-15, miR-16, miR-17-92 cluster, miR-33, miR-146a, miR-150, miR-155, miR-181a, miR-222, miR-223, and miR-339. MiRNAs were first analyzed by miRNA microarray and thereafter validated by qRT-PCR. Sufficient RNA for qRT-PCR was available for 42 pediatric and 19 adult patients. RESULTS: Of the studied miRNAs, only miR-18a differed significantly in microarray analysis between adult and pediatric ALL, being lower in children (FC, -3.74; P, 0.0037). Results were confirmed by qRT-PCR (down-regulated in pediatric patients, P 0.003161). The other members of the miR-17-92 cluster did not differ significantly. CONCLUSIONS: Pediatric and adult patients with ALL have remarkably similar patterns of immune-cell-associated miRNAs in their bone marrow at diagnosis. However, the low expression of miR-18a in pediatric ALL is interesting and demands further study.

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro.

Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated "Focal adhesion" and "ECM-receptor interaction" as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren't significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.

Downregulation of Plasma MiR-142-3p and MiR-26a-5p in Patients With Colorectal Carcinoma.

BACKGROUND: Colorectal cancer is one of the most commonly diagnosed cancers and cancer- related death worldwide. Identification of new specific biomarkers could be helpful to detection of this malignancy. Altered plasma microRNA expression has been identified in many cancers, including colorectal cancer. OBJECTIVES: The main objective of this study was to identify the circulating microRNAs with the most expression changes in colorectal cancer patients compared with neoplasm free healthy individuals. MATERIALS AND METHODS: MicroRNA expression profiling was performed on plasma samples of 37 colorectal cancer patients and 8 normal subjects using microRNA microarray. Quantitative real-time reverse transcription polymerase chain reaction was used to validate the two selected altered microR NAs. Plasma samples from 61 colorectal cancer patients and 24 normal subjects were used in our validation study. RESULTS: In profiling study we found a panel of six plasma microRNAs with significant downregulation. MicroRNA-142-3p and microRNA-26a-5p were selected and validated by polymerase chain reaction. Our results demonstrated that expression levels of plasma microRNA-142-3p and microRNA-26a-5p were significantly downregulated in patients with colorectal cancer when compared to control group. CONCLUSIONS: Our findings suggest that downregulation of plasma microRNA-142-3p and microRNA-26a-5p might serve as novel noninvasive biomarkers in the diagnosis of colorectal cancer, although more studies are needed to highlight the theoretical strengths.

Simultaneous Underexpression of let-7a-5p and let-7f-5p microRNAs in Plasma and Stool Samples from Early Stage Colorectal Carcinoma.

Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer death worldwide. Early detection of CRC can improve patient survival rates; thus, the identification of noninvasive diagnostic markers is urgently needed. MicroRNAs (miRNAs) have extensive potential to diagnose several diseases, including cancer. In this study, we compared the expression pattern of miRNAs from plasma and stool samples of patients with early stages of CRC (I, II) with that of healthy subjects. We performed miRNA profiling using microarrays on plasma and stool samples of eight patients with CRC and four healthy subjects. Seven miRNAs were found to be underexpressed in both plasma and stool samples of patients with CRC versus healthy subjects. Then, we aimed to verify two out of these seven differentially expressed miRNAs (let-7a-5p and let-7f-5p) by quantitative reverse transcriptase polymerase chain reaction on a larger set of plasma and stool samples of 51 patients with CRC and 26 healthy subjects. We confirmed the results of microarray analysis since their expression was significantly lower in stool and plasma samples of patients with CRC. Moreover, receiver operating characteristic curve analysis demonstrated that fecal let-7f expression levels have significant sensitivity and specificity to distinguish between patients with CRC and healthy subjects. In conclusion, if the results are confirmed in larger series of patients, underexpressed let-7a-5p and let-7f-5p miRNAs in both plasma and stool samples of patients with CRC may serve potentially as noninvasive molecular biomarkers for the early detection of CRC.

miRNA-34a underexpressed in Merkel cell polyomavirus-negative Merkel cell carcinoma.

Merkel cell polyomavirus (MCV) is frequently detectable in Merkel cell carcinoma (MCC) tumors, but the significance of MCV infection is not yet totally understood. Thus far, no key regulatory miRNA has been identified for MCC tumorigenesis. However, distinct miRNA expression profiles have been suggested for MCV-positive and MCV-negative tumors. We used microarray hybridization to identify miRNA expression differences in MCC tumor samples according to MCV status and further validated these results by quantitative reverse transcription polymerase chain reaction (qRT-PCR). When compared with MCV-negative tumors, we detected overexpression of miR-34a, miR-30a, miR-142-3p, and miR-1539 in those MCV positives. In addition, slight underexpression was detectable in MCV-positive tumors of miR-181d. We confirmed the distinct expression of miRNAs in MCV-positive and MCV-negative tumors and confirmed statistically significant underexpression of miR-34a in MCV-negative tumors by both array analysis and qRT-PCR. Neither tumor location nor development of metastases affected miRNA expression.

Decreased expression of fecal miR-4478 and miR-1295b-3p in early-stage colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a major cause of cancer-related deaths world-wide. Detection of molecular markers in stool samples is a promising strategy for CRC screening. MicroRNAs (miRNAs) are short, non-coding RNA molecules that are commonly dysregulated in neoplasia. OBJECTIVE: The objective of this study was to evaluate the fecal miRNAs differentiation between early-stage CRC patients and healthy subjects. METHODS: Stool samples were collected from 40 patients with early stage (I, II) CRC and 16 healthy controls. RNA was extracted from all samples using miRNAeasy Mini Kits. MiRNA microarray expression profiling was performed with Agilent's miRNA Microarray system on 12 CRC and 8 normal stool samples. The expression levels of miR-4478 and miR-1295b-3p were determined by the SYBR Green miScript PCR system. RESULTS: In profiling study, we found 215 down-regulated miRNAs in CRC group. Furthermore, in validation study we found that the expression levels of fecal miR-4487 and miR-1295b-3p were significantly decreased in CRC patients compared to healthy controls. CONCLUSIONS: The expression of miR-4478 and miR-1295b-3p were significantly diminished in stool samples of CRC patients with early stage (I, II) in comparison with normal group. These miRNAs maybe use as potential non-invasive molecular markers for CRC diagnosis, but further studies are needed.

Differentiating soft tissue leiomyosarcoma and undifferentiated pleomorphic sarcoma: A miRNA analysis.

The rare and highly aggressive adult soft tissue sarcomas leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS) contain complex genomes characterized by a multitude of rearrangements, amplifications, and deletions. Differential diagnosis remains a challenge. MicroRNA (miRNA) profiling was conducted on a series of LMS and UPS samples to initially investigate the differential expression and to identify specific signatures useful for improving the differential diagnosis. Initially, 10 high-grade LMS and 10 high-grade UPS were profiled with a miRNA microarray. Two cultured human mesenchymal stem cell samples were used as controls. 38 and 46 miRNAs classified UPS and LMS samples, respectively, into separate groups compared to control samples. When comparing the two profiles, miR-199b-5p, miR-320a, miR-199a-3p, miR-126, miR-22 were differentially expressed. These were validated by RT-PCR on a further series of 27 UPS and 21 LMS for a total of 68 cases. The levels of miR-199-5p and miR-320a, in particular, confirmed the microarray data, the former highly expressed in UPS and the latter in LMS. Immunohistochemistry was performed on all 68 cases to confirm original diagnosis. Recently reported LMS- and UPS-associated genes were correlated with miRNA targets based on target algorithms of three databases. Several genes including IMP3, ROR2, MDM2, CDK4, and UPA, are targets of differentially expressed miRNAs. We identified miRNA expression patterns in LMS and UPS, linking them to chromosomal regions and mRNA targets known to be involved in tumor development/progression of LMS and UPS.

Hsa-miR-31-3p expression is linked to progression-free survival in patients with KRAS wild-type metastatic colorectal cancer treated with anti-EGFR therapy.

PURPOSE: To identify microRNAs (miRNA) that predict response to anti-EGFR antibodies in patients with wild-type KRAS metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: miRNA profiling was performed in a training set of 87 patients with mCRC refractory to chemotherapy treated with anti-EGFR antibodies. This included 33 fresh-frozen (FF) and 35 formalin-fixed paraffin-embedded (FFPE) samples retrospectively collected and 19 prospectively collected FF samples. An independent validation cohort consisting of 19 FF and 26 FFPE prospectively collected samples from patients with mCRC treated with anti-EGFR antibodies was used to confirm our findings. RESULTS: After screening the expression of 1,145 miRNAs in FF samples from the training set, we identified that hsa-miR-31-3p expression level was significantly associated with progression-free survival (PFS). Statistical models based on miRNA expression discriminated between high and low risk of progression for both FF and FFPE samples. These models were confirmed in the validation cohort for both FF [HR, 4.1; 95% confidence interval (CI), 1.1-15.3; P < 0.04] and FFPE samples (HR, 2.44; 95% CI, 1.1-5.4; P = 0.028). The percentage of variation of RECIST criteria in the validation series was significantly associated with the expression level of hsa-miR-31-3p (r(2) = 0.49; P = 0.0035) and risk status determined by hsa-miR-31-3p expression level (P = 0.02, Kruskal-Wallis rank test). Nomograms were built and validated to predict PFS-depending on hsa-miR-31-3p expression level. Following in vitro studies, we identified 47 genes regulated by hsa-miR-31-3p. CONCLUSION: Hsa-miR-31-3p seems to be a new mCRC biomarker whose expression level allows for the identification of patients with wild-type KRAS mCRC who are more likely to respond to anti-EGFR therapy.

Down-regulation of miR-181c in imatinib-resistant chronic myeloid leukemia.

The association of microRNA alterations with progression and treatment outcome has been revealed in different types of cancers. To find miRNAs involved in imatinib response we performed miRNA microarray followed by RT-qPCR verification of 9 available diagnostic bone marrow core biopsies from 9 CML patients including 4 imatinib-resistant and 5 imatinib-responder patients. Only one differentially expressed miRNA, miR-181c, was found when the imatinib-resistant group was compared with imatinib-responders. Significant down-regulation of miR-181c in imatinib-resistant versus imatinib-responders was confirmed by qRT-PCR. Some miR-181c target genes such as PBX3, HSP90B1, NMT2 and RAD21 have been associated with drug response.

MicroRNA expression profiles in metastatic and non-metastatic giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a skeletal neoplasm, a locally aggressive tumor that occasionally metastasizes to the lungs. To identify novel biomarkers associated with GCTB progression and metastasis, we performed a miRNA microarray on ten primary tumors of GCTB, of which five developed lung metastases and the rest remained metastasis-free. Between metastatic and non-metastatic GCTB, 12 miRNAs were differentially expressed (such as miR-136, miR-513a-5p, miR-494, miR-224, and miR-542-5p). A decreased level of miR-136 in metastatic versus non-metastatic GCTB was significantly confirmed by the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (p=0.04). To identify potential target genes for the differentially expressed miRNAs, we used three target prediction databases. Then, to functionally validate the potential target genes of the differentially expressed miRNAs, we re-analyzed our previous gene expression data from the same ten patients. Eight genes such as NFIB, TNC, and FLRT2 were inversely expressed relative to their predicted miRNA regulators. NFIB expression correlated in metastatic GCTB with no or low expression of miR-136, and this gene was selected for further verification with qRT-PCR and immunohistochemistry. Verification of NFIB mRNA and protein by qRT-PCR showed elevated expression levels in metastatic GCTBs. Further, the protein expression level of NFIB was tested in an independent validation cohort of 74 primary archival GCTB specimens. In the primary tumors that developed metastases compared to the disease-free group, NFIB protein was moderately to strongly expressed at a higher frequency. Thus, in GCTB, miR-136 and NFIB may serve as prognostic makers.

MicroRNA profiling predicts survival in anti-EGFR treated chemorefractory metastatic colorectal cancer patients with wild-type KRAS and BRAF.

Anti-EGFR monoclonal antibodies (anti-EGFRmAb) serve in the treatment of metastatic colorectal cancer (mCRC), but patients with a mutation in KRAS/BRAF and nearly one-half of those without the mutation fail to respond. We performed microRNA (miRNA) analysis to find miRNAs predicting anti-EGFRmAb efficacy. Of the 99 mCRC patients, we studied differential miRNA expression by microarrays from primary tumors of 33 patients who had wild-type KRAS/BRAF and third- to sixth-line anti-EGFRmAb treatment, with/without irinotecan. We tested the association of each miRNA with overall survival (OS) by the Cox proportional hazards regression model. Significant miR-31* up-regulation and miR-592 down-regulation appeared in progressive disease versus disease control. miR-31* expression and down-regulation of its target genes SLC26A3 and ATN1 were verified by quantitative reverse transcriptase polymerase chain reaction. Clustering of patients based on miRNA expression revealed a significant difference in OS between patient clusters. Members of the let-7 family showed significant up-regulation in the patient cluster with poor OS. Additionally, miR-140-5p up-regulation and miR-1224-5p down-regulation were significantly associated with poor OS in both cluster analysis and the Cox proportional hazards regression model. In mCRC patients with wild-type KRAS/BRAF, miRNA profiling can efficiently predict the benefits of anti-EGFRmAb treatment. Larger series of patients are necessary for application of these miRNAs as predictive/prognostic markers.

An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma.

BACKGROUND: Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers. METHOD: Microarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods. RESULTS: The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers. CONCLUSION: In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

MicroRNA profiling differentiates colorectal cancer according to KRAS status.

Recent studies have shown the important role of microRNAs (miRNAs) in a variety of biological processes, and in its ability to distinguish tumors according to their prognostic and predictive properties. To identify miRNA signatures associated with colorectal carcinoma (CRC) and with KRAS status, we studied, using Agilent's miRNA microarrays, miRNA expression in primary tumors from 55 metastatic CRC patients, including 15 with mutant and 40 with wild-type KRAS. Comparing these with normal colon tissue, we identified 49 miRNAs--including 19 novel miRNAs--significantly deregulated in tumor tissue. The presence of the KRAS mutation was associated with up-regulation of miR-127-3p, miR-92a, and miR-486-3p and down-regulation of miR-378. Increased expression of miR-127-3p and miR-92a in KRAS mutant tumors was significantly confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (P < 0.05). We identified some predicted target genes of differentially expressed miRNAs between mutated and wild-type KRAS, such as RSG3 and TOB1, which are involved in apoptosis and proliferation. Target prediction and pathway analysis suggest a possible role for deregulated miRNAs in nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and G protein-coupled receptor signaling pathways.

Unique microRNA profile in Dupuytren's contracture supports deregulation of ß-catenin pathway.

Dupuytren's contracture, a proliferative disease of unknown origin, is characterized by an abnormal fibroblast proliferation process. Evidence from numerous microRNA (miRNA) studies shows that miRNAs have a vital function in many biological processes, for instance, in cellular signaling networks, cell growth, tissue differentiation, and cell proliferation. Our aim was to characterize, to our knowledge for the first time, the miRNA-expression profile of Dupuytren's contracture. The miRNAs identified may have a function in the pathogenesis of Dupuytren's contracture by targeting and regulating important pathways. We compared the miRNA-expression profile of 29 Dupuytren's contracture patients with that of control samples (fibroblast cells and palmar fascia). Some of the miRNAs identified in our Dupuytren's contracture samples, including miR-29c, miR-130b, miR-101, miR-30b, and miR-140-3p, were found to regulate important genes related to the ß-catenin pathway: WNT5A, ZIC1, and TGFB1. Expression profiles of these genes reanalyzed from published gene-expression data from similar patient material correlated with our miRNA results. Analysis was also performed for groups of patients with recurrent/non-recurrent and patients with hereditary/non-hereditary Dupuytren's contracture, but no significant differences appeared in miRNA-expression profiles of these groups. Identification of unique miRNA expression in Dupuytren's contracture may lead to the development of novel molecular therapy for its treatment.