microRNA Expression In Tissues Research Articles

LGG-33. NETWORK ANALYSIS INTEGRATING MICRORNAS AND CLINICAL DATA TO DETERMINE PROGNOSTIC OUTCOME IN PEDIATRIC LOW-GRADE GLIOMA PATIENTS

Abstract BACKGROUND Low-grade pediatric gliomas (pLGGs) are the most common pediatric brain tumors. Surgical resection, when feasible, represents the standard of care. If complete resection cannot be achieved, disease progression occurs in about 30% of the cases, and these patients receive adjuvant therapy. The identification of predictive biomarkers to predict tumors that will progress after partial resection, requiring systemic treatment (Chemotherapy or target therapy), could support clinicians in the decision-making strategy. MicroRNAs have been related to tumorigenesis and tumor maintenance and can be used as important biomarkers. This study aims to develop a predictive model able to determine prognostic outcomes and to better stratify pLGG patients according to disease progression. METHODS MicroRNAs levels have been assessed in 76 human tumor tissue samples. Clinical data have been collected for statistical and network-based analyses. Using a network-based approach, we integrated clinical data and tissue microRNA expression to identify a microRNA signature able to predict disease progression. RESULTS Our preliminary microRNA expression analysis displayed new promising signatures correlating with prognostic features, such as disease progression and embryological origin of the tumor. CONCLUSION The predictive model we developed integrating biomarkers and clinical data could be crucial to support clinicians in the decision-making strategy and in advancing personalized medicine.

Changes in subcutaneous adipose tissue microRNA expression in response to exercise training in African women with obesity

The mechanisms that underlie exercise-induced adaptations in adipose tissue have not been elucidated, yet, accumulating studies suggest an important role for microRNAs (miRNAs). This study aimed to investigate miRNA expression in gluteal subcutaneous adipose tissue (GSAT) in response to a 12-week exercise intervention in South African women with obesity, and to assess depot-specific differences in miRNA expression in GSAT and abdominal subcutaneous adipose tissue (ASAT). In addition, the association between exercise-induced changes in miRNA expression and metabolic risk was evaluated. Women underwent 12-weeks of supervised aerobic and resistance training (n = 19) or maintained their regular physical activity during this period (n = 12). Exercise-induced miRNAs were identified in GSAT using Illumina sequencing, followed by analysis of differentially expressed miRNAs in GSAT and ASAT using quantitative real-time PCR. Associations between the changes (pre- and post-exercise training) in miRNA expression and metabolic parameters were evaluated using Spearman’s correlation tests. Exercise training significantly increased the expression of miR-155-5p (1.5-fold, p = 0.045), miR-329-3p (2.1-fold, p < 0.001) and miR-377-3p (1.7-fold, p = 0.013) in GSAT, but not in ASAT. In addition, a novel miRNA, MYN0617, was identified in GSAT, with low expression in ASAT. The exercise-induced differences in miRNA expression were correlated with each other and associated with changes in high-density lipoprotein concentrations. Exercise training induced adipose-depot specific miRNA expression within subcutaneous adipose tissue depots from South African women with obesity. The significance of the association between exercise-induced miRNAs and metabolic risk warrants further investigation.

MicroRNA expression in tissue and circulating tumor cells of patients with stage II-IV colon cancer.

e15502 Background: The purpose of this study was a comparative analysis of the expression of miRNAs in the tumor and circulating tumor cells (CTCs) in colon cancer (CC). Methods: Expression of seven miRNAs (hsa-let-7i-5p, hsa-miR-126-5p, hsa-miR-143-3p, hsa-miR-21-5p, hsa-miR-25-3p, hsa-miR-26a-5p, hsa-miR-92a-3p) were determined by real-time PCR in tumors of 200 patients with stage II-IV CC compared with normal colon tissue; levels of CTCs were determined by the CellSearch, and at CTC &gt; 3, CTCs were isolated and the expression of the same miRNAs was studied in them. Results: Tumor tissues showed statistically significant (p &lt; 0.0005) changes, compared to normal tissues, in the expression of hsa-let-7i-5p (increased by 4.2 times in stage IV), hsa-miR-126-5p (increased by 2.0; 2.1 and 2.9 times in stages II, III and IV, respectively), hsa-miR-143-3p (decreased by 3.3 times in stage IV), hsa-miR-21-5p (increased by 3.9 and 4.8 times in stages III and IV), hsa-miR-25-3p (increased by 3.2 times in stage IV), hsa-miR-26a-5p (decreased by 10.0; 5.0 and 6.7 times in stages II, III and IV, respectively) and hsa-miR-92a-3p (increased by 2.2; 5.1 and 9.5 times in stages II, III and IV, respectively). We observed changes in the expression of hsa-let-7i-5p (increased by 3.4 times), hsa-miR-143-3p (decreased by 3.4 times), hsa-miR-21-5p (increased by 3.2 times) and hsa-miR-92a-3p (increased by 4.3 times) in tumors of patients with stage IV disease, compared to stage II (p &lt; 0.005). CTC expression of miRNAs hsa-miR-143-3p, hsa-miR-21-5p, hsa-miR-26a-5p in patients with lymph node metastases, compared to patients without metastases, was decreased by 2.5; 3.6; 5.0 times (p &lt; 0.05) respectively, and expression of hsa-miR-92a-3p was elevated by 3.0 times (p &lt; 0.05). In patients with liver metastases, CTC expression of hsa-miR-143-3p, hsa-miR-21-5p, hsa-miR-25-3p, hsa-miR-26a-5p was statistically significantly (p &lt; 0.05) lower by 4.6; 5.5; 1.7; 5.3 times, respectively, compared to the CTC expression in patients without metastases, and expression of hsa-miR-126-5p and hsa-miR-92a-3p was higher by 2.6 and 5.0 times, respectively, compared to the CTC expression in patients without metastases (p &lt; 0.05). CTC expression of hsa-miR-143-3p was 1.8 times lower (p &lt; 0.05), and expression of hsa-miR-92a-3p was 1.7 times (p &lt; 0.05) higher in patients with distant metastases, compared to patients with regional metastases. Conclusions: On the whole, the miRNA expression profile in the tumor and CTCs in CC were similar, although there were some differences. Tumor tissues in stage IV patients were characterized by overexpression of hsa-let-7i-5p, which is not typical for CTCs. The levels of the tumor suppressor hsa-miR-26a-5p were reduced in tumors of different stages to similar values, but differed in CTCs, which allows differentiation between non-metastatic CC and metastatic CRC.

Expression of specific microRNAs in tissue and plasma in colorectal cancer.

MicroRNAs (miRNA/miR) play significant roles in the regulation of cell differentiation, cell cycle progression, and apoptosis. They become dysregulated during carcinogenesis and are eventually released into the circulation, enabling their detection in body fluids. Thus, this study compared the miRNA expression in tissue and plasma samples of colorectal cancer (CRC) patients and clinically healthy controls and determined miRNA expression as a potential CRC biomarker. Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), miR-21-5p, miR-29a-3p, miR-92a-3p, miR-135b-5p, miR-196b-5p, and miR-197-3p, expression was analyzed and compared between the malignant (n = 41) and the adjacent neoplasm free mucosal tissues (n = 41) of CRC patients. The findings were validated in plasma samples (n = 36) collected from the same CRC patients prior to surgery or any form of treatment and compared to plasma from their age and sex-matched controls (n = 36). MiR-21-5p, miR-29a-3p, miR-92a-3p, and miR- 196b-5p were upregulated and miR-135b-5p was downregulated in CRC malignant tissues compared to their expression in adjacent neoplasm-free tissue. This was further observed in the plasma of the same CRC cases compared to controls. MiR-92a-3p showed itself the most sensitive (0.93; p < .001) and most specific (0.95; p < .001) in detecting CRC in tissue. In plasma, miR-196b-5p was the most sensitive (0.97; p < .001) and specific (0.94; p < .001) in detecting CRC. Plasma miR-92a-3p and miR-196b-5p were the most sensitive (0.95; p < .001) and specific (0.94; p < .001) in the early detection of CRC. Results show that specific miRNAs dysregulated in malignant tissues are released and can be detected in the circulation, supporting their potential as non-invasive biomarkers of CRC.

Changes in adipose tissue microRNA expression across the menstrual cycle in regularly menstruating females: a pilot study.

Cyclical changes in hormone profiles across the menstrual cycle are associated with alterations in metabolic control. MicroRNAs (miRNAs) contribute to regulating metabolic control, including adipose tissue metabolism. How fluctuations in hormonal profiles across the menstrual cycle affect adipose tissue miRNA expression remains unknown. Eleven healthy, regularly menstruating females underwent four sampling visits across their menstrual cycle. Subcutaneous abdominal adipose tissue and venous blood samples were collected at each sampling visit. Luteinizing hormone (LH) tests, calendar counting, and serum hormone concentrations were used to determine menstrual cycle phases: early-follicular (EF), late-follicular (LF), postovulatory (PO), and midluteal (ML). Serum follicle-stimulating hormone, LH, estrogen, progesterone, and testosterone were determined using multiplex magnetic bead panels and enzyme-linked immunosorbent assays. Global adipose tissue miRNA expression levels were determined via microarray in a subset of participants (n = 8) and 17 candidate miRNAs were validated by RT-qPCR in the whole cohort (n = 11). Global analysis of adipose tissue miRNA expression identified 33 miRNAs significantly altered across the menstrual cycle; however, no significant differences remained after correcting for multiple testing (P > 0.05). RT-qPCR analysis of candidate miRNAs revealed miR-497-5p expression was significantly altered across the menstrual cycle ([Formula: see text] = 0.18, P = 0.03); however, post hoc tests did not reveal any significant differences between menstrual cycle phases (P > 0.05). miR-30c-5p was associated with testosterone concentration (R2 = 0.13, P = 0.033). These pilot data indicate differences in adipose tissue miRNAs in healthy women across the menstrual cycle and a weak association with ovarian hormones. Further research in larger sample sizes is required to confirm regulation of miRNA expression across the menstrual cycle.

Tissue microRNA expression in aortic aneurysm dissection

Abstract Background Dissection and other complications of ascending aortic aneurysms are potentially life-threatening. Several factors may be implicated in aneurysm progression and dissection. The role of tissue microRNAs may be of interest. Purpose To examine how serum biomarkers and tissue expression of microRNAs are associated with thoracic aortic aneurysms and dissection. Methods We compared three groups of patients; 21 patients with aneurysm of the aortic root, ascending aorta or aortic arch undergoing scheduled repair, 11 patients with acute Stanford type A aortic dissection who underwent emergency surgery and 18 patients with normal aortic diameter undergoing other cardiac surgery (control group). Prior to surgery, peripheral blood samples were obtained from patients, to assess osteoprotegerin and adiponectin levels with the ELISA method. Tissue samples from ascending aortic wall were obtained from patients during surgery. Following appropriate storage and homogenization, tissue Matrix Metalloproteinases (MMPs) 2 and 9 were measured with the ELISA method, while tissue microRNAs 29 and 195 were measured using qrtPCR, after RNA extraction. Results There was no significant difference among control, aneurysm and dissection groups in terms of age (62±10 years vs 66±12 years vs 59±12 years, p=0.052), gender distribution (77.8% male vs 81% male vs 90% male, p=0.28) or BMI (28.51±2.92 kg/m2 vs 25.72±3.09 kg/m2 vs 27.02±3.2 kg/m2, p=0.76). There was also no difference among control, aneurysm and dissection groups regarding hypertension (72% vs 62% vs 73%, p=0.73), diabetes mellitus (22% vs 19% vs 36%, p=0.54), smoking (44% vs 29% vs 46%, p=0.09) or dyslipidemia (78% vs 43% vs 55%, p=0.08). The groups of control subjects, aneurysms and dissections did not differ in osteoprotegerin [44 (28, 52) pmol/l vs 31 (28, 37) pmol/l vs 45 (24, 71) pmol/l, p=0.17], adiponectin [6,65 (2,39, 9,79) μg/ml vs 5,28 (2,34, 6,98) μg/ml vs 4,13 (2,49, 7,52) μg/ml, p=0.43], tissue MMP2 [0.97 (0.42, 27.66) ng/ml vs 9.12 (1.72, 61.49) ng/ml vs 2.51 (0.22, 235.72) ng/ml, p=0.34] and tissue MMP9 levels [0.96 (0.29, 8.56) ng/ml vs 10.31 (1.18, 25.58) ng/ml vs 2.76 (0.63, 54.83) ng/ml, p=0.09] (Figure 1). Importantly, tissue expression of mir29 was 2.11-fold higher in the dissection group (p=0.001) and 2.99-fold higher in the aneurysm group (p&amp;lt;0.001) compared to the control group. Tissue expression of mir195 was 2.72-fold higher in the dissection group (p&amp;lt;0.001) and 2.00-fold lower in the aneurysm group (p=0.08) compared to the control group (Figure 2). Conclusions These findings highlight the role of epigenetic modifications through altered microRNA tissue expression in aortic wall synthesis, extracellular matrix degradation and progress of aneurysm formation and dissection. The exact role of microRNA expression in aortic dilatation and dissection, as well as their role as potential biomarkers, merit further validation. Funding Acknowledgement Type of funding sources: None. Figure 1Figure 2

MicroRNA profile and iron-related gene expression in hepatitis C-related hepatocellular carcinoma: a preliminary study.

IntroductionHepatocellular carcinoma (HCC) is very difficult to diagnose, especially in its early stages. Non-invasive diagnostic and prognostic factors for this cancer are urgently needed. The purpose of our study was to investigate whether the microRNAs (miRNAs) regulating genes involved in iron homeostasis, whose disruption is a hallmark of HCC, offer potential as diagnostic or prognostic factors of HCV-related hepatocellular carcinoma.Material and methodsSerum and tumor samples, and adjacent liver specimens, were obtained from 65 HCC patients. Additionally, serum samples were obtained from 65 healthy controls. In total, 28 circulating and eight tissue microRNA expression profiles were estimated by TaqMan qPCR.ResultsThe expression profiles of all tested miRNAs were altered in the hepatocellular carcinoma patients. Iron level was negatively related to serum miR-96 level in healthy controls. Although the expression of iron metabolism proteins correlated with the level of serum miRNA in the controls, this was not observed in cancer patients. In the group of cancer patients, Let-7a, miR-29b, and miR-133a were positively related to ferroportin, transferrin and ferritin levels, while miR-31, miR-221 and miR-532 were negatively related to ferroportin, transferrin receptor 1 and ferritin levels. According to ROC curve analyses, 15 miRNAs are able to discriminate with 100% sensitivity and specificity between hepatocellular carcinoma patients and healthy subjects, which is more efficient than α-fetoprotein.ConclusionsCirculating miRNAs that regulate the expression of iron metabolism proteins should be evaluated as promising candidates for HCV-related HCC diagnostic agents.

Human Prostate Tissue MicroRNAs and Their Predicted Target Pathways Linked to Prostate Cancer Risk Factors.

Simple SummaryProstate cancer is a major medical issue in men worldwide. In 2018, prostate cancer was the most frequently diagnosed cancer among men in developed countries. MicroRNAs seem to be important regulators of prostate cancer, but better understanding of their role in this disease is still needed to develop novel diagnostic tools and treatments. In this study, we aim to achieve insight on the microRNA profile of prostatic tissue environment in men with prostate cancer and to correlate this microRNA expression profile with risk factors of prostate cancer. We also examined the effects of cholesterol-lowering atorvastatin on the prostatic tissue microRNA profile. Our results provide new evidence from the effects of prostate cancer-related factors on the expression of prostate tissue microRNAs. Notably, this can reveal new pathways that may link risk factors to prostate cancer and pinpoint new therapeutic possibilities.MicroRNAs are important in prostate cancer development, progression and metastasis. The aim of this study was to test microRNA expression profile in prostate tissue obtained from prostate cancer patients for associations with various prostate cancer related factors and to pinpoint the predicted target pathways for these microRNAs. Prostate tissue samples were obtained at prostatectomy from patients participating in a trial evaluating impact of pre-operative atorvastatin on serum prostate specific antigen (PSA) and Ki-67 expression in prostate tissue. Prostate tissue microRNA expression profiles were analyzed using OpenArray® MicroRNA Panel. Pathway enrichment analyses were conducted for predicted target genes of microRNAs that correlated significantly with studied factors. Eight microRNAs correlated significantly with studied factors of patients after Bonferroni multiple testing correction. MiR-485-3p correlated with serum HDL-cholesterol levels. In atorvastatin-treated subjects, miR-34c-5p correlated with a change in serum PSA and miR-138-3p with a change in total cholesterol. In the placebo arm, both miR-576-3p and miR-550-3p correlated with HDL-cholesterol and miR-627 with PSA. In pathway analysis, these eight microRNAs related significantly to several pathways relevant to prostate cancer. This study brings new evidence from the expression of prostate tissue microRNAs and related pathways that may link risk factors to prostate cancer and pinpoint new therapeutic possibilities.

Differences in plasma microRNA content impair microRNA-based signature for breast cancer diagnosis in cohorts recruited from heterogeneous environmental sites

Circulating microRNAs are non-invasive biomarkers that can be used for breast cancer diagnosis. However, differences in cancer tissue microRNA expression are observed in populations with different genetic/environmental backgrounds. This work aims at checking if a previously identified diagnostic circulating microRNA signature is efficient in other genetic and environmental contexts, and if a universal circulating signature might be possible. Two populations are used: women recruited in Belgium and Rwanda. Breast cancer patients and healthy controls were recruited in both populations (Belgium: 143 primary breast cancers and 136 healthy controls; Rwanda: 82 primary breast cancers and 73 healthy controls; Ntot = 434), and cohorts with matched age and cancer subtypes were compared. Plasmatic microRNA profiling was performed by RT-qPCR. Random Forest was used to (1) evaluate the performances of the previously described breast cancer diagnostic tool identified in Belgian-recruited cohorts on Rwandan-recruited cohorts and vice versa; (2) define new diagnostic signatures common to both recruitment sites; (3) define new diagnostic signatures efficient in the Rwandan population. None of the circulating microRNA signatures identified is accurate enough to be used as a diagnostic test in both populations. However, accurate circulating microRNA signatures can be found for each specific population, when taken separately.

Corrigendum] A serum microRNA signature as a prognostic factor for patients with advanced NSCLC and its association with tissue microRNA expression profiles

Subsequently to the publication of this paper, an interested reader drew to the authors' attention that, in Fig.5B on p.4651, the images selected to represent the miR‑486/mimic and miR‑221/inhibitor conditions for the migration experiments with the H358cell line bore some striking similarities. After having examined their original data, the authors realized that they uploaded an incorrect image to represent the miR‑486/mimic data panel during the process of compiling this figure. The corrected version of Fig.5, showing all the correct data for Fig.5B, is shown on the next page. Note that the replacement of the erroneous data does not affect either the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize to the readership for any inconvenience caused [the original article was published in Molecular Medicine Reports13: 4643‑4653, 2016; DOI: 10.3892/mmr.2016.5114].

Tissue MicroRNA Expression as a Predictor of Response to Immunotherapy in NSCLC Patients.

IntroductionExpression of PD-L1 protein on tumor cells, which is so far the only validated predictive factor for immunotherapy, is regulated by epigenetic and genetic factors. Among the most important ones that regulate gene expression are microRNAs.Materials and MethodsThe study included 60 patients with NSCLC who underwent first or second line immunotherapy with pembrolizumab or nivolumab. FFPE materials were collected before the start of immunotherapy. We examined relative expression of microRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-429, miR-508-3p, miR-1184, miR-1255a) and PD-L1 mRNA expression. Copy number variation (CNV) of PD-L1 gene by qPCR and FISH methods were assessed. Two single nucleotide polymorphisms (SNPs) in promoter region of PD-L1 gene (rs822335 and rs822336) were examined. Expression of PD-L1 protein on tumor cells was assessed by immunohistochemistry (IHC). The response rate to immunotherapy and progression free survival (PFS) measured in weeks and overall survival (OS) measured in months from the start of immunotherapy were evaluated.ResultsResponse to immunotherapy was observed in nine patients (15%, including one complete response), disease stabilization in 22 patients (36.7%), and progression in 29 patients (48.3%). Significantly higher (p=0.015) expression of miR-200b and significantly lower (p=0.043) expression of miR-429 were observed in responders compared to patients who did not respond to immunotherapy. The median PFS in the whole group of patients was 16 weeks, and the median OS was 10.5 month. In univariate analysis, the median PFS was significantly higher in patients with high miR-200b expression (HR=0.4253, 95%CI: 0.1737–1.0417, p=0.05) and high miR-508 expression (HR=0.4401, 95%CI: 0.1903–1.0178, p=0.05) and with low expression of miR-429 (HR=0.1288, 95%CI: 0.01727–0.9606, p=0.0456) compared to patients with low and high expression of these molecules, respectively. The median OS was higher in patients with low expression of miR-429 (HR=0,6288, 95%CI: 0,3053–1,2949, p=0.06) compared with patients with high expression of this microRNA. In multivariate analysis, we found that patients with PD-L1 expression on ≥1% of tumor cells compared to patients without PD-L1 expression on cancer cells had a significantly lower risk of progression (HR=0.3857, 95%CI: 0.1612–0.9226, p=0.0323) and death (HR=0.377, 95%CI: 0.1636–0.8688, p=0.022).ConclusionThe miR-200b and miR-429 molecules in tumor cells seem to have greatest impact on the effectiveness of immunotherapy in NSCLC patients.

MiRNAs may play a major role in the control of gene expression in key pathobiological processes in Chagas disease cardiomyopathy.

Chronic Chagas disease cardiomyopathy (CCC), an especially aggressive inflammatory dilated cardiomyopathy caused by lifelong infection with the protozoan Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Although chronic myocarditis may play a major pathogenetic role, little is known about the molecular mechanisms responsible for its severity. The aim of this study is to study the genes and microRNAs expression in tissues and their connections in regards to the pathobiological processes. To do so, we integrated for the first time global microRNA and mRNA expression profiling from myocardial tissue of CCC patients employing pathways and network analyses. We observed an enrichment in biological processes and pathways associated with the immune response and metabolism. IFNγ, TNF and NFkB were the top upstream regulators. The intersections between differentially expressed microRNAs and differentially expressed target mRNAs showed an enrichment in biological processes such as Inflammation, inflammation, Th1/IFN-γ-inducible genes, fibrosis, hypertrophy, and mitochondrial/oxidative stress/antioxidant response. MicroRNAs also played a role in the regulation of gene expression involved in the key cardiomyopathy-related processes fibrosis, hypertrophy, myocarditis and arrhythmia. Significantly, a discrete number of differentially expressed microRNAs targeted a high number of differentially expressed mRNAs (>20) in multiple processes. Our results suggest that miRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue. This may have a bearing on pathogenesis, biomarkers and therapy.

Role of microRNAs in neuroendocrine neoplasms of the stomach

Neuroendocrine neoplasms (NENs) are a heterogeneous group of rare epithelial tumors that arise from cells with a neuroendocrine phenotype. NENs are found in the gastrointestinal tract and pancreas – 60 % of all localities. The incidence of gastric NENs is about 9 % of all neuroendocrine tumors of the gastrointestinal tract and 0.3 % of all stomach tumors. Stomach neuroendocrine tumors (NETs) are classified into three clinico-pathological types, based on etiology, pathogenesis and morphology. There are also separate neuroendocrine cancers: small- and large-cell. The prognosis and approach to treatment of various types of gastric NENs differs significantly. Modern methods of instrumental diagnostics, immunohistochemical methods of morphological research, along with light microscopy, do not always allow us to accurately assess the malignant potential of a tumor and individualize the treatment process. One of the promising directions in the study of NETs is to determine the molecular mechanism underlying their development, in particular the role of microRNAs. This direction can open a new vector of understanding the pathogenesis, determining the prognosis of the disease, as well as finding new application points for the drug treatment of NETs. MicroRNAs are a class of short non-coding RNA molecules (18–25 nucleotides). MicroRNAs can be involved in the regulation of all major cellular processes, including proliferation and differentiation, metabolism, signaling pathways, and apoptosis. A study of microRNA expression in tissues revealed tumor-specific microRNAs. In contrast to a number of other malignant tumors, microRNA expression in patients diagnosed with NENs is poorly understood. MicroRNA-222 and microRNA-202 are among the few microRNAs that have been demonstrated in the NETs of the stomach.

Tissue microRNA expression profiling in hepatic and pulmonary metastatic melanoma.

Malignant melanoma has a propensity for the development of hepatic and pulmonary metastases. MicroRNAs (miRs) are small, noncoding RNA molecules containing about 22 nucleotides that mediate protein expression and can contribute to cancer progression. We aim to identify clinically useful differences in miR expression in metastatic melanoma tissue. RNA was extracted from formalin-fixed, paraffin-embedded samples of hepatic and pulmonary metastatic melanoma, benign, nevi, and primary cutaneous melanoma. Assessment of miR expression was performed on purified RNA using the NanoString nCounter miRNA assay. miRs with greater than twofold change in expression when compared to other tumor sites (P value ≤ 0.05, modified t-test) were identified as dysregulated. Common gene targets were then identified among dysregulated miRs unique to each metastatic site. Melanoma metastatic to the liver had differential expression of 26 miRs compared to benign nevi and 16 miRs compared to primary melanoma (P < 0.048). Melanoma metastatic to the lung had differential expression of 19 miRs compared to benign nevi and 10 miRs compared to primary melanoma (P < 0.024). Compared to lung metastases, liver metastases had greater than twofold upregulation of four miRs, and 4.2-fold downregulation of miR-200c-3p (P < 0.0081). These findings indicate that sites of metastatic melanoma have unique miR profiles that may contribute to their development and localization. Further investigation of the utility of these miRs as diagnostic and prognostic biomarkers and their impact on the development of metastatic melanoma is warranted.

Combined MicroRNA In Situ Hybridization and Immunohistochemical Detection of Protein Markers.

MicroRNAs are short (18-23 nucleotides) noncoding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3'UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The characterization of microRNA expression in tissues by in situ detection is often crucial following a microRNA biomarker discovery phase in order to validate the candidate microRNA biomarker and allow better interpretation of its molecular functions and derived cellular interactions. The in situ hybridization data provides information about contextual distribution and cellular origin of the microRNA. By combining microRNA in situ hybridization with immunohistochemical staining of protein markers, it is possible to precisely characterize the microRNA-expressing cells and to identify the potential microRNA targets. This combined technology can also help to monitor changes in the level of potential microRNA targets in a therapeutic setting. In this chapter, we present a fluorescence-based detection method that allows the combination of microRNA in situ hybridization with immunohistochemical staining of one and, in this updated version of the paper, two protein markers detected with primary antibodies raised in the same host species.

Sensitive fluorescent hybridisation protocol development for simultaneous detection of microRNA and cellular marker proteins (in the retina)

Nowadays, increasing number of microRNAs are found to have crucial roles in various physiological processes through gene expression regulation via RNA silencing as a result of base pairing with complementary mRNA sequences. To reveal the spatial distribution of microRNA expression in tissues, in situ hybridisation is the only method developed to date. This work aims to provide a novel approach to obtain information on the possible involvement of microRNA-s in regulatory processes under experimental conditions by enhancing fluorescent detection of microRNA labelling. Developing Wistar rats were used as a model system to analyse retinal microRNA expression in the first 3 postnatal weeks. Using cryosections, the crucial elements of optimal labels were (1) the concentration and duration of proteinase K treatment, (2) hybridisation temperature of microRNA probes and (3) temperature of stringency washes. Further improvements made possible to combine our in situ hybridisation protocol with double-label immunofluorescence allowing for the simultaneous detection of microRNA-s with high sensitivity and a neuronal cell marker and/or a synaptic marker protein. Thus, the regulatory microRNA-s can be localised in an identified cell type along with its potential target protein. We believe that our protocol can be easily adapted for a variety of tissues of different origins, developmental stages and experimental conditions.

Preliminary identification of key miRNAs, signaling pathways, and genes associated with Hirschsprung's disease by analysis of tissue microRNA expression profiles.

Hirschsprung's disease (HSCR) is a congenital gut motility disorder of infants, and if left untreated, it is fatal to the affected infants. This study aimed to identify key microRNAs (miRNAs), signaling pathways and genes involved in the pathogenesis of HSCR. The miRNA microarray dataset GSE77296 was downloaded. Nine colon tissue samples were available: six from HSCR patients and three matched control samples. Differentially expressed miRNAs (DEMs) were identified after data preprocessing. Target genes of the selected upregulated and downregulated DEMs were predicted. In addition, functional enrichment analyses for the selected DEMs and target genes were conducted. Finally, interaction networks between the DEMs and target genes were constructed. A total of 162 DEMs (73 upregulated and 89 downregulated) were obtained. A total of 2511 DEM-target gene pairs for the 40 selected DEMs were identified, including 1645 pairs for the upregulated DEMs and 866 pairs for the downregulated DEMs. The upregulated DEM miR-141-3p and down-regulated DEM miR-30a-3p were identified as key miRNAs by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and network analyses. Besides, KEGG pathway enrichment analysis revealed that pathways in cancer and the mitogen-activated protein kinase (MAPK) signaling pathway were key pathways. The key genes frizzled class receptor 3 (FZD3) and docking protein 6 (DOK6) were obtained through the DEM-target gene interaction networks. Two key miRNAs (miR-141-3p and miR-30a-3p), the MAPK signaling pathway and two key genes (FZD3 and DOK6) were implicated in the pathogenesis of HSCR.

Transcriptome analysis reveals non-identical microRNA profiles between arterial and venous plasma.

Circulating microRNAs presented in venous plasma have been demonstrated as powerful biomarkers for the complex diseases like cancer. Nevertheless, those presented in arterial plasma remained largely unexplored. Here, using microarray technique, we compared microRNA expression profiles of the matched arterial and venous plasma samples from the same male rats. Though the microRNA profiles were largely similar, we identified 24 differentially expressed microRNAs, including 10 arterial highly expressed microRNAs and 14 venous highly expressed microRNAs. The differentially expressed microRNAs were validated by qRT-PCR. Computational analysis of these microRNAs and their targets indicated that arterial highly expressed microRNAs were overrepresented for functional terms like hematopoiesis and diseases like Crohn's Disease and leukemia; while venous highly expressed microRNAs were enriched for cell differentiation function, and diseases like distal myopathies and heart failure. Our analysis also suggested significant correlations between plasma microRNA expression and tissue microRNA expression. Four arterial highly expressed microRNAs also showed enriched expression in specific tissues and would be novel biomarker candidates.

A serum microRNA signature as a prognostic factor for patients with advanced NSCLC and its association with tissue microRNA expression profiles.

The aim of the present study was to detect microRNA (miRNA) signatures in advanced non-small cell lung cancer (NSCLC), and to study the association between miRNA expression levels in serum and tissue. A cohort of patients who had previously been diagnosed with advanced NSCLC was enrolled in the present study. miRNAs associated with prognosis, which had previously been detected in early stage NSCLC samples, were measured in the serum of the patient groups using a cross-validation method. In addition, serum miRNAs associated with progression-free survival (PFS) were detected in paired fresh tissue samples, in order to analyze the correlation between serum and tissue expression levels. A risk-score analysis was used to develop a four-miRNA signature to predict PFS. miR-1, miR-30d, miR-221 and miR-486 were identified as having a significant correlation with PFS in advanced NSCLC. miR-221 and miR-486 exhibited significant positive correlations between serum and tissue expression. Furthermore, overexpression of miR-221 and reduced expression of miR-486 increased cell proliferation, migration and invasion in vitro. In conclusion, the miRNA signature identified in the present study may be considered an independent prognostic factor of PFS in advanced NSCLC. In addition, the expression levels of miR-221 and miR-486 were significantly correlated between serum and tissue. miR-221 was identified as an oncogenic risk factor, whereas miR-486 exerted protective effects against cancer cell proliferation, migration and invasion.

P100: MicroRNA-21, -155, -200c and -205 in carcinogenesis of larynx

The purpose of the study was to access pattern of microRNA expression in tissue of precancerous lesions and larynx cancer. A total of 25 people with a diagnosis of dysplasia II–III (n = 10), control group (dysplasia 0, n = 15) and larynx cancer patients (n = 46) were examined. Fresh frozen biopsies and adjacent normal epithelium were used. The diagnosis was verified by histology. All qRT-PCR data were analyzed using the Pfaffl analysis. Multiplex RT-PCR on miRNA templates were performed as described by Chen et al. (2005). TaqMan miRNA assays (Iyevleva et al., 2012) were used to quantify expression of mature miRNAs of interest (miR-18a, -21, -155, -200a, -200c, -205, -221, -494). Data were analyzed using Welsh t-test with a 5% FDR correction. According to our results, aberrant expression of some miRNAs was showed. There was no significant differences in miRNA expression in a total group (n = 25) although a trend towards overexpression of oncogenic miRNA-21 and -155 according to severity of dysplastic changes in a tissue were present. Detailed analysis showed significant overexpression of that miRNAs as well as miRNA-200c and -205 in a group of dysplasia II- III against control group (15 vs 10, p = 0.019, p = 0.045 and p = 0.020, p = 0.038, respectively) but these results did not meet 5% FDR correction. Data showed overexpression of the same microRNA (-205, -155, -200 and -21) in larynx cancer patients compared to control group (46 vs 15, p = 0.0002, p = 0.008, p = 0.009, p = 0.013, respectively). It should be pointed that microRNA pattern both in larynx cancer and patients with dysplasia II-III was very close showing similarity of these groups at molecular level. Frequency of cases with microRNA-205 overexpression was 2.98 times higher in cancer patients than in those who had no malignant transformation (CI 95%, 1.41–16.26, p = 0.007). Data showed that up regulation of microRNA-205 could be a marker of disease progression (OR = 4.79). Three other microRNAs did not show any promising results as biomarkers of cancer progression but data obtained has interesting fundamental value. These miRs (-21, -155, -205 and -200c) are known to be regulators of processes that play an emergent role in carcinogenesis and our results obtained in vivo highlight and expand knowledge about some aspects of larynx cell transformation. Data suggest that miRNAs changing its expression according to dysplasia progress and could be important players in complex process of carcinogenesis as well as could be potential markers of disease progression.