Relative Expression Of miRNAs Research Articles

Serum microRNA Expression Profiling: Potential Diagnostic Implications of a Panel of Serum microRNAs for Clear Cell Renal Cell Cancer

To study the expression profiles of 5 microRNAs in tissue and serum of patients with clear cell renal cell cancer (ccRCC) and evaluate their diagnostic and prognostic potential. We prospectively analyzed 30 patients of histologically proven ccRCC and collected 3 mL of serum preoperatively and small pieces of tumor and adjacent non-tumor renal tissue intraoperatively. Control serum samples were obtained from 15 patients of non-renal benign diseases. We analyzed 5 miRNAs-miR-34a, miR-141, miR-200c, miR-1233, and miR-21-2. Freshly collected samples were immediately frozen in liquid nitrogen and total RNA was extracted. cDNA was synthesized by reverse transcription, and quantitative polymerase chain reaction was performed to determine relative miRNA expression. In the renal tissue and serum samples, 3 out of 5 miRNAs were differentially expressed; that is, the expression levels of miR-34a and miR-141 were significantly decreased, whereas that of miR-1233 was significantly increased. Serum miR-34a, miR-141, and miR-1233 were able to diagnose ccRCC with a sensitivity of 80.76%, 75%, and 93.33%, and specificity of 80%, 73.33%, and 100%, respectively, as compared to histopathology. Using a panel of 2 serum miRNAs (miR-141 and miR-1233) ccRCC can be diagnosed with 100% sensitivity and 73.3% specificity. miRNAs are differentially expressed in serum of patients with ccRCC and can be used to diagnose ccRCC with high sensitivity and specificity. Diagnostic sensitivity can be further improved by using a panel of miRNAs and has the potential to serve as novel diagnostic markers of ccRCC.

MicroRNA Expression Analysis of Centenarians and Rheumatoid Arthritis Patients Reveals a Common Expression Pattern.

Micro-RNA (miRNA) are a family of small non-coding ribonucleic acids that inhibits post-transcriptionally the expression of their target messenger RNA (mRNA). We are interested in studying the involvement of miRNA in longevity and autoimmune diseases. In this study we compared the different expression of seven microRNAs between human plasma healthy controls, plasma samples of centenarians and samples from patients with rheumatoid arthritis. We used the Life Technologies' protocol to quantify seven miRNAs from 62 plasma samples: 20 healthy human controls, 14 centenarians, 28 patients with rheumatoid arthritis. TaqMan MicroRNA assays were used to analyze the expression profiles of miR-125b-5p, miR-425-5p, miR-200b5p, miR-200c-3p, miR-579-3p, miR-212-3p, miR-21-5p and miR-126-3p. The relative expression of mature miRNAs was analyzed using software REST. Our results show that miR-425-5p, miR-21 and miR-212 significantly decreased in centenarians and in patients with rheumatoid arthritis compared with controls. Furthermore in this work we highlight a connection between corticosteroid treatment and miRNAs expression.

MicroRNA profiling in human breast cancer cell lines exposed to the anti-neoplastic drug cediranib.

Cediranib, a pan-tyrosine kinase inhibitor is showing promising results for the treatment of several solid tumours. In breast cancer, its effects remain unclear, and there are no predictive biomarkers. Several studies have examined the expression profiles of microRNAs(miRNAs) in response to different chemotherapy treatments and found that the expression patterns may be associated with the treatment response. Therefore, our aim was to evaluate the cellular behaviour and differential expression profiles of miRNAs in breast cancer cell lines exposed to cediranib. The biological effect of this drug was measured by viability, migration, invasion and cell death in invitro assays. Signaling pathways were assessed using a human phospho-receptor tyrosine kinase array. Furthermore, using a miRNA array and quantitative real-time PCR(qRT‑PCR), we assessed the relative expression of miRNAs following cediranib treatment. The breast cancer cell lines exhibited a distinct cytotoxic response to cediranib treatment. Cediranib exposure resulted in a decrease in the cell migration and invasion of all the breast cancer cell lines. Treatment with cediranib appeared to be able to modulate the activation of several RTKs that are targets of cediranib such as EGFR and a new potential target ROR2. Furthermore, this drug was able to modulate the expression profile of different microRNAs such as miR-494, miR-923, miR-449a, miR-449b and miR-886-3 in breast cancer cell lines. These miRNAs are reported to regulate genes involved in important molecular processes, according to bioinformatics prediction tools.

MicroRNA in sperm from Duroc, Landrace and Yorkshire boars

Sperm contain microRNAs (miRNAs), which may have roles in epigenetic control. Regarding phylogenetic relationships among various swine breeds, Yorkshire and Landrace, are considered phenotypically and genetically very similar, but distinctly different from Duroc. The objective of the present study was to compare abundance of boar sperm miRNAs in these three breeds. Overall, 252 prioritized miRNAs were investigated using real-time PCR; relative expression of miRNAs in sperm was similar in Yorkshire and Landrace boars, but significantly different compared to Duroc. Seventeen miRNAs (hsa-miR-196a-5p, hsa-miR-514a-3p, hsa-miR-938, hsa-miR-372-3p, hsa-miR-558, hsa-miR-579-3p, hsa-miR-595, hsa-miR-648, hsa-miR-524-3p, hsa-miR-512-3p, hsa-miR-429, hsa-miR-639, hsa-miR-551a, hsa-miR-624-5p, hsa-miR-585-3p, hsa-miR-508-3p and hsa-miR-626) were down-regulated (P < 0.05; fold regulation ≤−2) in Yorkshire and Landrace sperm, compared to Duroc sperm. Furthermore, three miRNAs (hsa-miR-9-5p, hsa-miR-150-5p, and hsa-miR-99a-5p) were significantly up-regulated in Yorkshire and Landrace sperm compared to Duroc sperm, However, 240 miRNAs were not significantly different (within + 2 fold) between Yorkshire and Landrace sperm. We concluded that miRNAs in sperm were not significantly different between Yorkshire and Landrace boars, but there were significant differences between those two breeds and Duroc boars. Furthermore, integrated target genes for selected down-regulated miRNAs (identified via an in-silico method) appeared to participate in spermatogenesis and sperm functions.

Comprehensive profiling of functional Epstein-Barr virus miRNA expression in human cell lines.

BackgroundEpstein-Barr virus (EBV) establishes lifelong infections in its human host. The virus is associated with a broad range of malignancies of lymphoid and epithelial origin, including Burkitt’s lymphoma, post-transplant lymphoproliferative disease, nasopharyngeal carcinoma and gastric carcinoma. During the latent phase of its life cycle, EBV expresses more than 40 mature miRNAs that are highly abundant in tumor cells and may contribute to oncogenesis. Although multiple studies have assessed the relative expression profiles of EBV miRNAs in tumor cells, data linking these expression levels to functional target knockdown are mostly lacking. Therefore we set out to systematically assess the EBV miRNA expression levels in EBV+ tumor cell lines, and correlate this to their functional silencing capacity in these cells.ResultsWe provide comprehensive EBV miRNA expression profiles of the EBV+ cell lines C666-1 (nasopharyngeal carcinoma), SNU-719 (gastric carcinoma), Jijoye (Burkitt’s lymphoma), and AKBM (Burkitt’s lymphoma) and of EBV− cells ectopically expressing the BART miRNA cluster. By deep sequencing the small RNA population and conducting miRNA-reporter experiments to assay miRNA potency, we were able to compare the expression profiles of the EBV miRNAs with their functional silencing efficacy. We observe a strong correlation between miRNA expression levels and functional miRNA activity. There is large variation in expression levels between EBV miRNAs in a given cell line, whereas the relative expression profiles are well maintained between cell lines. Furthermore, we show that miRNA arm selection bias is less pronounced for gamma-herpesvirus miRNAs than for human miRNAs.ConclusionWe provide an in depth assessment of the expression levels and silencing activity of all EBV miRNAs in B- and epithelial cell lines of different latency stages. Our data show a good correlation between relative EBV miRNA expression levels and silencing capacity, and suggest preferential processing of particular EBV miRNAs irrespective of cell-type. In addition to encoding the largest number of precursor miRNAs of all human herpesviruses, EBV expresses many miRNAs precursors that yield two functional miRNA strands, rather than one guide strand and a non-functional passenger strand. This reduced strand bias may increase the size of the EBV miRNA targetome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2978-6) contains supplementary material, which is available to authorized users.

MiRNA Profile in Three Different Normal Human Ocular Tissues by miRNA-Seq.

PurposeBecause microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human ciliary body (CB), cornea, and trabecular meshwork (TM) using miRNA-Seq to provide a foundation for better understanding of miRNA function and disease involvement in these tissues.MethodsTotal RNAs were extracted from seven normal human CB, seven cornea, and seven TM samples using mirVana total RNA isolation kit. miRNA-Seq was done with Illumina MiSeq. Bowtie software was used to trim and align generated sequence reads, and only exact matches to mature miRNAs from miRBase were included. The miRTarBase database was used to analyze miRNA target interactions, and the expression of five selected miRNAs was validated using droplet digital PCR (ddPCR).ResultsUsing the miRNA extracted from 21 human samples, we found 378 miRNAs collectively expressed, of which the 11 most abundant miRNAs represented 80% of the total normalized reads. We also identified uniquely expressed miRNAs, of which five share 18 highly validated gene targets, and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we validated the expression profile of five miRNAs from miRNA-Seq.ConclusionsFor the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Defining the relative expression of miRNAs in nondiseased eye tissues could help uncover changes in miRNA expression that accompany diseases such as glaucoma and keratoconus.

114 - Expression of hypoxia related miRNAs in laryngeal squamous cell carcinoma

114 - Expression of hypoxia related miRNAs in laryngeal squamous cell carcinoma

Decreased Expression of Bioinformatically Predicted piwil2-targetting microRNAs, miR-1267 and miR-2276 in Breast Cancer.

Human Piwil2, a member of Piwi subfamily of Argonaute proteins, is primarily expressed in testis, where it regulates self-renewal of germ cells. However, its ectopic expression has been reported with several tumors, including breast cancer. The upregulation of piwil2 in various stages of breast cancer suggested its suitability as a novel tumor biomarker. Considering the vital role of microRNAs (miRNAs) in regulating the expression of most human genes, we hypothesized a concomitant downregulation of the bioinformatically-predicted piwil2-targetting microRNAs in breast cancer. We employed different bioinformatic tools to predict piwil2-targeting miRNAs. Then, from the list of predicted miRNAs, we chose two less studied miRNAs (miR-1267 and miR-2276) for experimental validation. Using a real-time RT-PCR approach, we quantified the relative expression of the miRNAs in 31 pairs of formalin-fixed paraffin-embedded tumor/non-tumor tissue samples. Our data revealed a noticeable but not statistically significant (P = 0.133) downregulation of miR-1267 in tumor samples, compared to non-tumor samples obtained from the same patients. Downregulation of miR-1267 was more significant in higher grades of malignancies (fold change = 2.39, P = 0.033) and also in lymph nodes containing high-grade tumor cells (fold change = 6.66, P = 0.02). Interestingly, a significant upregulation of miR-1267 was observed in tumors at high stages (stage 3a, 3b), compared to low stages (stage 2a, 2b) (fold change = 8.05, P = 0.048). Similar patterns of expression alteration were also observed for miR-2276. Altogether, our findings suggest a probable tumor suppressor role for miR-1267 and miR-2276 in breast tumor initiation and progression, but a probable promoting role for them in invasion and metastasis.

739. rAAV Delivered MicroRNA Therapeutics Towards Efficacious Treatment of Corneal Neovascularization

Corneal diseases are the second major cause of blindness worldwide. Corneal neovascularization (NV) is one of the most common pathologies in corneal diseases, leading to vision loss or even blindness. However, even though different treatments for corneal NV are available, a safe and effective therapy remains to be an unmet medical challenge. MicroRNAs (miRNAs) play roles in regulating more than half of all protein-coding genes in mammals including those involved with angiogenesis. We hypothesize that modulating expression of angiogenesis related miRNAs might be an effective approach for treating corneal NV. To this end, we first set out to identify target miRNAs that play roles in corneal NV using Nanostring technologies and the classic alkali-burn induced corneal NV mouse model. Among 618 mouse miRNAs profiled, we found 35 up-regulated and 3 down-regulated miRNAs in the neovascularized corneas, which were confirmed by qRT-PCR. We selected miR-184 and miR-204, two miRNAs that were down-regulated > 10-folds in neovascularized corneas, as our candidate targets to test the concept of in vivo gene delivery for therapeutic miRNA to treat corneal NV. As recombinant adeno-associated virus (rAAV) holds promise for highly efficient and safe in vivo gene delivery to different target tissues in a serotype dependent manner, we opted to evaluate 14 serotypes of rAAV.EGFP for gene transfer efficiency in mouse corneas by eye drops (ED), intra-stromal (IS) and subconjunctival (SC) injections to search for the suitable vector(s) for delivering miRNA therapeutics to the corneas. Among the 4 leading serotype vectors (i.e. rAAVrh.8, rh.10, rh.39 and rh.43) identified, we chose rAAVrh.10 for overexpressing pri miR-184 and pri miR-204 in the corneas with alkali-burn induced corneal NV. rAAVrh.10-mediated overexpression of miR-184 or miR-204 by IS and SC injections but not ED efficiently inhibited angiogenesis and was efficacious and safe for either prevention (IS) or treatment (SC) of corneal NV. We further invested potential target genes and pathways to elucidate the anti-angiogenic mechanism of these two miRNA therapeutics. We revealed that the anti-angiogenic effect of rAAV.pri-miR-184 was achieved by targeting Fzd4 gene, thus suppressing the canonical Wnt signaling, a well-known pathway playing vital roles in angiogenesis; while rAAV.pri-miR-204 exerted its anti-angiogenic effect by targeting angiopoietin-1 gene (Angpt1), a well-acknowledged pro-angiogenic factor. Meanwhile, the transcriptome analysis is under the way to search for novel gene targets of the miRNA therapeutics. Our study has great clinical relevance and demonstrated, for the first time, that miRNA-targeted novel therapeutics that can be delivered as either rAAV or synthetic nucleic acid drugs (i.e. miRNA mimics and antagomir) might offer an additional efficacious and safe clinical option for treating corneal NV and other angiogenesis-related diseases including age-related macular edema (AMD) and cancer.

Lactobacillus acidophilus Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells.

Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis.

Comparative microRNA profiling in relation to urinary albumin excretion in newly diagnosed hypertensive patients.

Microalbuminuria is an established early marker of endothelial dysfunction and damage. MicroRNAs (miRNAs) are emerging as essential modulators of cardiovascular physiology and disease. In the present study, we sought an association between the differential expression of related miRNAs in the peripheral blood mononuclear cells of untreated patients with newly diagnosed essential hypertension and the levels of urinary albumin excretion. We assessed the expression of the miRNAs miRNA-1, miRNA-133a, miRNA-26b, miRNA-208b, miRNA-499 and miRNA-21 in consecutive subjects with untreated newly diagnosed essential hypertension (aged 62.5±9.7 years) and with no indications of other organic heart disease. MiRNA expression levels in peripheral blood mononuclear cells were quantified by real-time reverse transcription-polymerase chain reaction. The prevalence of microalbuminuria was 9.8%. miRNA-208b and miRNA-133a were independently correlated with 24-h urinary albumin excretion. More specifically, a strong association was found between the gene expression levels of miRNA-208b in our patients' peripheral blood cells and urinary albumin (r=0.72, P<0.001). A similar association was found for miRNA-133a (r=0.372, P<0.001). In conclusion, miRNA-208b and miRNA-133a show distinct profiling in peripheral blood cells isolated from untreated patients with recently diagnosed essential hypertension. Their gene expression levels reveal a strong correlation with urinary albumin excretion levels. Our findings provide new perspectives on the development of a new generation of biomarkers for the better monitoring of end-organ damage in hypertension.

The Pathway Analysis of Micrornas Regulated Drug-Resistant Responses in HeLa Cells.

Chemotherapy is the main strategy in the treatment of cancer; however, the development of drug-resistance is the obstacle in long-term treatment of cervical cancer. Cisplatin is one of the most common drugs used in cancer therapy. Recently, accumulating evidence suggests that miRNAs are involved in various bioactivities in oncogenesis. It is not unexpected that miRNAs play a key role in acquiring of drug-resistance in the progression of tumor. In this study, we induced and maintained four levels of cisplatin-resistant HeLa cell lines (HeLa/CR1, HeLa/CR2, HeLa/CR3, and HeLa/CR4). According to the previous studies and existing evidence, we selected five miRNAs (miR-183, miR-182, miR-30a, miR-15b, and miR-16) and their potential target mRNAs as our research targets. The real-time RT-PCR was adopted to detect the relative expression of miRNAs and their mRNAs. The results show that miR-182 and miR-15b were up-regulated in resistant cell lines, while miR-30a was significantly down-regulated. At the same time, their targets are related to drug resistance. Compared to their parent HeLa cell line, the expression of selected miRNAs in resistant cell lines altered. The alteration suggests that HeLa cell drug resistance is associated with distinct miRNAs, which indicates that miRNAs may be one of the therapy targets in the treatment of cervical cancer by sensitizing cell to chemotherapy. We suggested a possible network diagram based on the existing theory and the preliminary results of candidate miRNAs and their targets in HeLa cells during development of drug resistance.

MicroRNAs in CD4+ T cell subsets are markers of disease risk and T cell dysfunction in individuals at risk for type 1 diabetes

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4+ FOXP3+ T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4+ T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4+ T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4+ T cell subsets are markers of risk and T cell dysfunction in T1D.

MicroRNA analysis in model species based on evolutionary rates.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression. In an attempt to gain insights into miRNAs at the macroevolutionary level, we performed a systematic analysis of miRNAs in six model organisms based on their evolutionary rates. First, we calculated their miRNA evolutionary rates, and found that they did not correlate with the complexity of the organisms. A correlation between evolutionary rates and single nucleotide polymorphisms (SNPs) in the miRNA sequence suggested that slow-evolving miRNAs in humans tolerate more SNPs than miRNAs with similar evolutionary rates in other species. However, fast-evolving miRNAs had lower SNP densities in humans than in the fruit fly. We also found that evolutionary rates were correlated with the proportion of parasite or clustered miRNAs. This correlation exhibited a different pattern in zebrafish, which may be related to significant genome duplication in the early vertebrates. The minimized free energy of the miRNA stem-loop structure was not correlated with the evolutionary rates of any species in our analysis. After evaluating relative miRNA expression levels, we observed that newly emerged miRNAs in complex species would integrate into the gene network at a faster pace and be functionally important; therefore, miRNAs may have accelerated human evolution.

SPIRE, a modular pipeline for eQTL analysis of RNA-Seq data, reveals a regulatory hotspot controlling miRNA expression in C. elegans.

The interpretation of genome-wide association study is difficult, as it is hard to understand how polymorphisms can affect gene regulation, in particular for trans-regulatory elements located far from their controlling gene. Using RNA or protein expression data as phenotypes, it is possible to correlate their variations with specific genotypes. This technique is usually referred to as expression Quantitative Trait Loci (eQTLs) analysis and only few packages exist for the integration of genotype patterns and expression profiles. In particular, tools are needed for the analysis of next-generation sequencing (NGS) data on a genome-wide scale, which is essential to identify eQTLs able to control a large number of genes (hotspots). Here we present SPIRE (Software for Polymorphism Identification Regulating Expression), a generic, modular and functionally highly flexible pipeline for eQTL processing. SPIRE integrates different univariate and multivariate approaches for eQTL analysis, paying particular attention to the scalability of the procedure in order to support cis- as well as trans-mapping, thus allowing the identification of hotspots in NGS data. In particular, we demonstrated how SPIRE can handle big association study datasets, reproducing published results and improving the identification of trans-eQTLs. Furthermore, we employed the pipeline to analyse novel data concerning the genotypes of two different C. elegans strains (N2 and Hawaii) and related miRNA expression data, obtained using RNA-Seq. A miRNA regulatory hotspot was identified in chromosome 1, overlapping the transcription factor grh-1, known to be involved in the early phases of embryonic development of C. elegans. In a follow-up qPCR experiment we were able to verify most of the predicted eQTLs, as well as to show, for a novel miRNA, a significant difference in the sequences of the two analysed strains of C. elegans. SPIRE is publicly available as open source software at , together with some example data, a readme file, supplementary material and a short tutorial.

Initial research on the relationship between let-7 family members in the serum and massive cerebral infarction

Eighty-eight ischemic stroke patients with massive cerebral infarction (MCI) who met our selection criteria were included in this study. MCI was assessed using the Glasgow Coma Scale (GCS) at hospital admission and at 2weeks. The sera of all patients and controls were sampled at 48h after the patients' attacks, and the sera of patients with MCI who had no severe cardiopulmonary complications, including those with hemorrhagic transformation (HT), were sampled again at 2weeks. The relative expression of let-7 miRNA in the serum was determined by real-time qRT-PCR, and the blood levels of lipids, glucose, high-sensitivity C-reactive protein (hs-CRP), homocysteine and blood pressure were measured at admission. Interleukin-6 (IL-6) levels were detected by ELISA, and a luciferase assay was performed to confirm that IL-6 was a gene target of let-7. The relative expression of let-7f was significantly down-regulated in MCI without HT patients compared with controls (P<0.001), and it was positively correlated with GCS (P<0.01) and negatively correlated with hs-CRP (P<0.01). The relative expression of let-7f was significantly up-regulated in MCI patients with HT (P<0.01). IL-6 is a direct target gene for let-7f, and IL-6 expression was increased in MCI without HT patients compared to controls (P<0.01). The expression of let-7f in serum is associated with MCI without HT, which specifically inhibits IL-6. This suggests that let-7f may control inflammation in patients with MCI without HT.

Identification of a STAT3-miRNA Axis in T-LGL Leukemia

IntroductionT large granular lymphocytes leukemia (T-LGLL) is a rare disease characterized by the abnormal expansion of T-large granular lymphocytes (T-LGLs) in the peripheral blood. The etiology of this disease is still largely unknown. LGL proliferation is maintained through an impairment of the apoptotic machinery due to the activation of many survival signals. Among these, JAK/STAT signaling represents one of the most important deregulated pathways in T-LGLL. In particular, leukemic LGLs are equipped with STAT3 constitutively over-expressed and over-activated. Moreover, in 30-40% of patients, STAT3 has been demonstrated carrying hot-spot mutations, likely resulting in STAT3 activation. Although STAT3 is an inducer of transcription of a large number of oncogenes, its relationship with microRNAs (miRNAs) has not yet been extensively evaluated in T-LGLL patients. As a matter of fact, several miRNAs contribute to normal hematopoietic processes and many miRNAs act both as tumor suppressors and oncogenes in the pathology of hematological malignancies, including acute and chronic leukemias and lymphomas, where they contribute to lymphomagenesis acting in various cellular functions, such as the regulation of cell survival and proliferation.AimsWe investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. A high throughput quantitative and qualitative analysis of the miRNA expression profile in leukemic LGLs compared to healthy controls was performed with the aim to investigate whether STAT3 activation and/or mutation were correlated to some miRNAs in leukemic LGLs.MethodsSix patients (3 characterized by STAT3 mutations and 3 with wild type STAT3) and three healthy controls were enrolled in a pilot study. STAT3 mRNA expression and protein activation levels were analyzed by Real Time-PCR and Western Blot, respectively. The expression level of 756 mature miRNAs was assessed by using a TaqMan-based Low Density Array on purified LGLs. Experimental data were analyzed by ViiA7 RUO software and the relative miRNA expression values were calculated using U6 as endogenous control. miRNA array data underwent hierarchical cluster analysis (HCL) by using MEV. miRNAs with a 2 or 0.5 fold change and p value < 0.05 in samples as compared to controls were considered as differentially expressed. Results of this pilot study were validated on additional 12 T-LGLL and 3 healthy controls subjects.ResultsTwo clusters were identified by HCL analysis: cluster A included healthy controls and LGL patients characterized by comparably low levels of STAT3 activation (S3low) and absence of STAT3 mutations. Cluster B included four patients characterized by high levels of STAT3 activation (S3high). Remarkably, three out of four LGL patients in cluster B shared STAT3 mutation. Comparative analysis of the miRNAs expressed identified 33 miRNAs upregulated and 9 miRNAs downregulated in S3high as compared to S3low. Interestingly, the level of expression of these selected miRNAs correlated with the level of STAT3 expression/activation in LGL. Among these, three miRNAs, miR-484, miR-501 and miR-1249, have been validated and confirmed in the validation cohort.ConclusionsThese data firstly describe the miRNA pattern in T-LGLL, providing evidence that a series of miRNAs are correlated with relevant key factors in T-LGLL pathogenesis, including STAT3 activation/expression and mutations. Our results suggest the hypothesis that STAT3 could mediate its role through some defined miRNAs. DisclosuresNo relevant conflicts of interest to declare.

Expression profiling of iron deficiency responsive microRNAs and gene targets in rice seedlings of Madhukar x Swarna recombinant inbred lines with contrasting levels of iron in seeds

To test if microRNAs are involved in iron (Fe) homeostasis in Oryza sativa. Recombinant inbred lines (RILs) of rice with contrasting levels of iron in seeds (high iron line HL, low iron line LL) and parent Swarna were grown in Fe sufficient (+Fe) and deficient (−Fe) environment. miRNAs whose target genes underlie the QTLs mapped for iron concentration (mapped in our previous study) were identified using bioinformatics. The expression analysis of these miRNAs and their targets along with few other miRNAs involved in nutrient homeostasis was done in root and shoot tissue. Real time PCR was used to study the relative expression of miRNAs and their target genes. Out of nine miRNAs used in this study, 7 miRNAs-miR156, 168, 172, 162, 167, 171, and 398 showed down-regulation under Fe deficiency in root and shoot of high iron line when compared with Fe sufficient condition. Further, most of the miRNAs showed down-regulation while their target genes showed up-regulation under Fe deficiency in roots of all three genotypes (HL, LL and Swarna) suggesting roots are more responsive to Fe deficiency. Important role of miRNAs in iron homeostasis was analyzed by comparing the expression of these miRNAs in HL, LL and Swarna under + Fe and –Fe. MicroRNAs showed differential expression in + Fe and –Fe environment. Further, their expression is more effectively regulated in root under Fe deficiency. This indicates that miRNAs might be playing regulatory roles in iron homeostasis in rice. This study suggests that Fe deficiency responsive miRNAs are involved in cross talk between other nutrients stress.

Assessment of miRNA expression profile and differential expression pattern of target genes in cold-tolerant and cold-sensitive tomato cultivars

MircroRNAs (miRNAs) are small non-coding RNAs about 21 nt in length. These short transcripts regulate developmental and stress responses in plants. Cold stress is one of the most restraining abiotic factors adversely affecting the plant yield. In the present study, some cold stress-related miRNAs (miR167, miR169, miR172, miR393 and miR397) in tomato (Solanum lycopersicum) were assessed at early time points (0, 1, 4 and 16 h) of cold exposure. Relative expression of miRNAs was measured by stem–loop quantitative reverse transcription polymerase chain reaction. The results showed that miR167, miR169, miR172 and miR393 were activated in the early time points of cold treatment. Especially, miR172 was found to have highest expression level. Furthermore, target genes of selected miRNAs were identified and their expression profiles were assessed between cold-sensitive and cold-tolerant cultivars of tomato. It was found that inferred expression patterns of target genes were differentiated between the cultivars. Analysis of cis-acting elements showed that miRNAs had stress-responsive elements. Meanwhile, since no miR393 sequence is available, putative miR393 sequence and its secondary structure were predicted in tomato. These results may provide a framework for further analysis in terms of understanding the response of miRNAs against cold stress in tomato.

Mango polyphenolics suppressed tumor growth in breast cancer xenografts in mice: role of the PI3K/AKT pathway and associated microRNAs

The cytotoxic and anti-inflammatory properties of mango polyphenolics including gallic acid and gallotannins have been demonstrated in numerous types of cancers. We hypothesized that the phosphoinositide 3-kinase (PI3K)/AKT pathway and the expression of related miRNAs are involved in the chemotherapeutic activities of mango polyphenolics in a mouse xenograft model for breast cancer. The objectives of this research were to determine the tumor-cytotoxic activities of mango polyphenolics and the underlying molecular mechanisms involving posttranscriptional targets in BT474 breast cancer cells and xenografts in mice. In vitro findings showed cytotoxic effects of mango polyphenolics in BT474 breast cancer cells within a concentration range of 2.5 to 20 mg/L gallic acid equivalents. Mango polyphenolics suppressed the expression of PI3K, AKT, hypoxia inducible factor–1α, and vascular endothelial growth factor (VEGF) mRNA, and pAKT, AKT, pPI3K (p85), VEGF and nuclear factor–kappa B protein levels. The involvement of miR-126 was verified by using antagomiR for miR-126, where mango reversed the effect of the antagomiR of miR-126. In vivo, the intake of mango polyphenolics decreased the tumor volume by 73% in BT474 xenograft-bearing mice compared with the control group. In addition, mango reduced the expression of nuclear factor–kappa B (p65), pAKT, pPI3K, mammalian target of rapamycin, hypoxia inducible factor–1α, and VEGF protein in athymic nude mice. A screening for miRNA expression changes confirmed that mango polyphenolics modulated the expression of cancer-associated miRNAs including miR-126 in the xenografted tumors. In summary, mango polyphenolics have a chemotherapeutic potential against breast cancer that at least in part is mediated through the PI3K/AKT pathway and miR-126.