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1.  microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma 
A moderate loss of miR-122 function correlates with up-regulation of seed-matched genes and down-regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti-miR-122 antagomir.Putative direct targets up-regulated with loss of miR-122 and secondary targets down-regulated with loss of miR-122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR-122 over- and under-expression.Loss of miR-122 secondary target expression in either tumorous or adjacent non-tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis-B or -C viruses (HBV or HCV) (But et al, 2008). Globally, the 5-year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)-expression profiles of tumor and adjacent non-tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR-122 was the most highly expressed miRNA in liver, and its expression was decreased almost two-fold in HCC tissue relative to adjacent non-tumor tissue, confirming earlier observations (Lagos-Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti-correlated with miR-122 expression. Consistent with the idea that transcripts anti-correlated with miR-122 are potential miR-122 targets, the most highly anti-correlated transcripts were highly enriched for the presence of the miR-122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell-cycle genes, the subset of seed-matched genes had no significant KEGG Pathway annotation, suggesting that miR-122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR-122 were not enriched for 3′UTR seed matches to miR-122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR-122 in vivo, silencing of miR-122 was performed by antisense inhibition (anti-miR-122) in wild-type mice (Figure 3). As with the genes negatively correlated with miR-122 in HCC patients, no significant biological annotation was associated with the seed-matched genes up-regulated by anti-miR-122 in mouse livers. The most significantly enriched biological annotation for anti-miR-122 down-regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down-regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR-122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti-correlation of proposed primary and secondary targets in non-healthy tissues.
To validate the direct correlation between miR-122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR-122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT-1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC-1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over-expressing miR-122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC-1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. To further validate the link of miR-122 and PGC-1α protein, we transfected PLC/PRF/5 cells with miR-122-expression vector, and observed an increase in PGC-1α protein levels. Importantly, transfection of both miR-122 mimetic and miR-122-expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti-miR-122 treatment increased lactate production. Together, the data support the function of miR-122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR-122-expression levels. However, miR-122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non-tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR-122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR-122 in liver physiology. We observed decreased expression of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 seemed to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC and to test whether increasing miR-122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
doi:10.1038/msb.2010.58
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival
2.  The expression of microRNA-375 in plasma and tissue is matched in human colorectal cancer 
BMC Cancer  2014;14(1):714.
Background
MicroRNAs (miRNAs) offer great potential as cancer biomarkers. The importance of miRNAs profiling in tissue and body fluids in colorectal cancer (CRC) have been addressed respectively in many studies. The purpose of our study is to systematically assess the expression of miRNAs in cancer tissue and matched plasma samples and to evaluate their usefulness as minimally invasive diagnostic biomarkers for the detection of CRC.
Methods
The study was divided into two phases: firstly, qRT-PCR based TaqMan Low Density MiRNA Arrays (TLDAs) was used to screen the differentially expressed miRNAs in 6 plasma samples of CRC patients and 6 healthy controls. Secondly, marker validation by stem-loop reverse transcription real-time PCR using an independent set of paired cancer tissues (n = 88) and matched plasma samples (CRC, n = 88; control, n = 40). Correlation analysis was determined by Pearson’s test. Receiver operating characteristic curve analyses were applied to obtain diagnostic utility of the differentially expressed miRNAs. Target gene prediction and signal pathway analyses were used to predict the function of miRNAs.
Results
TLDAs identified 42 miRNAs, which were differentially expressed in patients and healthy individuals. Five of them (miR-375, miR-150, miR-206, miR-125b and miR-126*) were chosen to be validated in plasma and tissue samples. The results indicated that for plasma sample, miR-375 (p < 0.0001) and miR-206 (p = 0.0002) were dysregulated and could discriminate CRC patients from healthy controls. For tissue samples, miR-375 (p < 0.0001), miR-150 (p < 0.0001), miR-125b (p = 0.0065) and miR-126*(p = 0.0009) were down-regulated. miR-375 was significantly down-regulated and positively correlated in both tissue and plasma samples (r = 0.4663, p = 0.0007). Gene ontology and signal pathway analyses showed that most of the target genes that were regulated by miR-375 were involved in some critical pathways in the development and progression of cancer.
Conclusions
Our results indicate that the down-regulation of miR-375 in plasma and tissue is matched in CRC. Moreover, bioinformatics prediction revealed miR-375 association with some critical signal pathways in the development and progression of CRC. Therefore, plasma miR-375 holds great promise to be an alternative tissue biomarker for CRC detection.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-714) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-714
PMCID: PMC4181388  PMID: 25255814
Colorectal cancer; MicroRNA; Plasma; Tissue; Biomarker; Diagnosis
3.  A signature of circulating microRNAs differentiates takotsubo cardiomyopathy from acute myocardial infarction 
European Heart Journal  2013;35(15):999-1006.
Aims
Takotsubo cardiomyopathy (TTC) remains a potentially life-threatening disease, which is clinically indistinguishable from acute myocardial infarction (MI). Today, no established biomarkers are available for the early diagnosis of TTC and differentiation from MI. MicroRNAs (miRNAs/miRs) emerge as promising sensitive and specific biomarkers for cardiovascular disease. Thus, we sought to identify circulating miRNAs suitable for diagnosis of acute TTC and for distinguishing TTC from acute MI.
Methods and results
After miRNA profiling, eight miRNAs were selected for verification by real-time quantitative reverse transcription polymerase chain reaction in patients with TTC (n = 36), ST-segment elevation acute myocardial infarction (STEMI, n = 27), and healthy controls (n = 28). We quantitatively confirmed up-regulation of miR-16 and miR-26a in patients with TTC compared with healthy subjects (both, P < 0.001), and up-regulation of miR-16, miR-26a, and let-7f compared with STEMI patients (P < 0.0001, P < 0.05, and P < 0.05, respectively). Consistent with previous publications, cardiac specific miR-1 and miR-133a were up-regulated in STEMI patients compared with healthy controls (both, P < 0.0001). Moreover, miR-133a was substantially increased in patients with STEMI compared with TTC (P < 0.05). A unique signature comprising miR-1, miR-16, miR-26a, and miR-133a differentiated TTC from healthy subjects [area under the curve (AUC) 0.835, 95% CI 0.733–0.937, P < 0.0001] and from STEMI patients (AUC 0.881, 95% CI 0.793–0.968, P < 0.0001). This signature yielded a sensitivity of 74.19% and a specificity of 78.57% for TTC vs. healthy subjects, and a sensitivity of 96.77% and a specificity of 70.37% for TTC vs. STEMI patients. Additionally, we noticed a decrease of the endothelin-1 (ET-1)-regulating miRNA-125a-5p in parallel with a robust increase of ET-1 plasma levels in TTC compared with healthy subjects (P < 0.05).
Conclusion
The present study for the first time describes a signature of four circulating miRNAs as a robust biomarker to distinguish TTC from STEMI patients. The significant up-regulation of these stress- and depression-related miRNAs suggests a close connection of TTC with neuropsychiatric disorders. Moreover, decreased levels of miRNA125a-5p as well as increased plasma levels of its target ET-1 are in line with the microvascular spasm hypothesis of the TTC pathomechanism.
doi:10.1093/eurheartj/eht392
PMCID: PMC3985061  PMID: 24046434
Takotsubo cardiomyopathy; Biomarker; Endothelin-1; MicroRNA
4.  MicroRNA-125a is over-expressed in insulin target tissues in a spontaneous rat model of Type 2 Diabetes 
BMC Medical Genomics  2009;2:54.
Background
MicroRNAs (miRNAs) are non-coding RNA molecules involved in post-transcriptional control of gene expression of a wide number of genes, including those involved in glucose homeostasis. Type 2 diabetes (T2D) is characterized by hyperglycaemia and defects in insulin secretion and action at target tissues. We sought to establish differences in global miRNA expression in two insulin-target tissues from inbred rats of spontaneously diabetic and normoglycaemic strains.
Methods
We used a miRNA microarray platform to measure global miRNA expression in two insulin-target tissues: liver and adipose tissue from inbred rats of spontaneously diabetic (Goto-Kakizaki [GK]) and normoglycaemic (Brown-Norway [BN]) strains which are extensively used in genetic studies of T2D. MiRNA data were integrated with gene expression data from the same rats to investigate how differentially expressed miRNAs affect the expression of predicted target gene transcripts.
Results
The expression of 170 miRNAs was measured in liver and adipose tissue of GK and BN rats. Based on a p-value for differential expression between GK and BN, the most significant change in expression was observed for miR-125a in liver (FC = 5.61, P = 0.001, Padjusted = 0.10); this overexpression was validated using quantitative RT-PCR (FC = 13.15, P = 0.0005). MiR-125a also showed over-expression in the GK vs. BN analysis within adipose tissue (FC = 1.97, P = 0.078, Padjusted = 0.99), as did the previously reported miR-29a (FC = 1.51, P = 0.05, Padjusted = 0.99). In-silico tools assessing the biological role of predicted miR-125a target genes suggest an over-representation of genes involved in the MAPK signaling pathway. Gene expression analysis identified 1308 genes with significantly different expression between GK and BN rats (Padjusted < 0.05): 233 in liver and 1075 in adipose tissue. Pathways related to glucose and lipid metabolism were significantly over-represented among these genes. Enrichment analysis suggested that differentially expressed genes in GK compared to BN included more predicted miR-125a target genes than would be expected by chance in adipose tissue (FDR = 0.006 for up-regulated genes; FDR = 0.036 for down-regulated genes) but not in liver (FDR = 0.074 for up-regulated genes; FDR = 0.248 for down-regulated genes).
Conclusion
MiR-125a is over-expressed in liver in hyperglycaemic GK rats relative to normoglycaemic BN rats, and our array data also suggest miR-125a is over-expressed in adipose tissue. We demonstrate the use of in-silico tools to provide the basis for further investigation of the potential role of miR-125a in T2D. In particular, the enrichment of predicted miR-125a target genes among differentially expressed genes has identified likely target genes and indicates that integrating global miRNA and mRNA expression data may give further insights into miRNA-mediated regulation of gene expression.
doi:10.1186/1755-8794-2-54
PMCID: PMC2754496  PMID: 19689793
5.  MicroRNA expression after ionizing radiation in human endothelial cells 
Background
Endothelial cells (EC) in tumor and normal tissue constitute critical radiotherapy targets. MicroRNAs have emerged as master switchers of the cellular transcriptome. Here, we seek to investigate the role of miRNAs in primary human dermal microvascular endothelial cells (HDMEC) after ionizing radiation.
Methods
The microRNA status in HDMEC after 2 Gy radiation treatment was measured using oligo-microarrays covering 361 miRNAs. To functionally analyze the role of radiation-induced differentially regulated miRNAs, cells were transfected with miRNA precursor or inhibitor constructs. Clonogenic survival and proliferation assays were performed.
Results
Radiation up-regulated miRNA expression levels included let-7g, miR-16, miR-20a, miR-21 and miR-29c, while miR-18a, miR-125a, miR-127, miR-148b, miR-189 and miR-503 were down-regulated. We found that overexpression or inhibition of let-7g, miR-189, and miR-20a markedly influenced clonogenic survival and cell proliferation per se. Notably, the radiosensitivity of HDMEC was significantly influenced by differential expression of miR-125a, -127, -189, and let-7g. While miR-125a and miR-189 had a radioprotective effect, miR-127 and let-7g enhanced radiosensitivity in human endothelial cells.
Conclusion
Our data show that ionizing radiation changes microRNA levels in human endothelial cells and, moreover, exerts biological effects on cell growth and clonogenicity as validated in functional assays. The data also suggest that the miRNAs which are differentially expressed after radiation modulate the intrinsic radiosensitivity of endothelial cells in subsequent irradiations. This indicates that miRNAs are part of the innate response mechanism of the endothelium to radiation.
doi:10.1186/1748-717X-5-25
PMCID: PMC2859352  PMID: 20346162
6.  Identification of microRNAs from Amur grape (vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics 
BMC Genomics  2012;13:122.
Background
MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species.
Results
A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism.
Conclusions
Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.
doi:10.1186/1471-2164-13-122
PMCID: PMC3353164  PMID: 22455456
Amur grape; microRNA; Sequences evolution; Solexa sequencing; miR-RACE; qRT-PCR
7.  Comprehensive microRNA Profiling of Prostate Cancer 
Journal of Cancer  2013;4(5):350-357.
MicroRNAs are small non-coding RNA molecules that have been shown to regulate the expression of genes linked to cancer. The relevance of microRNAs in the development, progression and prognosis of prostate cancer is not fully understood. It is also possible that these specific molecules may assist in the recognition of aggressive tumors and the development of new molecular targets. Our study investigated the importance of several microRNAs in cases of prostate cancer from 37 patients that were manually microdissected to obtain pure populations of tumor cells, normal epithelium and adjacent stroma. MicroRNA was extracted for PCR array profiling. Differentially expressed miRNAs for each case were used to compare tumor vs. normal epithelium and tumor-adjacent stroma samples.
Loss of 18 miRNAs (e.g.miR-34c, miR-29b, miR-212 and miR-10b) and upregulation of miR-143 and miR-146b were significantly found in all the tumors in comparison with normal epithelium and/or stroma (p≤ 0.001). A different signature was found in the high grade tumors (Gleason score ≥ 8) when compared with tumors Gleason score 6. Upregulation of miR-122, miR-335, miR-184, miR-193, miR-34, miR-138, miR-373, miR-9, miR-198, miR-144 and miR-215 and downregulation of miR-96, miR-222, miR-148, miR-92, miR-27, miR-125, miR-126, miR-27 were found in the high grade tumors.
MicroRNA profiling in prostate cancer appears to have unique expression patterns in comparison with normal tissue. These differential expressed miRNAs may provide novel diagnostic and prognostic tools that will assist in the recognition of prostate cancers with aggressive behavior.
doi:10.7150/jca.6394
PMCID: PMC3677622  PMID: 23781281
microRNA; Prostate Cancer; biomarkers.
8.  Hormonal Regulation of MicroRNA Expression in Steroid Producing Cells of the Ovary, Testis and Adrenal Gland 
PLoS ONE  2013;8(10):e78040.
Background
Given the emerging roles of miRNAs as potential posttranscriptional/posttranslational regulators of the steroidogenic process in adrenocortical and gonadal cells, we sought to determine miRNA profiles in rat adrenals from animals treated with vehicle, ACTH, 17α-E2 or dexamethasone. Key observations were also confirmed using hormone (Bt2cAMP)-treated mouse Leydig tumor cells, MLTC-1, and primary rat ovarian granulosa cells.
Methodology
RNA was extracted from rat adrenal glands and miRNA profiles were established using microarray and confirmed with qRT-PCR. The expression of some of the hormone-sensitive miRNAs was quantified in MLTC-1 and granulosa cells after stimulation with Bt2cAMP. Targets of hormonally altered miRNAs were explored by qRT-PCR and Western blotting in adrenals and granulosa cells.
Results
Adrenals from ACTH, 17α-E2 and dexamethasone treated rats exhibited miRNA profiles distinct from control animals. ACTH up-regulated the expression of miRNA-212, miRNA-182, miRNA-183, miRNA-132, and miRNA-96 and down-regulated the levels of miRNA-466b, miRNA-214, miRNA-503, and miRNA-27a. The levels of miR-212, miRNA-183, miRNA-182, miRNA-132, miRNA-370, miRNA-377, and miRNA-96 were up-regulated, whereas miR-125b, miRNA-200b, miR-122, miRNA-466b, miR-138, miRNA-214, miRNA-503 and miRNA27a were down-regulated in response to 17α-E2 treatment. Dexamethasone treatment decreased miRNA-200b, miR-122, miR-19a, miRNA-466b and miRNA27a levels, but increased miRNA-183 levels. Several adrenal miRNAs are subject to regulation by more than one hormone. Significant cAMP-induced changes in certain miRNAs were also noted in MLTC-1 and granulosa cells. Some of the hormone-induced miRNAs in steroidogenic cells were predicted to target proteins involved in lipid metabolism/steroidogenesis. We also obtained evidence that miR-132 and miRNA-214 inhibit the expression of SREBP-1c and LDLR, respectively.
Conclusion
Our results demonstrate that expression of a number of miRNAs in steroidogenic cells of the testis, ovary and adrenal glands is subject to hormonal regulation and that miRNAs and their regulation by specific hormones are likely to play a key role in posttranscriptional/posttranslational regulation of steroidogenesis.
doi:10.1371/journal.pone.0078040
PMCID: PMC3810252  PMID: 24205079
9.  Conserved Regulation of p53 Network Dosage by MicroRNA–125b Occurs through Evolving miRNA–Target Gene Pairs 
PLoS Genetics  2011;7(9):e1002242.
MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate homologue of the Caenorhabditis elegans microRNA lin-4, has been implicated in the regulation of neural and hematopoietic stem cell homeostasis, analogous to how lin-4 regulates stem cells in C. elegans. Depending on the cell context, miR-125b has been proposed to regulate both apoptosis and proliferation. Because the p53 network is a central regulator of both apoptosis and proliferation, the dual roles of miR-125b raise the question of what genes in the p53 network might be regulated by miR-125b. By using a gain- and loss-of-function screen for miR-125b targets in humans, mice, and zebrafish and by validating these targets with the luciferase assay and a novel miRNA pull-down assay, we demonstrate that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1, and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l, in the p53 network. We found that, although each miRNA–target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network level. Our results lead us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.
Author Summary
MicroRNAs (miRNAs) are tiny endogenous RNAs that can regulate the expression of hundreds of genes simultaneously, thus orchestrating changes in gene networks and mediating cellular functions in both plants and animals. Although the identification of individual targets of miRNAs is of major importance, to date few studies have sought to uncover miRNA targets at the gene network level and general principles of miRNA regulation at the network level. Here we describe how miR-125b targets 20 apoptosis and proliferation genes in the p53 network. We found that, although each miRNA-target pair evolves rapidly across vertebrates, regulation of the p53 pathway by miR-125b is conserved at the network level. The structure of the miR-125b regulatory network suggests that miR-125b buffers and fine-tunes p53 network activity. This buffering feature of miR-125b has implications for our understanding of how miR-125b regulates oncogenesis and tissue stem cell homeostasis. We believe these findings on miR-125b support a new fundamental principle for how miRNAs regulate gene networks in general.
doi:10.1371/journal.pgen.1002242
PMCID: PMC3174204  PMID: 21935352
10.  Cardio-miRNAs and onco-miRNAs: circulating miRNA-based diagnostics for non-cancerous and cancerous diseases 
Cardiovascular diseases and cancers are the leading causes of morbidity and mortality in the world. MicroRNAs (miRNAs) are short non-coding RNAs that primarily repress target mRNAs. Here, miR-24, miR-125b, miR-195, and miR-214 were selected as representative cardio-miRs that are upregulated in human heart failure. To bridge the gap between miRNA studies in cardiology and oncology, the targets and functions of these miRNAs in cardiovascular diseases and cancers will be reviewed. ACVR1B, BCL2, BIM, eNOS, FGFR3, JPH2, MEN1, MYC, p16, and ST7L are miR-24 targets that have been experimentally validated in human cells. ARID3B, BAK1, BCL2, BMPR1B, ERBB2, FGFR2, IL6R, MUC1, SITR7, Smoothened, STAT3, TET2, and TP53 are representative miR-125b targets. ACVR2A, BCL2, CCND1, E2F3, GLUT3, MYB, RAF1, VEGF, WEE1, and WNT7A are representative miR-195 targets. BCL2L2, ß-catenin, BIM, CADM1, EZH2, FGFR1, NRAS, PTEN, TP53, and TWIST1 are representative miR-214 targets. miR-125b is a good cardio-miR that protects cardiomyocytes; miR-195 is a bad cardio-miR that elicits cardiomyopathy and heart failure; miR-24 and miR-214 are bi-functional cardio-miRs. By contrast, miR-24, miR-125b, miR-195, and miR-214 function as oncogenic or tumor suppressor miRNAs in a cancer (sub)type-dependent manner. Circulating miR-24 is elevated in diabetes, breast cancer and lung cancer. Circulating miR-195 is elevated in acute myocardial infarction, breast cancer, prostate cancer and colorectal adenoma. Circulating miR-125b and miR-214 are elevated in some cancers. Cardio-miRs and onco-miRs bear some similarities in functions and circulation profiles. miRNAs regulate WNT, FGF, Hedgehog and other signaling cascades that are involved in orchestration of embryogenesis and homeostasis as well as pathogenesis of human diseases. Because circulating miRNA profiles are modulated by genetic and environmental factors and are dysregulated by genetic and epigenetic alterations in somatic cells, circulating miRNA association studies (CMASs) within several thousands of cases each for common non-cancerous diseases and major cancers are necessary for miRNA-based diagnostics.
doi:10.3389/fcell.2014.00061
PMCID: PMC4207049  PMID: 25364765
Alzheimer's disease; early diagnosis; gastric cancer; hypertension; pancreatic cancer; personalize medicine; rheumatoid arthritis; stem cells
11.  Comparative Analysis Reveals Dynamic Changes in miRNAs and Their Targets and Expression during Somatic Embryogenesis in Longan (Dimocarpus longan Lour.) 
PLoS ONE  2013;8(4):e60337.
Somatic embryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development.
doi:10.1371/journal.pone.0060337
PMCID: PMC3623967  PMID: 23593197
12.  MicroRNA Profiling Implies New Markers of Chemoresistance of Triple-Negative Breast Cancer 
PLoS ONE  2014;9(5):e96228.
Objective
Triple-negative breast cancer (TNBC) patients with truly chemosensitive disease still represent a minority among all TNBC patients. The aim of the present study is to identify microRNAs (miRNAs) that correlate with TNBC chemoresistance.
Methods
In this study, we conducted miRNAs profile comparison between triple-negative breast cancer (TNBCs) and normal breast tissues by microRNA array. Quantitative real-time PCR (qRT-PCR) was utilized to confirm the specific deregulated miRNAs change trend. We used starBase 2.1 and GOrilla to predict the potential targets of the specific miRNAs. Cells viability and apoptosis assays were employed to determine the effect of alteration of the specific miRNAs in TNBC cells on the chemosensitivity.
Results
We identified 11 specific deregulated miRNAs, including 5 up-regulated miRNAs (miR-155-5p, miR-21-3p, miR-181a-5p, miR-181b-5p, and miR-183-5p) and 6 down-regulated miRNAs (miR-10b-5p, miR-451a, miR-125b-5p, miR-31-5p, miR-195-5p and miR-130a-3p). Thereafter, this result was confirmed by qRT-PCR. We predicted the potential targets of the candidate miRNAs and found that they are involved in cancer-associated pathways. For the first time, we found that miR-130a-3p and miR-451a were down-regulated in TNBC. 9 of the 11 specific deregulated miRNAs were found to be associated with chemoresistance. In vitro assays, we found that up-regulation of either miR-130a-3p or miR-451a in MDA-MB-231 cells significantly changed the cells sensitivity to doxorubicin. The results suggest that TNBC chemotherapy might be affected by a cluster of miRNAs.
Conclusion
The abnormal expression miRNAs in TNBC are mainly chemoresistance related. This might be part of reason that TNBC likely to evade from chemotherapy resulting in early relapse and high risk of death. To alter their expression status might be a potential therapeutic strategy to improve the outcome of chemotherapy for TNBC patients.
doi:10.1371/journal.pone.0096228
PMCID: PMC4008525  PMID: 24788655
13.  MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis 
BMC Genomics  2012;13:552.
Background
Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI.
Results
The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed.
Conclusion
Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.
doi:10.1186/1471-2164-13-552
PMCID: PMC3532181  PMID: 23066896
Human myocardial infarction; Expression; SERCA2; miRNA; Bioinformatics
14.  Serum microRNA profiles in children with autism 
Molecular Autism  2014;5:40.
Background
As regulators of gene expression, microRNAs (miRNAs) play a key role in the transcriptional networks of the developing human brain. Circulating miRNAs in the serum and plasma are remarkably stable and are suggested to have promise as noninvasive biomarkers for neurological and neurodevelopmental disorders. We examined the serum expression profiles of neurologically relevant miRNAs in autism spectrum disorder (ASD), a complex neurodevelopmental disorder characterized by multiple deficits in communication, social interaction and behavior.
Methods
Total RNA, including miRNA, was extracted from the serum samples of 55 individuals with ASD and 55 age- and sex-matched control subjects, and the mature miRNAs were selectively converted into cDNA. Initially, the expression of 125 mature miRNAs was compared between pooled control and ASD samples. The differential expression of 14 miRNAs was further validated by SYBR Green quantitative PCR of individual samples. Receiver-operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of miRNAs. The target genes and pathways of miRNAs were predicted using DIANA mirPath software.
Results
Thirteen miRNAs were differentially expressed in ASD individuals compared to the controls. MiR-151a-3p, miR-181b-5p, miR-320a, miR-328, miR-433, miR-489, miR-572, and miR-663a were downregulated, while miR-101-3p, miR-106b-5p, miR-130a-3p, miR-195-5p, and miR-19b-3p were upregulated. Five miRNAs showed good predictive power for distinguishing individuals with ASD. The target genes of these miRNAs were enriched in several crucial neurological pathways.
Conclusions
This is the first study of serum miRNAs in ASD individuals. The results suggest that a set of serum miRNAs might serve as a possible noninvasive biomarker for ASD.
doi:10.1186/2040-2392-5-40
PMCID: PMC4132421  PMID: 25126405
Autism spectrum disorder; microRNA; complementary DNA; microarray; quantitative PCR
15.  Expression profiling of cancerous and normal breast tissues identifies microRNAs that are differentially expressed in serum from patients with (metastatic) breast cancer and healthy volunteers 
Introduction
MicroRNAs (miRNAs) are a group of small noncoding RNAs involved in the regulation of gene expression. As such, they regulate a large number of cellular pathways, and deregulation or altered expression of miRNAs is associated with tumorigenesis. In the current study, we evaluated the feasibility and clinical utility of circulating miRNAs as biomarkers for the detection and staging of breast cancer.
Methods
miRNAs were extracted from a set of 84 tissue samples from patients with breast cancer and eight normal tissue samples obtained after breast-reductive surgery. After reverse transcription and preamplification, 768 miRNAs were profiled by using the TaqMan low-density arrays. After data normalization, unsupervised hierarchical cluster analysis (UHCA) was used to investigate global differences in miRNA expression between cancerous and normal samples. With fold-change analysis, the most discriminating miRNAs between both tissue types were selected, and their expression was analyzed on serum samples from 20 healthy volunteers and 75 patients with breast cancer, including 16 patients with untreated metastatic breast cancer. miRNAs were extracted from 200 μl of serum, reverse transcribed, and analyzed in duplicate by using polymerase chain reaction (qRT-PCR).
Results
UHCA showed major differences in miRNA expression between tissue samples from patients with breast cancer and tissue samples from breast-reductive surgery (P < 0.0001). Generally, miRNA expression in cancerous samples tends to be repressed when compared with miRNA expression in healthy controls (P = 0.0685). The four most discriminating miRNAs by fold-change (miR-215, miR-299-5p, miR-411, and miR-452) were selected for further analysis on serum samples. All miRNAs at least tended to be differentially expressed between serum samples from patients with cancer and serum samples from healthy controls (miR-215, P = 0.094; miR-299-5P, P = 0.019; miR-411, P = 0.002; and miR-452, P = 0.092). For all these miRNAs, except for miR-452, the greatest difference in expression was observed between serum samples from healthy volunteers and serum samples from untreated patients with metastatic breast cancer.
Conclusions
Our study provides a basis for the establishment of miRNAs as biomarkers for the detection and eventually staging of breast cancer through blood-borne testing. We identified and tested a set of putative biomarkers of breast cancer and demonstrated that altered levels of these miRNAs in serum from patients with breast cancer are particularly associated with the presence of metastatic disease.
doi:10.1186/bcr3127
PMCID: PMC3496152  PMID: 22353773
16.  Microrna profiling analysis of differences between the melanoma of young adults and older adults 
Background
This study represents the first attempt to perform a profiling analysis of the intergenerational differences in the microRNAs (miRNAs) of primary cutaneous melanocytic neoplasms in young adult and older age groups. The data emphasize the importance of these master regulators in the transcriptional machinery of melanocytic neoplasms and suggest that differential levels of expressions of these miRs may contribute to differences in phenotypic and pathologic presentation of melanocytic neoplasms at different ages.
Methods
An exploratory miRNA analysis of 666 miRs by low density microRNA arrays was conducted on formalin fixed and paraffin embedded tissues (FFPE) from 10 older adults and 10 young adults including conventional melanoma and melanocytic neoplasms of uncertain biological significance. Age-matched benign melanocytic nevi were used as controls.
Results
Primary melanoma in patients greater than 60 years old was characterized by the increased expression of miRs regulating TLR-MyD88-NF-kappaB pathway (hsa-miR-199a), RAS/RAB22A pathway (hsa-miR-204); growth differentiation and migration (hsa-miR337), epithelial mesenchymal transition (EMT) (let-7b, hsa-miR-10b/10b*), invasion and metastasis (hsa-miR-10b/10b*), hsa-miR-30a/e*, hsa-miR-29c*; cellular matrix components (hsa-miR-29c*); invasion-cytokinesis (hsa-miR-99b*) compared to melanoma of younger patients. MiR-211 was dramatically downregulated compared to nevi controls, decreased with increasing age and was among the miRs linked to metastatic processes. Melanoma in young adult patients had increased expression of hsa-miR-449a and decreased expression of hsa-miR-146b, hsa-miR-214*. MiR-30a* in clinical stages I-II adult and pediatric melanoma could predict classification of melanoma tissue in the two extremes of age groups. Although the number of cases is small, positive lymph node status in the two age groups was characterized by the statistically significant expression of hsa-miR-30a* and hsa-miR-204 (F-test, p-value < 0.001).
Conclusions
Our findings, although preliminary, support the notion that the differential biology of melanoma at the extremes of age is driven, in part, by deregulation of microRNA expression and by fine tuning of miRs that are already known to regulate cell cycle, inflammation, Epithelial-Mesenchymal Transition (EMT)/stroma and more specifically genes known to be altered in melanoma. Our analysis reveals that miR expression differences create unique patterns of frequently affected biological processes that clearly distinguish old age from young age melanomas. This is a novel characterization of the miRnomes of melanocytic neoplasms at two extremes of age and identifies potential diagnostic and clinico-pathologic biomarkers that may serve as novel miR-based targeted modalities in melanoma diagnosis and treatment.
doi:10.1186/1479-5876-8-27
PMCID: PMC2855523  PMID: 20302635
17.  miRNA Profiling of Naïve, Effector and Memory CD8 T Cells 
PLoS ONE  2007;2(10):e1020.
microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific naïve, effector and memory CD8+ T cells using 3 different methods-small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for ∼60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to naïve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to naïve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3′end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.
doi:10.1371/journal.pone.0001020
PMCID: PMC2000354  PMID: 17925868
18.  MicroRNAs in bovine adipogenesis: genomic context, expression and function 
BMC Genomics  2014;15:137.
Background
MicroRNAs (miRNAs) are small non-coding RNAs found to regulate several biological processes including adipogenesis. Understanding adipose tissue regulation is critical for beef cattle as fat is an important determinant of beef quality and nutrient value. This study analyzed the association between genomic context characteristics of miRNAs with their expression and function in bovine adipose tissue. Twenty-four subcutaneous adipose tissue biopsies were obtained from eight British-continental crossbred steers at 3 different time points. Total RNA was extracted and miRNAs were profiled using a miRNA microarray with expression further validated by qRT-PCR.
Results
A total of 224 miRNAs were detected of which 155 were expressed in all steers (n = 8), and defined as the core miRNAs of bovine subcutaneous adipose tissue. Core adipose miRNAs varied in terms of genomic location (59.5% intergenic, 38.7% intronic, 1.2% exonic, and 0.6% mirtron), organization (55.5% non-clustered and 44.5% clustered), and conservation (49% highly conserved, 14% conserved and 37% poorly conserved). Clustered miRNAs and highly conserved miRNAs were more highly expressed (p < 0.05) and had more predicted targets than non-clustered or less conserved miRNAs (p < 0.001). A total of 34 miRNAs were coordinately expressed, being part of six identified relevant networks. Two intronic miRNAs (miR-33a and miR-1281) were confirmed to have coordinated expression with their host genes, transcriptional factor SREBF2 and EP300 (a transcriptional co-activator of transcriptional factor C/EBPα), respectively which are involved in lipid metabolism, suggesting these miRNAs may also play a role in regulation of bovine lipid metabolism/adipogenesis. Furthermore, a total of 17 bovine specific miRNAs were predicted to be involved in the regulation of energy balance in adipose tissue.
Conclusions
These findings improve our understanding on the behavior of miRNAs in the regulation of bovine adipogenesis and fat metabolism as it reveals that miRNA expression patterns and functions are associated with miRNA genomic location, organization and conservation.
doi:10.1186/1471-2164-15-137
PMCID: PMC3930007  PMID: 24548287
Adipogenesis; Adipose tissue; Bovine; Fat metabolism; Genomic context; microRNA; Cluster; Co-expression; Species specific
19.  Identification of Nitrogen Starvation-Responsive MicroRNAs in Arabidopsis thaliana 
PLoS ONE  2012;7(11):e48951.
microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.
doi:10.1371/journal.pone.0048951
PMCID: PMC3498362  PMID: 23155433
20.  Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis 
Introduction
MicroRNAs (miRNAs), endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes, are present in human plasma in a stable form. In this study, we investigated whether miRNAs are also stably present in synovial fluids and whether plasma and synovial fluid miRNAs could be biomarkers of rheumatoid arthritis (RA) and osteoarthritis (OA).
Methods
We measured concentrations of miR-16, miR-132, miR-146a, miR-155 and miR-223 in synovial fluid from patients with RA and OA, and those in plasma from RA, OA and healthy controls (HCs) by quantitative reverse transcription-polymerase chain reaction. Furthermore, miRNAs in the conditioned medium of synovial tissues, monolayer fibroblast-like synoviocytes, and mononuclear cells were examined. Correlations between miRNAs and biomarkers or disease activities of RA were statistically examined.
Results
Synovial fluid miRNAs were present and as stable as plasma miRNAs for storage at -20°C and freeze-thawing from -20°C to 4°C. In RA and OA, synovial fluid concentrations of miR-16, miR-132, miR-146a, and miR-223 were significantly lower than their plasma concentrations, and there were no correlation between plasma and synovial fluid miRNAs. Interestingly, synovial tissues, fibroblast-like synoviocytes, and mononuclear cells secreted miRNAs in distinct patterns. The expression patterns of miRNAs in synovial fluid of OA were similar to miRNAs secreted by synovial tissues. Synovial fluid miRNAs of RA were likely to originate from synovial tissues and infiltrating cells. Plasma miR-132 of HC was significantly higher than that of RA or OA with high diagnosability. Synovial fluid concentrations of miR-16, miR-146a miR-155 and miR-223 of RA were significantly higher than those of OA. Plasma miRNAs or ratio of synovial fluid miRNAs to plasma miRNAs, including miR-16 and miR-146a, significantly correlated with tender joint counts and 28-joint Disease Activity Score.
Conclusions
Plasma miRNAs had distinct patterns from synovial fluid miRNAs, which appeared to originate from synovial tissue. Plasma miR-132 well differentiated HCs from patients with RA or OA, while synovial fluid miRNAs differentiated RA and OA. Furthermore, plasma miRNAs correlated with the disease activities of RA. Thus, synovial fluid and plasma miRNAs have potential as diagnostic biomarkers for RA and OA and as a tool for the analysis of their pathogenesis.
doi:10.1186/ar3013
PMCID: PMC2911870  PMID: 20470394
21.  Identification and characteristics of microRNAs from Bombyx mori 
BMC Genomics  2008;9:248.
Background
MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression by targeting messenger RNAs (mRNAs) and causing mRNA cleavage or translation blockage. Of the 355 Arthropod miRNAs that have been identified, only 21 are B. mori miRNAs that were predicted computationally; of these, only let-7 has been confirmed by Northern blotting.
Results
Combining a computational method based on sequence homology searches with experimental identification based on microarray assays and Northern blotting, we identified 46 miRNAs, an additional 21 plausible miRNAs, and a novel small RNA in B. mori. The latter, bmo-miR-100-like, was identified using the known miRNA aga-miR-100 as a probe; bmo-miR-100-like was detected by microarray assay and Northern blotting, but its precursor sequences did not fold into a hairpin structure. Among these identified miRNAs, we found 12 pairs of miRNAs and miRNA*s. Northern blotting revealed that some B. mori miRNA genes were expressed only during specific stages, indicating that B. mori miRNA genes (e.g., bmo-miR-277) have developmentally regulated patterns of expression. We identified two miRNA gene clusters in the B. mori genome. bmo-miR-2b, which is found in the gene cluster bmo-miR-2a-1/bmo-miR-2a-1*/bmo-miR-2a-2/bmo-miR-2b/bmo-miR-13a*/bmo-miR-13b, encodes a newly identified member of the mir-2 family. Moreover, we found that methylation can increase the sensitivity of a DNA probe used to detect a miRNA by Northern blotting. Functional analysis revealed that 11 miRNAs may regulate 13 B. mori orthologs of the 25 known Drosophila miRNA-targeted genes according to the functional conservation. We predicted the binding sites on the 1671 3'UTR of B. mori genes; 547 targeted genes, including 986 target sites, were predicted. Of these target sites, 338 had perfect base pairing to the seed region of 43 miRNAs. From the predicted genes, 61 genes, each of them with multiple predicted target sites, should be considered excellent candidates for future functional studies. Biological classification of predicted miRNA targets showed that "binding", "catalytic activity" and "physiological process" were over-represented for the predicted genes.
Conclusion
Combining computational predictions with microarray assays, we identified 46 B. mori miRNAs, 13 of which were miRNA*s. We identified a novel small RNA and 21 plausible B. mori miRNAs that could not be located in the available B. mori genome, but which could be detected by microarray. Thirteen and 547 target genes were predicted according to the functional conservation and binding sites, respectively. Identification of miRNAs in B. mori, particularly those that are developmentally regulated, provides a foundation for subsequent functional studies.
doi:10.1186/1471-2164-9-248
PMCID: PMC2435238  PMID: 18507836
22.  MicroRNAs Located in the Hox Gene Clusters Are Implicated in Huntington's Disease Pathogenesis 
PLoS Genetics  2014;10(2):e1004188.
Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntington's disease (HD). MicroRNAs (miRNAs) represent a major system of post-transcriptional regulation, by either preventing translational initiation or by targeting transcripts for storage or for degradation. Using next-generation miRNA sequencing in prefrontal cortex (Brodmann Area 9) of twelve HD and nine controls, we identified five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-615-3p and miR-1247-5p) up-regulated in HD at genome-wide significance (FDR q-value<0.05). Three of these, miR-196a-5p, miR-196b-5p and miR-615-3p, were expressed at near zero levels in control brains. Expression was verified for all five miRNAs using reverse transcription quantitative PCR and all but miR-1247-5p were replicated in an independent sample (8HD/8C). Ectopic miR-10b-5p expression in PC12 HTT-Q73 cells increased survival by MTT assay and cell viability staining suggesting increased expression may be a protective response. All of the miRNAs but miR-1247-5p are located in intergenic regions of Hox clusters. Total mRNA sequencing in the same samples identified fifteen of 55 genes within the Hox cluster gene regions as differentially expressed in HD, and the Hox genes immediately adjacent to the four Hox cluster miRNAs as up-regulated. Pathway analysis of mRNA targets of these miRNAs implicated functions for neuronal differentiation, neurite outgrowth, cell death and survival. In regression models among the HD brains, huntingtin CAG repeat size, onset age and age at death were independently found to be inversely related to miR-10b-5p levels. CAG repeat size and onset age were independently inversely related to miR-196a-5p, onset age was inversely related to miR-196b-5p and age at death was inversely related to miR-615-3p expression. These results suggest these Hox-related miRNAs may be involved in neuroprotective response in HD. Recently, miRNAs have shown promise as biomarkers for human diseases and given their relationship to disease expression, these miRNAs are biomarker candidates in HD.
Author Summary
Huntington's disease (HD) is an inherited fatal neurological disorder that commonly affects people in midlife. Past studies have implicated abnormal patterns of gene expression as a candidate for causing the death of the brain cells affected in HD. MicroRNAs (miRNAs) are small molecules that regulate and target transcripts for either storage or destruction. We measured the levels of miRNAs, as well as the levels of gene expression (mRNAs) in twelve HD and nine control brain samples. We found five miRNAs that had greatly increased expression in the HD brains, including three that were not expressed in the normal samples. Four of these were related to important characteristics of the disease expression, including the age at disease onset, and the age at death of the individual. The genes that these miRNAs target for regulation were also altered in their expression with most being increased, suggesting they may have been targeted for storage. One of the miRNAs, miR-196a-5p was previously implicated in enhancing the survival of brain cells in HD. When we overexpressed miR-10b-5p in an HD cell model, the cells survived longer than untreated cells, suggesting these miRNAs may promote neuron survival and may hold new clues for treatments in HD.
doi:10.1371/journal.pgen.1004188
PMCID: PMC3937267  PMID: 24586208
23.  Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development 
PLoS Biology  2007;5(8):e203.
Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.
Author Summary
The striking tissue-specific expression patterns of microRNAs (miRNAs) suggest that they play a role in tissue development. These small RNA molecules (∼22 bases in length) are processed from long primary transcripts (pri-miRNA) and regulate gene expression at the posttranscriptional level. There are hundreds of different miRNAs, many of which are strongly conserved. Vertebrate embryonic development is most easily studied in zebrafish, but genetically disrupting miRNA genes to see which miRNA does what is technically challenging. In this study, we interfere with miRNA function during the first few days of zebrafish embryonic development by introducing specific antisense morpholino oligonucleotides (morpholinos have been used previously to interfere with the synthesis of the much larger mRNAs). We show that morpholinos targeting the miRNA precursor can block processing of the pri-miRNA or directly inhibit the activity of the mature miRNA. We also used morpholinos to study the developmental effects of miRNA knockdown. Although we did not observe gross phenotypic defects for many miRNAs, we found that zebrafish miR-375 is essential for formation of the insulin-secreting pancreatic islet. Loss of miR-375 results in dispersed islet cells by 36 hours postfertilization, representing one of the first vertebrate miRNA loss-of-function phenotypes.
The authors show that morpholinos can be used to knock down zebrafish miRNAs, revealing that miR-375 is important for vertebrate pancreas development.
doi:10.1371/journal.pbio.0050203
PMCID: PMC1925136  PMID: 17676975
24.  The miR-15/107 group of microRNA genes: evolutionary biology, cellular functions, and roles in human diseases 
Journal of molecular biology  2010;402(3):491-509.
The miR-15/107 group of microRNA (miRNA) genes is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease, and neurodegenerative diseases including Alzheimer’s disease. Here, we provide an overview of (1) the evolution of miR-15/107 group member genes, (2) the expression levels of the miRNAs in mammalian tissues, (3) evidence for overlapping gene regulatory functions by the different miRNAs, (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs, and (5) the roles played by these miRNAs in human diseases. Membership in this group is defined on the basis of sequence similarity near the mature miRNAs’ 5′ end: all include the sequence AGCAGC. Phylogeny of this group of miRNAs is incomplete so a definitive taxonomic classification (for example, designation as a “superfamily”) is currently not possible. While all vertebrates studied to date express miR-15a, -15b, -16, -103, and -107, mammals alone are known to express miR-195, -424, -497, -503, and -646. Multiple different miRNAs in the miR-15/107 group are expressed at moderate-to-high levels in human tissues. We present data on the expression of all known miR-15/107 group members in human cerebral cortical gray and white matter using new miRNA profiling microarrays. There is extensive overlap in the mRNAs targeted by miR-15/107 group members. We show new data from cultured H4 cancer cells that demonstrate similarities in mRNAs targeted by miR-16 and miR-103, and also support the importance of the mature miRNAs’ 5′ seed region in mRNA target recognition. In conclusion, the miR-15/107 group of miRNA genes is a fascinating topic of study for evolutionary biologists, miRNA biochemists, and clinically-oriented translational researchers alike.
doi:10.1016/j.jmb.2010.07.051
PMCID: PMC2978331  PMID: 20678503
25.  Identification and characterization of microRNAs in the flag leaf and developing seed of wheat (Triticum aestivum L.) 
BMC Genomics  2014;15:289.
Background
MicroRNAs (miRNAs) regulate various biological processes in plants. Considerable data are available on miRNAs involved in the development of rice, maize and barley. In contrast, little is known about miRNAs and their functions in the development of wheat. In this study, five small RNA (sRNA) libraries from wheat seedlings, flag leaves, and developing seeds were developed and sequenced to identify miRNAs and understand their functions in wheat development.
Results
Twenty-four known miRNAs belonging to 15 miRNA families were identified from 18 MIRNA loci in wheat in the present study, including 15 miRNAs (9 MIRNA loci) first identified in wheat, 13 miRNA families (16 MIRNA loci) being highly conserved and 2 (2 MIRNA loci) moderately conserved. In addition, fifty-five novel miRNAs were also identified. The potential target genes for 15 known miRNAs and 37 novel miRNAs were predicted using strict criteria, and these target genes are involved in a wide range of biological functions. Four of the 15 known miRNA families and 22 of the 55 novel miRNAs were preferentially expressed in the developing seeds with logarithm (log2) of the fold change of 1.0 ~ 7.6, and half of them were seed-specific, suggesting that they participate in regulating wheat seed development and metabolism. From 5 days post-anthesis to 20 days post-anthesis, miR164 and miR160 increased in abundance in the developing seeds, whereas miR169 decreased, suggesting their coordinating functions in the different developmental stages of wheat seed. Moreover, 8 known miRNA families and 28 novel miRNAs exhibited tissue-biased expression in wheat flag leaves, with the logarithm of the fold changes of 0.1 ~ 5.2. The putative targets of these tissue-preferential miRNAs were involved in various metabolism and biological processes, suggesting complexity of the regulatory networks in different tissues. Our data also suggested that wheat flag leaves have more complicated regulatory networks of miRNAs than developing seeds.
Conclusions
Our work identified and characterised wheat miRNAs, their targets and expression patterns. This study is the first to elucidate the regulatory networks of miRNAs involved in wheat flag leaves and developing seeds, and provided a foundation for future studies on specific functions of these miRNAs.
doi:10.1186/1471-2164-15-289
PMCID: PMC4029127  PMID: 24734873
MicroRNA; Triticum aestivum; Flag leaf; Seed development; Small RNA sequencing; Expression profile; miRNA target

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