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1.  IL-10Rα expression is post-transcriptionally regulated by miR-15a, miR-185, and miR-211 in melanoma 
BMC Medical Genomics  2015;8:81.
IL-10 is an immunoregulatory cytokine that increases during malignant diseases. The purpose of this study was to: i) determine the mRNA amounts of IL-10, IL-10Rα, and IL-10Rβ in cutaneous and uveal melanoma cells and specimens; ii) evaluate their post-transcriptional regulation by miRNAs; iii) ascertain whether miRNA dysregulation may affect IL-10-induced proliferation.
Genome-wide miRNA expression profiling was performed using a human microarray platform. The reference gene mRNA was measured through qPCR. miRNAs/mRNAs interactions were predicted by TargetScan, microRNA, and PITA. Transfections of specific miRNA mimics/inhibitors were carried out. Cell proliferation was assessed by MTT assay in the presence of IL-10 after transfection with miRNA mimics/inhibitors.
There were no differences in IL-10 mRNA levels between any of the 3 melanoma cell lines tested and normal melanocytes. However, lower IL-10Rα expression was found in G361 and OCM-1 cells, and higher levels of IL-10Rβ were observed in G361 cells compared with normal melanocytes. GR-M cells did not exhibit any modifications in IL-10Rα and IL-10Rβ expression. miR-15a, miR-185, miR-211, and miR-30d were upregulated in G361 and OCM-1 cells, remaining at similar levels in GR-M cells. miR-409-3p and miR-605were down-regulated exclusively in G361 cells. Prediction tools revealed that miR-15a, miR-185, and miR-211 targeted IL-10Rα whereas none of the miRNAs exclusively downregulated in G361 cells targeted IL-10Rβ. Luciferase reporter and western blot assays showed that IL-10Rα expression is directly regulated by miR-15a, miR-185, and miR-211, either alone or in combination. An inverse expression pattern between IL-10Rα, on one side, and miR-15a, miR-185, and miR-211 on the other one was also shown in melanoma samples. Ectopic expression of individual miR-15a, miR-185, and miR-211, and even more their co-expression, caused a marked decrease in the proliferation rate of all the cell lines. Likewise, inhibition of any specific miRNA promoted cell growth, an effect that further increased when inhibition concerned all three miRNA. Moreover, specific knockdown of IL-10Rα prevented the proliferative effect of miRNA inhibitors.
Our results support a key role of IL-10Rα in the development and progression of melanoma and suggest that the IL-10/IL-10 receptor system may become a new therapeutic target for melanoma treatment.
PMCID: PMC4668633  PMID: 26631117
IL-10; IL-10Rα; IL-10Rβ; miRNAs; Cutaneous melanoma; Uveal melanoma
2.  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.
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival
3.  The expression of microRNA-375 in plasma and tissue is matched in human colorectal cancer 
BMC Cancer  2014;14:714.
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.
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.
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.
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.
PMCID: PMC4181388  PMID: 25255814
Colorectal cancer; MicroRNA; Plasma; Tissue; Biomarker; Diagnosis
4.  A signature of circulating microRNAs differentiates takotsubo cardiomyopathy from acute myocardial infarction 
European Heart Journal  2013;35(15):999-1006.
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).
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.
PMCID: PMC3985061  PMID: 24046434
Takotsubo cardiomyopathy; Biomarker; Endothelin-1; MicroRNA
5.  Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels 
MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P < 0.05), which was 16%–54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%–70% of control), while only expression of miR-138 was significantly upregulated (2.91 ± 0.8-fold of the control, P < 0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked to distinct biological roles in WAT and BAT during hibernation and may involve the regulation of signaling cascades.
PMCID: PMC4411486  PMID: 25526980
Non-coding RNA; Dicer; Ground squirrel; Stress adaptation; Hypometabolism
6.  MicroRNA-125a is over-expressed in insulin target tissues in a spontaneous rat model of Type 2 Diabetes 
BMC Medical Genomics  2009;2:54.
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.
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.
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).
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.
PMCID: PMC2754496  PMID: 19689793
7.  In silico analysis of polymorphisms in microRNAs that target genes affecting aerobic glycolysis 
Cancer cells preferentially metabolize glucose through aerobic glycolysis, an observation known as the Warburg effect. Recently, studies have deciphered the role of oncogenes and tumor suppressor genes in regulating the Warburg effect. Furthermore, mutations in glycolytic enzymes identified in various cancers highlight the importance of the Warburg effect at the molecular and cellular level. MicroRNAs (miRNAs) are non-coding RNAs that posttranscriptionally regulate gene expression and are dysregulated in the pathogenesis of various types of human cancers. Single nucleotide polymorphisms (SNPs) in miRNA genes may affect miRNA biogenesis, processing, function, and stability and provide additional complexity in the pathogenesis of cancer. Moreover, mutations in miRNA target sequences in target mRNAs can affect expression.
In silico analysis and cataloguing polymorphisms in miRNA genes that target genes directly or indirectly controlling aerobic glycolysis was carried out using different publically available databases.
miRNA SNP2.0 database revealed several SNPs in miR-126 and miR-25 in the upstream and downstream pre-miRNA flanking regions respectively should be inserted after flanking regions and miR-504 and miR-451 had the fewest. These miRNAs target genes that control aerobic glycolysis indirectly. SNPs in premiRNA genes were found in miR-96, miR-155, miR-25 and miR34a by miRNASNP. Dragon database of polymorphic regulation of miRNA genes (dPORE-miRNA) database revealed several SNPs that modify transcription factor binding sites (TFBS) or creating new TFBS in promoter regions of selected miRNA genes as analyzed by dPORE-miRNA.
Our results raise the possibility that integration of SNP analysis in miRNA genes with studies of metabolic adaptations in cancer cells could provide greater understanding of oncogenic mechanisms.
PMCID: PMC4779782  PMID: 27004216
MicroRNAs (miRNA); single nucleotide polymorphism (SNP); cancer
8.  Identification and bioinformatics analysis of microRNAs associated with stress and immune response in serum of heat-stressed and normal Holstein cows 
Cell Stress & Chaperones  2014;19(6):973-981.
MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that have an important regulatory function in animal growth and developmental processes. However, the differential expression of miRNA and the role of these miRNAs in heat-stressed Holstein cows are still unknown. In this study, the profile of differentially expressed miRNAs and the target genes analysis in the serum of heat-stressed and normal Holstein cows were investigated by a Solexa deep-sequencing approach and bioinformatics. The data identified 52 differentially expressed miRNAs in 486 known miRNAs which were changed significantly between heat-stressed and normal Holstein cows (fold change >2, P < 0.001). Target genes analysis showed that at least 7 miRNAs (miR-19a, miR-19b, miR-146a, miR-30a-5p, miR-345-3p, miR-199a-3p, and miR-1246) were involved in the response to stress, oxidative stress, development of the immune system, and immune response among the identified 52 differentially expressed miRNAs. Five miRNAs (miR-27b, miR-181a, miR-181b, miR-26a, and miR-146b) were involved in stress and immune responses and the expression of five miRNAs was striking (P < 0.001). In addition, RT-qPCR and deep-sequencing methods showed that 8 miRNAs among the 12 selected miRNAs (miR-19a, miR-19b, miR-27b, miR-30a-5p, miR-181a, miR-181b, miR-345-3p, and miR-1246) were highly expressed in the serum of heat-stressed Holstein cows. GO and KEGG pathway analysis showed that these differentially expressed miRNAs were involved in a pathway that may differentially regulate the expression of stress response and immune response genes. Our study provides an overview of miRNAs expression profile and the interaction between miRNAs and their target genes, which will lead to further understanding of the important roles of miRNAs in heat-stressed Holstein cows.
PMCID: PMC4389857  PMID: 24917036
Heat stress; Serum; MicroRNAs; Target genes; Cows
9.  MicroRNA expression after ionizing radiation in human endothelial cells 
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.
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.
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.
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.
PMCID: PMC2859352  PMID: 20346162
10.  Identification of microRNAs from Amur grape (vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics 
BMC Genomics  2012;13:122.
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.
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.
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.
PMCID: PMC3353164  PMID: 22455456
Amur grape; microRNA; Sequences evolution; Solexa sequencing; miR-RACE; qRT-PCR
11.  Expression of Dicer and Its Related MiRNAs in the Progression of Prostate Cancer 
PLoS ONE  2015;10(3):e0120159.
Dicer is aberrantly expressed in several types of malignancies. Cleaved by Dicer, the small noncoding microRNAs (miRNAs) are considered potential tools for the diagnosis and prognosis of cancer. This study investigated the expression of miRNAs thought to target Dicer. Expression of 1,205 human miRNAs and miRNA*s were examined in four patients with prostate cancer (PCa) by miRNA array in which the threshold was set as two-fold. Seventy-three miRNAs and miRNA*s were significantly down-regulated while 10 were up-regulated in PCa tissues compared with matched histologically normal glands. Of these, miR-29b-1, miR-200a, miR-370, and miR-31, which were the most down/up-regulated and closely potentially target to the Dicer 3′ UTR, were investigated further. Tissues of primary tumors and matched normal prostate glands from 185 patients with PCa were collected for further investigation. Dicer mRNA levels were negatively correlated with miR-29b-1 (ρs = −0.177, p = 0.017), miR-200a (ρs = -0.489, p < 0.0001) and miR-31 (ρs = −0.314, p < 0.0001) expression. Compared with adjacent normal glands, PCa tissues showed significantly lower miR-200a and miR-31 expression levels. Furthermore, in metastatic PCa, the expression levels of miR-200a, miR-370, and miR-31 were dramatically higher than in localized PCa. Additionally, elevated expression levels of miR-200a and miR-31 appeared to be associated with castration-resistant PCa. These findings suggest possibilities that miR-200a and miR-31 target Dicer and are involved in the carcinogenesis, migration, and behavior of castration-resistant PCa, indicating that they could be potential biomarkers for monitoring PCa progression.
PMCID: PMC4358996  PMID: 25768283
12.  Hormonal Regulation of MicroRNA Expression in Steroid Producing Cells of the Ovary, Testis and Adrenal Gland 
PLoS ONE  2013;8(10):e78040.
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.
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.
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.
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.
PMCID: PMC3810252  PMID: 24205079
13.  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.
PMCID: PMC3174204  PMID: 21935352
14.  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.
PMCID: PMC3677622  PMID: 23781281
microRNA; Prostate Cancer; biomarkers.
15.  Identification of microRNAs associated with allergic airway disease using a genetically diverse mouse population 
BMC Genomics  2015;16(1):633.
Allergic airway diseases (AADs) such as asthma are characterized in part by granulocytic airway inflammation. The gene regulatory networks that govern granulocyte recruitment are poorly understood, but evidence is accruing that microRNAs (miRNAs) play an important role. To identify miRNAs that may underlie AADs, we used two complementary approaches that leveraged the genotypic and phenotypic diversity of the Collaborative Cross (CC) mouse population. In the first approach, we sought to identify miRNA expression quantitative trait loci (eQTL) that overlap QTL for AAD-related phenotypes. Specifically, CC founder strains and incipient lines of the CC were sensitized and challenged with house dust mite allergen followed by measurement of granulocyte recruitment to the lung. Total lung RNA was isolated and miRNA was measured using arrays for CC founders and qRT-PCR for incipient CC lines.
Among CC founders, 92 miRNAs were differentially expressed. We measured the expression of 40 of the most highly expressed of these 92 miRNAs in the incipient lines of the CC and identified 18 eQTL corresponding to 14 different miRNAs. Surprisingly, half of these eQTL were distal to the corresponding miRNAs, and even on different chromosomes. One of the largest-effect local miRNA eQTL was for miR-342-3p, for which we identified putative causal variants by bioinformatic analysis of the effects of single nucleotide polymorphisms on RNA structure. None of the miRNA eQTL co-localized with QTL for eosinophil or neutrophil recruitment. In the second approach, we constructed putative miRNA/mRNA regulatory networks and identified three miRNAs (miR-497, miR-351 and miR-31) as candidate master regulators of genes associated with neutrophil recruitment. Analysis of a dataset from human keratinocytes transfected with a miR-31 inhibitor revealed two target genes in common with miR-31 targets correlated with neutrophils, namely Oxsr1 and Nsf.
miRNA expression in the allergically inflamed murine lung is regulated by genetic loci that are smaller in effect size compared to mRNA eQTL and often act in trans. Thus our results indicate that the genetic architecture of miRNA expression is different from mRNA expression. We identified three miRNAs, miR-497, miR-351 and miR-31, that are candidate master regulators of genes associated with neutrophil recruitment. Because miR-31 is expressed in airway epithelia and is predicted to target genes with known links to neutrophilic inflammation, we suggest that miR-31 is a potentially novel regulator of airway inflammation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1732-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4548451  PMID: 26303911
Allergic inflammation; Asthma; miRNA; Expression QTL
16.  MicroRNA Expression Profiling in Clear Cell Renal Cell Carcinoma: Identification and Functional Validation of Key miRNAs 
PLoS ONE  2015;10(5):e0125672.
This study aims to profile dysregulated microRNA (miRNA) expression in clear cell renal cell carcinoma (ccRCC) and to identify key regulatory miRNAs in ccRCC.
Methods and Results
miRNA expression profiles in nine pairs of ccRCC tumor samples at three different stages and the adjacent, non-tumorous tissues were investigated using miRNA arrays. Eleven miRNAs were identified to be commonly dysregulated, including three up-regulated (miR-487a, miR-491-3p and miR-452) and eight down-regulated (miR-125b, miR-142-3p, miR-199a-5p, miR-22, miR-299-3p, miR-29a, miR-429, and miR-532-5p) in tumor tissues as compared with adjacent normal tissues. The 11 miRNAs and their predicted target genes were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and three key miRNAs (miR-199a-5p, miR-22 and miR-429) were identified by microRNA-gene network analysis. Dysregulation of the three key miRNAs were further validated in another cohort of 15 ccRCC samples, and the human kidney carcinoma cell line 786-O, as compared with five normal kidney samples. Further investigation showed that over-expression of miR-199a-5p significantly inhibited the invasion ability of 786-O cells. Luciferase reporter assays indicated that miR-199a-5p regulated expression of TGFBR1 and JunB by directly interacting with their 3’ untranslated regions. Transfection of miR-199a-5p successfully suppressed expression of TGFBR1 and JunB in the human embryonic kidney 293T cells, further confirming the direct regulation of miR-199a-5p on these two genes.
This study identified 11 commonly dysregulated miRNAs in ccRCC, three of which (miR-199a-5p, miR-22 and miR-429) may represent key miRNAs involved in the pathogenesis of ccRCC. Further studies suggested that miR-199a-5p plays an important role in inhibition of cell invasion of ccRCC cells by suppressing expression of TGFBR1 and JunB.
PMCID: PMC4418764  PMID: 25938468
17.  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.
PMCID: PMC4207049  PMID: 25364765
Alzheimer's disease; early diagnosis; gastric cancer; hypertension; pancreatic cancer; personalize medicine; rheumatoid arthritis; stem cells
18.  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.
PMCID: PMC3623967  PMID: 23593197
19.  Uncovering miRNAs involved in crosstalk between nutrient deficiencies in Arabidopsis 
Scientific Reports  2015;5:11813.
Integrating carbon (C), nitrogen (N), and sulfur (S) metabolism is essential for the growth and development of living organisms. MicroRNAs (miRNAs) play key roles in regulating nutrient metabolism in plants. However, how plant miRNAs mediate crosstalk between different nutrient metabolic pathways is unclear. In this study, deep sequencing of Arabidopsis thaliana small RNAs was used to reveal miRNAs that were differentially expressed in response to C, N, or S deficiency. Comparative analysis revealed that the targets of the differentially expressed miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin signal transduction, nutrient homeostasis, and regulation of development. C, N, and S deficiency specifically induced miR169b/c, miR826 and miR395, respectively. In contrast, miR167, miR172, miR397, miR398, miR399, miR408, miR775, miR827, miR841, miR857, and miR2111 are commonly suppressed by C, N, and S deficiency. In particular, the miRNAs that are induced specifically by a certain nutrient deficiency are often suppressed by other nutrient deficiencies. Further investigation indicated that the modulation of nutrient-responsive miRNA abundance affects the adaptation of plants to nutrient starvation conditions. This study revealed that miRNAs function as important regulatory nodes of different nutrient metabolic pathways.
PMCID: PMC4488870  PMID: 26134148
20.  MicroRNA Profiling Implies New Markers of Chemoresistance of Triple-Negative Breast Cancer 
PLoS ONE  2014;9(5):e96228.
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.
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.
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.
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.
PMCID: PMC4008525  PMID: 24788655
21.  miR-203 and miR-221 regulate SOCS1 and SOCS3 in essential thrombocythemia 
Blood Cancer Journal  2016;6(3):e406-.
The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.
PMCID: PMC4817095  PMID: 26990535
22.  Identification of Genes Related to Growth and Lipid Deposition from Transcriptome Profiles of Pig Muscle Tissue 
PLoS ONE  2015;10(10):e0141138.
Transcriptome profiles established using high-throughput sequencing can be effectively used for screening genome-wide differentially expressed genes (DEGs). RNA sequences (from RNA-seq) and microRNA sequences (from miRNA-seq) from the tissues of longissimus dorsi muscle of two indigenous Chinese pig breeds (Diannan Small-ear pig [DSP] and Tibetan pig [TP]) and two introduced pig breeds (Landrace [LL] and Yorkshire [YY]) were examined using HiSeq 2000 to identify and compare the differential expression of functional genes related to muscle growth and lipid deposition. We obtained 27.18 G clean data through the RNA-seq and detected that 18,208 genes were positively expressed and 14,633 of them were co-expressed in the muscle tissues of the four samples. In all, 315 DEGs were found between the Chinese pig group and the introduced pig group, 240 of which were enriched with functional annotations from the David database and significantly enriched in 27 Gene Ontology (GO) terms that were mainly associated with muscle fiber contraction, cadmium ion binding, response to organic substance and contractile fiber part. Based on functional annotation, we identified 85 DEGs related to growth traits that were mainly involved in muscle tissue development, muscle system process, regulation of cell development, and growth factor binding, and 27 DEGs related to lipid deposition that were mainly involved in lipid metabolic process and fatty acid biosynthetic process. With miRNA-seq, we obtained 23.78 M reads and 320 positively expressed miRNAs from muscle tissues, including 271 known pig miRNAs and 49 novel miRNAs. In those 271 known miRNAs, 20 were higher and 10 lower expressed in DSP-TP than in LL-YY. The target genes of the 30 miRNAs were mainly participated in MAPK, GnRH, insulin and Calcium signaling pathway and others involved cell development, growth and proliferation, etc. Combining the DEGs and the differentially expressed (DE) miRNAs, we drafted a network of 46 genes and 18 miRNAs for regulating muscle growth and a network of 15 genes and 16 miRNAs for regulating lipid deposition. We identified that CAV2, MYOZ2, FRZB, miR-29b, miR-122, miR-145-5p and miR-let-7c, etc, were key genes or miRNAs regulating muscle growth, and FASN, SCD, ADORA1, miR-4332, miR-182, miR-92b-3p, miR-let-7a and miR-let-7e, etc, were key genes or miRNAs regulating lipid deposition. The quantitative expressions of eight DEGs and seven DE miRNAs measured with real-time PCR certified that the results of differential expression genes or miRNAs were reliable. Thus, 18,208 genes and 320 miRNAs were positively expressed in porcine longissimus dorsi muscle. We obtained 85 genes and 18 miRNAs related to muscle growth and 27 genes and 16 miRNAs related to lipid deposition, which provided new insights into molecular mechanism of the economical traits in pig.
PMCID: PMC4624711  PMID: 26505482
23.  MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis 
BMC Genomics  2012;13:552.
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.
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.
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.
PMCID: PMC3532181  PMID: 23066896
Human myocardial infarction; Expression; SERCA2; miRNA; Bioinformatics
24.  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 
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.
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).
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.
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.
PMCID: PMC3496152  PMID: 22353773
25.  Serum microRNA profiles in children with autism 
Molecular Autism  2014;5:40.
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.
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.
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.
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.
PMCID: PMC4132421  PMID: 25126405
Autism spectrum disorder; microRNA; complementary DNA; microarray; quantitative PCR

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