MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR-155 and miR-146a in rheumatoid arthritis (RA) and showed a repressive effect of miR-155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR-203 in RASFs and analyze its role in fibroblast activation.
Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real-time polymerase chain reaction (PCR)–based screening of 260 individual miRNA. Transfection of miR-203 precursor was used to analyze the function of miR-203 in RASFs. Levels of interleukin-6 (IL-6) and MMPs were measured by real-time PCR and enzyme-linked immunosorbent assay. RASFs were stimulated with IL-1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5-azacytidine (5-azaC). Activity of IκB kinase 2 was inhibited with SC-514.
Expression of miR-203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR-203 did not change upon stimulation with IL-1β, TNFα, or LPS; however, DNA demethylation with 5-azaC increased the expression of miR-203. Enforced expression of miR-203 led to significantly increased levels of MMP-1 and IL-6. Induction of IL-6 by miR-203 overexpression was inhibited by blocking of the NF-κB pathway. Basal expression levels of IL-6 correlated with basal expression levels of miR-203.
The current results demonstrate methylation-dependent regulation of miR-203 expression in RASFs. Importantly, they also show that elevated levels of miR-203 lead to increased secretion of MMP-1 and IL-6 via the NF-κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.
Several microRNA, which are ~22-nucleotide noncoding RNAs, exhibit tissue-specific or developmental stage–specific expression patterns and are associated with human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in synovial tissue from patients with rheumatoid arthritis (RA).
The expression of miR-146 in synovial tissue from 5 patients with RA, 5 patients with osteoarthritis (OA), and 1 normal subject was analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR) and by in situ hybridization and immunohistochemistry of tissue sections. Induction of miR-146 following stimulation with tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR.
Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFα, but the 2 microRNA were less highly expressed in OA and normal synovial tissue. In situ hybridization showed primary miR-146a expression in cells of the superficial and sublining layers in synovial tissue from RA patients. Cells positive for miR-146a were primarily CD68+ macrophages, but included several CD3+ T cell subsets and CD79a+ B cells. Expression of miR-146a/b was markedly up-regulated in RASFs after stimulation with TNFα and IL-1β.
This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFα and IL-1β. Further studies are required to elucidate the function of miR-146 in these tissues.
MicroRNAs (miRNAs), endogenous small non-coding RNAs, are stably detected in human plasma. Early diagnosis of gastric cancer (GC) is very important to improve the therapy effect and prolong the survival of patients. We aimed to identify whether four miRNAs (miR-223, miR-21, miR-218 and miR-25) closely associated with the tumorigenesis or metastasis of GC can serve as novel potential biomarkers for GC detection.
We initially measured the plasma levels of the four miRNAs in 10 GC patients and 10 healthy control subjects by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and then compared plasma miRNA results with the expressions in cancer tissues from eight GC patients. Finally, the presence of miR-223, miR-21 and miR-218 in the plasma was validated in 60 GC patients and 60 healthy control subjects, and the areas under the receiver operating characteristic (ROC) curves of these miRNAs were analyzed.
We found that the plasma levels of miR-223 (P<0.001) and miR-21 (P<0.001) were significantly higher in GC patients than in healthy controls, while miR-218 (P<0.001) was significantly lower. The ROC analyses yielded the AUC values of 0.9089 for miR-223, 0.7944 for miR-21 and 0.7432 for miR-218, and combined ROC analysis revealed the highest AUC value of 0.9531 in discriminating GC patients from healthy controls. Moreover, the plasma levels of miR-223 (P<0.001) and miR-21 (P = 0.003) were significantly higher in GC patients with stage I than in healthy controls. Furthermore, the plasma levels of miR-223 were significantly higher in GC patients with helicobacter pylori (Hp) infection than those without (P = 0.014), and significantly higher in healthy control subjects with Hp infection than those without (P = 0.016).
Plasma miR-223, miR-21 and miR-218 are novel potential biomarkers for GC detection.
Increasing evidence indicates that microRNAs (miRNAs) play a critical role in the pathogenesis of inflammatory diseases. The aim of the study was to investigate the expression pattern and function of miRNAs in CD4+ T cells from patients with rheumatoid arthritis (RA).
The expression profile of miRNAs in CD4+ T cells from synovial fluid (SF) and peripheral blood of 33 RA patients was determined by microarray assay and validated by qRT-PCR analysis. The correlation between altered expression of miRNAs and cytokine levels was determined by linear regression analysis. The role of miR-146a overexpression in regulating T cell apoptosis was evaluated by flow cytometry. A genome-wide gene expression analysis was further performed to identify miR-146a-regulated genes in T cells.
miRNA expression profile analysis revealed that miR-146a expression was significantly upregulated while miR-363 and miR-498 were downregulated in CD4+ T cells of RA patients. The level of miR-146a expression was positively correlated with levels of tumor necrosis factor-alpha (TNF-α), and in vitro studies showed TNF-α upregulated miR-146a expression in T cells. Moreover, miR-146a overexpression was found to suppress Jurkat T cell apoptosis. Finally, transcriptome analysis of miR-146a overexpression in T cells identified Fas associated factor 1 (FAF1) as a miR-146a-regulated gene, which was critically involved in modulating T cell apoptosis.
We have detected increased miR-146a in CD4+ T cells of RA patients and its close correlation with TNF-α levels. Our findings that miR-146a overexpression suppresses T cell apoptosis indicate a role of miR-146a in RA pathogenesis and provide potential novel therapeutic targets.
MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.
Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.
Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.
Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.
Several recent studies demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We hypothesised that plasma miRNAs concentrations contributed to potential biomarkers in patients with oesophageal squamous cell carcinoma (ESCC).
We selected three oncogenic miRNAs (miR-21, miR-184, miR-221) and one tumour suppressive miRNA (miR-375), which are frequently reported in squamous cell carcinoma, as candidate targets for this plasma miRNA assay. This study was divided into three steps: (1) Determination of appropriate plasma miRNAs in preliminary tests. (2) Evaluation of whether the plasma miRNA assays could monitor tumour dynamics. (3) Validation study on the clinical application of plasma miRNA assays in 50 ESCC patients and 20 healthy volunteers.
(1) In preliminary tests, the plasma level of miR-21 was significantly higher (P=0.0218) and that of miR-375 (P=0.0052) was significantly lower in ESCC patients than controls. (2) The high plasma miR-21 levels reflected tumour levels in all cases (100%). The plasma level of miR-21 was significantly reduced in postoperative samples (P=0.0058). (3) On validation analysis, the plasma level of miR-21 tended to be higher in ESCC patients (P=0.0649), while that of miR-375 was significantly lower (P<0.0001) and the miR-21/miR-375 ratio was significantly higher (P<0.0001) in ESCC patients than in controls. The value of the area under the receiver-operating characteristic curve (AUC) was 0.816 for the miR-21/miR-375 ratio assay. Patients with a high plasma level of miR-21 tended to have greater vascular invasion (P=0.1554) and to show a high correlation with recurrence (P=0.0164).
Detection of circulating miRNAs might provide new complementary tumour markers for ESCC.
oesophageal cancer; microRNA; plasma; biomarker
Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a poor prognosis. MicroRNAs (miRNAs) are a class of small non-coding RNAs, approximately 21–25 nucleotides in length. Recently, some researchers have demonstrated that plasma miRNAs are sensitive and specific biomarkers of various cancers. The primary aim of the study is to investigate whether miRNAs present in the plasma of GBM patients can be used as diagnostic biomarkers and are associated with glioma classification and clinical treatment.
Materials and Methods
Plasma samples were attained by venipuncture from 50 patients and 10 healthy donors. Plasma levels of miRNAs were determined by real-time quantitative polymerase chain reaction.
The plasma levels of miR-21, miR-128 and miR-342-3p were significantly altered in GBM patients compared to normal controls and could discriminate glioma from healthy controls with high specificity and sensitivity. However, these three miRNAs were not significantly changed in patients with other brain tumors such as meningioma or pituitary adenoma. Furthermore, the plasma levels of these three miRNAs in GBM patients treated by operation and chemo-radiation almost revived to normal levels. Finally, we also demonstrated that miR-128 and miR-342-3p were positively correlated with histopathological grades of glioma.
These findings suggest that plasma specific miRNAs have potential use as novel biomarkers of glioma and may be useful in clinical management for glioma patients.
Glioma; MicroRNA; Plasma; Biomarker
The purpose of this study was to identify and validate circulating microRNAs (miRNAs) in human plasma for use as breast cancer (BC) biomarkers and to analyze their relationship to clinicopathologic features and its preliminary biological function. Genome-wide expression profiling of miRNAs in BC was investigated by microarray analysis. miR-155 was up-regulated greater than two-fold in BC compared with Normal Adjacent Tissue (NAT), whereas let-7b, miR-381, miR-10b, miR-125a-5p, miR-335, miR-205 and miR-145 were down- regulated greater than two-fold. Our hypothesis was that circulating miRNAs are also present and differentially expressed in the serum of BC patients compared to controls. Using real-time PCR (RT-PCR), we analyzed miR-205 and miR-155 in archived serum from 30 participants, 20 with breast cancer and 10 healthy people. miR-205 was down-regulated in BC patient serum while miR-155 was up-regulated. Furthermore, we analyzed the relationship between the expression levels of these two miRNAs and the clinicopathologic parameters of BC patients. High expression of miR155 was associated with clinical stage, molecular type, Ki-67 and p53 in BC patients (P<0.05). By contrast, we found no significant correlation between miR-205 and BC patient clinicopathologic parameters. Functional analysis showed that ectopic expression of miR-205 significantly inhibits cell proliferation and promotes apoptosis. miR-205 was down-regulated and miR-155 was up-regulated in BC patient serum. miR-155 was positive correlated with clinical stage and ki-67 and negatively correlated with p53 status.
Breast cancer; microRNAs; miR-205; miR-155
Numerous studies have shown that aberrant microRNA (miRNA) expression is associated with the pathogenesis and progression of various human diseases. Hence, serum miRNAs are considered to be potential biomarkers for the diagnosis of human diseases. This study examined whether several miRNAs known to be commonly deregulated in liver diseases are deregulated in the serum of hosts with hepatic schistosomiasis, and thus whether they could serve as potential markers for detection of schistosome infection and evaluation of the effectiveness of chemotherapy.
We analyzed the serum levels of six selected candidate miRNA molecules (miR-146b, miR-122, miR-223, miR-199a-5p, miR-199a-3p, miR-34a) from mice, rabbits, buffalos and humans infected with Schistosoma japonicum using qPCR. We evaluated liver pathology by determining the hydroxyproline content in liver tissues. Primary resident liver cells were isolated to quantify the expression level of deregulated miRNAs. Bioinformatics analyses were also conducted to assess the potential function of miR-223.
Using a mouse model of Schistosoma japonicum infection, we found that the expression level of serum miR-223 was significantly elevated after infection, but returned to near normal levels after the treatment with praziquantel (PZQ). Importantly, the level of serum miR-223 reflected the extent of liver pathology post-infection. We validated the elevated level of the circulating miR-223 in serum samples of other host species including rabbits, buffalos and humans. In addition, our results showed that miR-223 was primarily located in the Kupffer cells, but its expression levels were significantly up-regulated in hepatocytes, hepatic stellate cells and Kupffer cells after infection. Bioinformatics analyses revealed a potential functional role of miR-223 in transcription regulator activity, transcription factor activity and DNA binding.
This study suggested that the circulating miR-223 could serve as a potential new biomarker for the detection of schistosome infection and the assessment of the response to chemotherapy.
Schistosomiasis; Serum miRNA; Biomarker; Schistosoma japonicum; Praziquantel
MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication.
IL-10−/− mice, an animal model of Th1-mediated inflammatory bowel disease, were screened for the expression of 600 microRNAs (miRNAs) using colonic tissues and peripheral blood leukocytes (PBLs) from animals having either mild inflammation or severe intestinal inflammation. The development of colonic inflammation in IL-10−/− mice was accompanied by upregulation in the expression of ten miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-223, miR-326, miR-142-3p, miR-142-5p, miR-146a, and miR-155). Notably, the expression of all of these miRNAs plus miR-375 was elevated in PBLs of IL-10−/− mice at a time when colonic inflammation was minimal, suggesting that changes in specific miRNAs in circulating leukocytes may be harbingers of ensuing colonic pathology. In vitro exposure of colonic intraepithelial lymphocytes to IL-10 resulted in down-regulation of miR-19a, miR-21, miR-31, miR-101, miR-223, and miR-155. Interestingly, unlike IL-10−/− mice, changes in miRNAs in PBL of dextran sulfate sodium-treated mice were minimal, but were selectively elevated in the colon after pathology was severe. We further show that miR-223 is a negative regulator of the Roquin ubiquitin ligase, that Roquin curtails IL-17A synthesis, and that the 3′ UTR of Roquin is a target for miR-223, thus defining a molecular pathway by which IL-10 modulates IL-17-mediated inflammation. To identify additional miRNAs that may be involved in the regulation of Roquin, transcriptome analysis was done using cDNAs from HeLa cells transfected with 90 miRNA mimics. Twenty-six miRNAs were identified as potential negative regulators of Roquin, thus demonstrating functional complexity in gene expression regulation by miRNAs.
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Diagnosis and management of PDAC are hampered by the absence of sensitive and specific disease biomarkers. MicroRNAs are non-coding regulatory RNAs involved in initiation and progression of human cancers. In this study we sought to determine whether miR-10b could serve as a biomarker for PDAC.
miRNA expression was characterized by fluorescence-based in situ hybridization (ISH) using Locked Nucleic Acid (LNA)-modified DNA probes against miR-10b, miR-21, miR-155, miR-196a, and miR-210, followed by co-detection of proteins by immunohistochemistry on the same tissue sections. miRNA expression in surgically resected PDAC tissues and in endoscopic ultrasonography (EUS)-guided fine needle aspirate (EUS-FNA) samples was analyzed in cytokeratin 19 (CK19)-positive epithelial cells using optical intensity analysis.
In 10 resected PDAC samples miR-10b was the most frequently and consistently overexpressed miRNA among characterized miRNAs, exhibiting a 4-fold increase in the cancer cells (p=0.012). Given this preferential overexpression of miR-10b, we sought to determine whether miR-10b expression was clinically relevant. Accordingly, miR-10b expression was examined in 106 EUS-FNA samples obtained from pancreatic lesions. miR-10b expression was increased in cancer cells compared to CK19-positive epithelial cells in benign lesions (p=0.0001). In patients with PDAC, lower levels of miR-10b were associated with improved response to multimodality neoadjuvant therapy, likelihood of surgical resection, delayed time to metastasis, and increased survival.
miR-10b is a novel diagnostic biomarker for PDAC when assessing pancreatic lesions. Expression of miR-10b is predictive of response to neoadjuvant therapy and outcome in this disease.
Pancreatic cancer; microRNA-10b; FNA; metastasis; neoadjuvant chemotherapy
Sepsis is a subtype of systemic inflammatory response syndrome (SIRS), which is characterized by infection. Circulating microRNAs (miRNAs), including miR-150, miR-146a and miR-223, are potential biomarkers of sepsis. In this study, we demonstrated that measuring the relative expression of miR-146a/U6 in plasma, using the 2−ΔΔCt method, provides a method for differentiating between sepsis and non-sepsis-SIRS. We observed a significant increase in miR-146a expression in the initial cohort of 6 non-sepsis-SIRS patients compared to the 4 sepsis patients (P=0.01) and in the second cohort of 8 non-sepsis-SIRS patients compared to the 10 sepsis patients (P=0.027). Additionally, we identified that sodium citrate and ethylenediaminetetraacetic acid (EDTA) K2 may be used as anticoagulant reagents. Generation of a standard curve is not necessary in these diagnostic tests, unless the standard of normalization is carefully selected. Thus we provide more detailed guidance for the clinical use of circulating miRNA biomarkers.
miR-146a; sepsis; diagnosis
Although the immune response is predominantly controlled at the transcriptional level, microRNA-mediated RNA interference is emerging as an important regulatory mechanism that operates at the translation level. Specifically, recent studies indicate that those miRNAs that are selectively and/or highly expressed in immune cells including the miR-17–92 cluster, miR-150, miR-155, miR-181 and miR-223 have a ‘permissive’ function in the maturation, proliferation and differentiation of myeloid and lymphoid cells. Importantly, these actions of miRNAs often involve interactions with transcription factors. In contrast, the rapid and transient induction of miR-9, miR-146a and miR-155 has been speculated to negatively regulate the acute responses following activation of innate immune through down-regulation of proteins involved in the receptor-induced signalling pathways.
Evidence underlines the importance of microRNAs (miRNAs) in the pathogenesis of multiple sclerosis (MS). Based on the fact that miRNAs are present in human biological fluids, we previously showed that miR-223, miR-23a and miR-15b levels were downregulated in the sera of MS patients versus controls. Here, the expression levels of these candidate miRNAs were determined in peripheral blood mononuclear cells (PBMCs) and the serum of MS patients, in addition to three genotyped single nucleotide polymorphisms (SNPs). Mapping in the genomic regions of miR-223, miR-23a and miR-15b genes, 399 cases and 420 controls were tested. Expression levels of miR-223 and miR-23a were altered in PBMCs from MS patients versus controls. Conversely, there were no differences in the expression levels of miR-15b. A significantly decreased genotypic frequency of miR-223 rs1044165 T/T genotype was observed in MS patients. Moreover, the allelic frequency of miR-23a rs3745453 C allele was significantly increased in patients versus controls. In contrast, there were no differences in the distribution of miR-15b SNP. In conclusion, our results suggest that miR-223 and miR-23a could play a role in the pathogenesis of MS. Moreover, miR-223 rs1044165 polymorphism likely acts as a protective factor, while miR-23a rs3745453 variant seems to act as a risk factor for MS.
multiple sclerosis; microRNA; gene expression; association analysis
The present study aimed to identify microRNA (miRNA) expression profiles associated with brain metastases of colorectal cancers. We conducted miRNA expression profiling of 3 primary colorectal cancers and 3 brain-metastatic carcinomas using Agilent Human miRNA Microarrays. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to validate the results obtained from the miRNA microarray analysis. Overexpression of miR-145, miR-1, miR-146a, miR-576-5p, miR-126*, HS287, miR-28-5p, miR-143, miR-199b-5p, miR-199a-5p, miR-10b, miR-22, miR-133b, miR-145*, miR-199a, miR-133a, miR-125b and downregulation of miR-31 and HS170 were observed in brain-metastatic carcinomas. Quantitative RT-PCR experiments with miR-125b confirmed the expression patterns we found in our microarray experiments. miRNAs are differentially expressed between colorectal cancers and matched brain-metastatic carcinomas. The miRNA variation trend is quite different in the process of metastasis compared to that of carcinogenesis. These miRNAs may therefore serve as potential diagnostic markers and therapeutic targets for colorectal cancers with brain metastases.
brain metastasis; colorectal cancer; microarray; microRNA
MicroRNAs (miRNAs) are endogenous noncoding RNA molecules that are involved in post-transcriptional gene silencing. Using global miRNA expression profiling, we found miR-21, -155, and 18a to be highly upregulated in rat kidneys following tubular injury induced by ischemia/reperfusion (I/R) or gentamicin administration. Mir-21 and -155 also showed decreased expression patterns in blood and urinary supernatants in both models of kidney injury. Furthermore, urinary levels of miR-21 increased 1.2-fold in patients with clinical diagnosis of acute kidney injury (AKI) (n = 22) as compared with healthy volunteers (n = 25) (p < 0.05), and miR-155 decreased 1.5-fold in patients with AKI (p < 0.01). We identified 29 messenger RNA core targets of these 3 miRNAs using the context likelihood of relatedness algorithm and found these predicted gene targets to be highly enriched for genes associated with apoptosis or cell proliferation. Taken together, these results suggest that miRNA-21 and -155 could potentially serve as translational biomarkers for detection of AKI and may play a critical role in the pathogenesis of kidney injury and tissue repair process.
MicroRNAs; kidney; biomarker; ischemia/reperfusion injury; nephrotoxicity.
Circulating microRNAs (miRNAs) may represent a potential noninvasive molecular biomarker for various pathological conditions. Moreover, the detection of circulating miRNAs can provide important novel disease-related information. In particular, inflammation-associated miR-155 and endothelial-enriched miR-126 are reported to be associated with vascular homeostasis. Vascular damage is a common event described in end-stage renal disease (ESRD). We hypothesized that miR-155 and miR-126 may be detectable in the circulation and serve as potential biomarkers for risk stratification. In this study, we assessed miR-155 and miR-126 in the plasma of 30 ESRD patients and 20 healthy controls using real-time quantification RT-PCR. The circulating levels of miR-155 and miR-126 were significantly reduced in patients with ESRD compared to healthy controls. However, there was no significant difference of circulating miR-155 and miR-126 levels between prehemodialysis and posthemodialysis patients. Furthermore, both circulating miR-126 and miR-155 correlated positively with estimated glomerular filtration rate (miR-126: r = 0.383, P = 0.037; miR-155: r = 0.494, P = 0.006) and hemoglobin (miR-126: r = 0.515, P = 0.004; miR-155: r = 0.598, P < 0.001) and correlated inversely with phosphate level (miR-126: r = -0.675, P < 0.001; miR-155: r = -0.399, P = 0.029). Pearson's correlation was used to compare circulating levels of miRNAs with clinical parameters. These results suggested that circulating miR-155 and miR-126 might be involved in the development of ESRD. Further studies are needed to demonstrate the role of circulating miR-155 and miR-126 as candidate biomarkers for risk estimation.
MicroRNAs; End-stage renal disease; Hemodialysis; MiR-126; MiR-155
Biomarkers for the diagnostic classification of pancreatic cysts are urgently needed. Deregulated microRNA (miRNAs) expression is widespread in pancreatic cancer. We assessed whether aberrant miRNAs in pancreatic cyst fluid could be used as potential biomarkers for cystic precursor lesions of pancreatic cancer.
Cyst fluid specimens were prospectively collected from 40 surgically resected pancreatic cysts, and small RNAs were extracted. The ‘mucinous’ cohort included 14 intraductal papillary mucinous neoplasms (including 3 with an associated adenocarcinoma) and 10 mucinous cystic neoplasms; the ‘nonmucinous’ cohort included 11 serous cystadenomas and 5 other benign cysts. Quantitative reverse transcription PCR was performed for five miRNAs (miR-21, miR-155, miR-221, miR-17-3p, miR-191), which were previously reported as overexpressed in pancreatic adenocarcinomas.
Significantly higher expression of miR-21, miR-221, and miR-17-3p was observed in the mucinous versus nonmucinous cysts (p < 0.01), with the mean relative fold differences being 7.0-, 7.9-, and 5.4-fold, respectively. Receiver operating characteristic curves demonstrated the highest median area under the curve for miR-21, with a median specificity of 76%, at a sensitivity of 80%.
This pilot study demonstrates that profiling miRNAs in pancreatic cyst fluid samples is feasible and can yield potential biomarkers for the classification of cystic lesions of the pancreas.
Pancreatic cyst; Intraductal papillary mucinous neoplasm; Mucinous cystic neoplasm; microRNA; Biomarker
Plasma microRNA (miRNA) has become a promising biomarker for detecting cancer; however, it remains uncertain whether miRNA expression levels in plasma reflect those in tumor cells. Our aim was to determine the biological relevance of miR-92a, which has been implicated as an oncomiR in both plasma and leukemia cells in patients with acute leukemia and to evaluate whether it could be a novel biomarker for monitoring these patients.
We quantified the expression level of miR-92a in both cells and plasma by reverse transcription polymerase chain reaction in 91 patients with acute leukemia. We also determined miR-92a expression levels in peripheral blood mononuclear cells (PBMNC) from normal controls. We compared miR-92a expression in plasma with its expression in leukemia cells. Synthetic anti-miR-92a inhibitor was transfected into Raji and OM9;22 cells, and apoptosis was assessed. For in vivo assessment, 6-week-old female nude mice were injected with U937 cells, and miR-92a expression in plasma and tumors was measured. The level of miR-92a expression in fresh leukemia cells was highly variable compared with PBMNC, but significantly lower compared with CD34-positive cells obtained from healthy volunteers. We also noticed that miR-92a was preferentially expressed in acute lymphoblastic leukemia (ALL) cells in comparison with acute myeloid leukemia (AML) cells. More specifically, cellular miR-92a expression was significantly increased in a subset of ALL cells, and ALL patients with overexpressed miR-92a had poor prognoses. The anti-miR-92a inhibitor-treated Raji and OM9;22 cells revealed an increase of apoptotic cells. Notably, the cell to plasma ratio of miR-92a expression was significantly higher in both AML and ALL cells compared with PBMNC from healthy volunteers. In tumor-bearing mice, the plasma miR-92a level was significantly decreased in accordance with tumor growth, while tumor tissue was strongly positive for miR-92a.
The miR-92a expression in leukemia cells could be a prognostic factor in ALL patients. The inverse correlation of miR-92a expression between cells and plasma and the cell to plasma ratio may be important to understanding the clinical and biological relevance of miR-92a in acute leukemia.
It has been found that aberrant expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. In the present study, we investigated the expression of miR-155 and miR-146a in diffuse large B-cell lymphoma (DLBCL) patients (n=90). The expression levels of miR-155 and miR-146a were significantly higher in de novo DLBCL patients. miR-146a expression levels were associated with miR-155, lactate dehydrogenase, β 2 microglobulin, c-myc, International Prognostic Index status and Eastern Cooperative Oncology Group performance status. We found that patients with low miR-155 and miR-146a expression levels achieved a higher complete remission rate, higher overall response rate and longer progression-free survival time. Moreover, a high expression level of miR-155, but not miR-146a, was an independent indicator for chemotherapy protocol selection in our study. Patients with high expression of miR-155 received more survival benefits from rituximab treatment. These data suggest that miR-155 and miR-146a have potential as diagnostic and prognostic markers in DLBCL.
diffuse large B-cell lymphoma; miR-155; miR-146a; diagnostic and prognostic marker
Gestational diabetes mellitus (GDM) is one type of diabetes that presents during pregnancy and significantly increases the risk of a number of adverse consequences for the fetus and mother. The microRNAs (miRNA) have recently been demonstrated to abundantly and stably exist in serum and to be potentially disease-specific. However, no reported study investigates the associations between serum miRNA and GDM.
We systematically used the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays to screen miRNAs in serum collected at 16–19 gestational weeks. The expression levels of three miRNAs (miR-132, miR-29a and miR-222) were significantly decreased in GDM women with respect to the controls in similar gestational weeks in our discovery evaluation and internal validation, and two miRNAs (miR-29a and miR-222) were also consistently validated in two-centric external validation sample sets. In addition, the knockdown of miR-29a could increase Insulin-induced gene 1 (Insig1) expression level and subsequently the level of Phosphoenolpyruvate Carboxy Kinase2 (PCK2) in HepG2 cell lines.
Serum miRNAs are differentially expressed between GDM women and controls and could be candidate biomarkers for predicting GDM. The utility of miR-29a, miR-222 and miR-132 as serum-based non-invasive biomarkers warrants further evaluation and optimization.
MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that post-transcriptionally inhibit gene expression. Many miRNAs have been implicated in several human cancers, including breast cancer. Here we describe the association between altered miRNA signatures and breast cancer tumorigenesis and metastasis. The loss of several tumor suppressor miRNAs (miR-206, miR-17-5p, miR-125a, miR-125b, miR-200, let-7, miR-34 and miR-31) and the overexpression of certain oncogenic miRNAs (miR-21, miR-155, miR-10b, miR-373 and miR-520c) have been observed in many breast cancers. The gene networks orchestrated by these miRNAs are still largely unknown, although key targets have been identified that may contribute to the disease phenotype. Here we report how the observed perturbations in miRNA expression profiles may lead to disruption of key pathways involved in breast cancer.
Analysis of molecular genetic markers in biological fluids has been proposed as a useful tool for cancer diagnosis. MicroRNAs (miRNAs) are small regulatory RNAs that are frequently dysregulated in lung cancer and have shown promise as tissue-based markers for its prognostication. The aim of this study was to determine whether aberrant miRNA expression can be used as a marker in sputum specimen for the diagnosis of non-small cell lung cancer (NSCLC). Experimental Design: Expressions of mature miRNAs, mir-21 and mir-155, were examined by real-time reverse transcription polymerase chain reaction (RT-PCR) and normalized to that of control miRNA, U6B, in sputum of 23 patients with NSCLC and 17 cancer-free subjects. The data was compared with conventional sputum cytology for the diagnosis of lung cancer. All endogenous miRNAs were present in sputum in a remarkably stable form and sensitively and specifically detected by real-time RT-PCR. Mir-21 expression in the sputum specimens was significantly higher in cancer patients (76.32 ± 9.79) than cancer-free individuals (62.24±3.82) (p<0.0001). Furthermore, overexpression of mir-21 showed highly discriminative receiver-operator characteristic (ROC) curve profile, clearly distinguishing cancer patients from cancer-free subjects with areas under the ROC curve at 0.902 ± 0.054. Detection of mir-21 expression produced 69.66% sensitivity and 100.00% specificity in diagnosis of lung cancer, as compared with 47.82% sensitivity and 100.00% specificity by sputum cytology. The measurement of altered miRNA expression in sputum could be a useful noninvasive approach for the diagnosis of lung cancer.
MicroRNA; sputum; lung cancer; real-time RT-PCR; diagnosis
MicroRNAs are fine tuners of diverse biological responses and are expressed in various cell types of the liver. Here we hypothesized that circulating microRNAs (miRNAs) may serve as biomarkers of liver damage and inflammation. We studied miRNA-122 that is abundant in hepatocytes and miR-155, -146a and -125b that regulate inflammation in immune cells in mouse models of alcoholic liver disease (ALD), drug (acetaminophen; APAP)-induced liver injury (DILI), and Toll-like receptor (TLR) 9+4 ligands-induced inflammatory cell-mediated liver damage. We found that serum/plasma miR-122 correlated with ALT increases in the liver damage caused by alcohol, APAP and TLR9 (CpG)+4 (LPS) ligands. MiR-155, a regulator of inflammation, was increased in serum/plasma in alcoholic and inflammatory liver injury. Alcohol failed to increase serum miR-122 in TLR4-deficient and p47phox-deficient mice that were protected from ALD. We found the most robust increase in plasma miR-122 in DILI and it correlated with the highest ALT levels. Consistent with the massive inflammatory cell infiltration in the liver, plasma miR-155 and miR-146a were significantly elevated after CpG+LPS administration. We show for the first time that, depending on the type of liver injury, circulating miRNAs show association either with the exosome-rich or protein-rich compartments. In ALD and in inflammatory liver injury, serum/plasma miR-122 and miR-155 were predominantly associated with exosome-rich fraction while in DILI/APAP injury these miRNAs were present in the protein-rich fraction. In conclusion, our results suggest that circulating miRNAs may serve as biomarkers to differentiate between hepatocyte injury and inflammation and the exosome versus protein association of miRNAs may provide further specificity to mechanisms of liver pathology.
Biomarker; miR-122; miR-155; TLR9; LPS; acetaminophen; ALT; TNF