mRNA expression profiles show differential regulatory effects of microRNAs between estrogen receptor-positive and estrogen receptor-negative breast cancer

Background

Recent studies have shown that the regulatory effect of microRNAs can be investigated by examining expression changes of their target genes. Given this, it is useful to define an overall metric of regulatory effect for a specific microRNA and see how this changes across different conditions.

Results

Here, we define a regulatory effect score (RE-score) to measure the inhibitory effect of a microRNA in a sample, essentially the average difference in expression of its targets versus non-targets. Then we compare the RE-scores of various microRNAs between two breast cancer subtypes: estrogen receptor positive (ER⁺) and negative (ER^-). We applied this approach to five microarray breast cancer datasets and found that the expression of target genes of most microRNAs was more repressed in ER^- than ER⁺; that is, microRNAs appear to have higher RE-scores in ER^- breast cancer. These results are robust to the microRNA target prediction method. To interpret these findings, we analyzed the level of microRNA expression in previous studies and found that higher microRNA expression was not always accompanied by higher inhibitory effects. However, several key microRNA processing genes, especially Ago2 and Dicer, were differentially expressed between ER^- and ER⁺ breast cancer, which may explain the different regulatory effects of microRNAs in these two breast cancer subtypes.

Conclusions

The RE-score is a promising indicator to measure microRNAs'' inhibitory effects. Most microRNAs exhibit higher RE-scores in ER^- than in ER⁺ samples, suggesting that they have stronger inhibitory effects in ER^- breast cancers.     

Serum microRNAs are promising novel biomarkers

Background

Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers.

Methods and Results

After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage.

Conclusions and Significance

Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.     

MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in type 2 diabetes mellitus

Background

Dysregulation of microRNA (miRNA) expression in various tissues and body fluids has been demonstrated to be associated with several diseases, including Type 2 Diabetes mellitus (T2D). Here, we compare miRNA expression profiles in different tissues (pancreas, liver, adipose and skeletal muscle) as well as in blood samples from T2D rat model and highlight the potential of circulating miRNAs as biomarkers of T2D. In parallel, we have examined the expression profiles of miRNAs in blood samples from Impaired Fasting Glucose (IFG) and T2D male patients.

Methodology/Principal Findings

Employing miRNA microarray and stem-loop real-time RT-PCR, we identify four novel miRNAs, miR-144, miR-146a, miR-150 and miR-182 in addition to four previously reported diabetes-related miRNAs, miR-192, miR-29a, miR-30d and miR-320a, as potential signature miRNAs that distinguished IFG and T2D. Of these microRNAs, miR-144 that promotes erythropoiesis has been found to be highly up-regulated. Increased circulating level of miR-144 has been found to correlate with down-regulation of its predicted target, insulin receptor substrate 1 (IRS1) at both mRNA and protein levels. We could also experimentally demonstrate that IRS1 is indeed the target of miR-144.

Conclusion

We demonstrate that peripheral blood microRNAs can be developed as unique biomarkers that are reflective and predictive of metabolic health and disorder. We have also identified signature miRNAs which could possibly explain the pathogenesis of T2D and the significance of miR-144 in insulin signaling.     

Curated MicroRNAs in Urine and Blood Fail to Validate as Predictive Biomarkers for High-Risk Prostate Cancer

Purpose

The purpose of this study was to determine if microRNA profiling of urine and plasma at radical prostatectomy can distinguish potentially lethal from indolent prostate cancer.

Materials and Methods

A panel of microRNAs was profiled in the plasma of 70 patients and the urine of 33 patients collected prior to radical prostatectomy. Expression of microRNAs was correlated to the clinical endpoints at a follow-up time of 3.9 years to identify microRNAs that may predict clinical response after radical prostatectomy. A machine learning approach was applied to test the predictive ability of all microRNAs profiled in urine, plasma, and a combination of both, and global performance assessed using the area under the receiver operator characteristic curve (AUC). Validation of urinary expression of miRNAs was performed on a further independent cohort of 36 patients.

Results

The best predictor in plasma using eight miRs yielded only moderate predictive performance (AUC = 0.62). The best predictor of high-risk disease was achieved using miR-16, miR-21 and miR-222 measured in urine (AUC = 0.75). This combination of three microRNAs in urine was a better predictor of high-risk disease than any individual microRNA. Using a different methodology we found that this set of miRNAs was unable to predict high-volume, high-grade disease.

Conclusions

Our initial findings suggested that plasma and urinary profiling of microRNAs at radical prostatectomy may allow prognostication of prostate cancer behaviour. However we found that the microRNA expression signature failed to validate in an independent cohort of patients using a different platform for PCR. This highlights the need for independent validation patient cohorts and suggests that urinary microRNA signatures at radical prostatectomy may not be a robust way to predict the course of clinical disease after definitive treatment for prostate cancer.     

MicroRNA profile predicts recurrence after resection in patients with hepatocellular carcinoma within the Milan Criteria

Objective

Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection.

Methods

We examined microRNA expression profiling in paired tumor and non-tumor liver tissues from 73 HCC patients who satisfied the Milan Criteria. We constructed prediction models of recurrence-free survival using the Cox proportional hazard model and principal component analysis. The prediction efficiency was assessed by the leave-one-out cross-validation method, and the time-averaged area under the ROC curve (ta-AUROC).

Results

The univariate Cox analysis identified 13 and 56 recurrence-related microRNAs in the tumor and non-tumor tissues, such as miR-96. The number of recurrence-related microRNAs was significantly larger in the non-tumor-derived microRNAs (N-miRs) than in the tumor-derived microRNAs (T-miRs, P<0.0001). The best ta-AUROC using the whole dataset, T-miRs, N-miRs, and clinicopathological dataset were 0.8281, 0.7530, 0.7152, and 0.6835, respectively. The recurrence-free survival curve of the low-risk group stratified by the best model was significantly better than that of the high-risk group (Log-rank: P = 0.00029). The T-miRs tend to predict early recurrence better than late recurrence, whereas N-miRs tend to predict late recurrence better (P<0.0001). This finding supports the concept of early recurrence by the dissemination of primary tumor cells and multicentric late recurrence by the ‘field effect’.

Conclusion

microRNA profiling can predict HCC recurrence in Milan criteria cases.     

Oligo- and Polymetastatic Progression in Lung Metastasis(es) Patients Is Associated with Specific MicroRNAs

Rationale

Strategies to stage and treat cancer rely on a presumption of either localized or widespread metastatic disease. An intermediate state of metastasis termed oligometastasis(es) characterized by limited progression has been proposed. Oligometastases are amenable to treatment by surgical resection or radiotherapy.

Methods

We analyzed microRNA expression patterns from lung metastasis samples of patients with ≤5 initial metastases resected with curative intent.

Results

Patients were stratified into subgroups based on their rate of metastatic progression. We prioritized microRNAs between patients with the highest and lowest rates of recurrence. We designated these as high rate of progression (HRP) and low rate of progression (LRP); the latter group included patients with no recurrences. The prioritized microRNAs distinguished HRP from LRP and were associated with rate of metastatic progression and survival in an independent validation dataset.

Conclusion

Oligo- and poly- metastasis are distinct entities at the clinical and molecular level.     

Genome-Wide Sequencing of Cellular microRNAs Identifies a Combinatorial Expression Signature Diagnostic of Sepsis

Rationale

Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity.

Objectives

We hypothesized changes in expression of microRNAs during sepsis may be of diagnostic value in the intensive care unit (ICU).

Methods

Massively parallel sequencing of microRNAs was utilised for screening microRNA candidates. Putative microRNAs were validated using quantitative real-time PCR (qRT-PCR). This study includes data from both a training cohort (UK) and an independent validation cohort (Sweden). A linear discriminant statistical model was employed to construct a diagnostic microRNA signature.

Results

A panel of known and novel microRNAs were detectable in the blood of patients with sepsis. After qRT-PCR validation, microRNA miR-150 and miR-4772-5p-iso were able to discriminate between patients who have systemic inflammatory response syndrome and patients with sepsis. This finding was also validated in independent cohort with an average diagnostic accuracy of 86%. Fractionating the cellular components of blood reveals miR-4772-5p-iso is expressed differentially in monocytes. Functional experiments using primary human monocytes demonstrate that it expressed in response to TLR ligation.

Conclusions

Taken together, these data provide a novel microRNA signature of sepsis that should allow rapid point-of-care diagnostic assessment of patients on ICU and also provide greater insight into the pathobiology of this severe disease.     

Identification of four functionally important microRNA families with contrasting differential expression profiles between drought-tolerant and susceptible rice leaf at vegetative stage

Background

Developing drought-tolerant rice varieties with higher yield under water stressed conditions provides a viable solution to serious yield-reduction impact of drought. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success of rice molecular breeding programmes. microRNAs have received tremendous attention recently due to its importance in negative regulation. In plants, apart from regulating developmental and physiological processes, microRNAs have also been associated with different biotic and abiotic stresses. Hence here we chose to analyze the differential expression profiles of microRNAs in three drought treated rice varieties: Vandana (drought-tolerant), Aday Sel (drought-tolerant) and IR64 (drought-susceptible) in greenhouse conditions via high-throughput sequencing.

Results

Twenty-six novel microRNA candidates involved in the regulation of diverse biological processes were identified based on the detection of miRNA*. Out of their 110 predicted targets, we confirmed 16 targets from 5 novel microRNA candidates. In the differential expression analysis, mature microRNA members from 49 families of known Oryza sativa microRNA were differentially expressed in leaf and stem respectively with over 28 families having at least a similar mature microRNA member commonly found to be differentially expressed between both tissues. Via the sequence profiling data of leaf samples, we identified osa-miR397a/b, osa-miR398b, osa-miR408-5p and osa-miR528-5p as being down-regulated in two drought-tolerant rice varieties and up-regulated in the drought-susceptible variety. These microRNAs are known to be involved in regulating starch metabolism, antioxidant defence, respiration and photosynthesis. A wide range of biological processes were found to be regulated by the target genes of all the identified differentially expressed microRNAs between both tissues, namely root development (5.3–5.7 %), cell transport (13.2–18.4 %), response to stress (10.5–11.3 %), lignin catabolic process (3.8–5.3 %), metabolic processes (32.1–39.5 %), oxidation-reduction process (9.4–13.2 %) and DNA replication (5.7–7.9 %). The predicted target genes of osa-miR166e-3p, osa-miR166h-5p*, osa-miR169r-3p* and osa-miR397a/b were found to be annotated to several of the aforementioned biological processes.

Conclusions

The experimental design of this study, which features rice varieties with different drought tolerance and tissue specificity (leaf and stem), has provided new microRNA profiling information. The potentially regulatory importance of the microRNA genes mentioned above and their target genes would require further functional analyses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1851-3) contains supplementary material, which is available to authorized users.     

Down-Regulation of miR-92 in Human Plasma Is a Novel Marker for Acute Leukemia Patients

Background

MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs that primarily function as gene regulators. Aberrant microRNA expression has been described for several human malignancies, and this new class of small regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this knowledge, there is little information regarding microRNAs in plasma especially because microRNAs in plasma, if exist, were thought to be digested by RNase. Recent studies, however, have revealed that microRNAs exist and escape digestion in plasma.

Methodology/Principal Findings

We performed microRNA microaray to obtain insight into microRNA deregulation in the plasma of a leukemia patient. We have revealed that microRNA-638 (miR-638) is stably present in human plasmas, and microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for distinguishing leukemia patients from healthy body.

Conclusions/Significance

The ratio of miR-92a/miR-638 in plasma has strong potential for clinical application as a novel biomarker for detection of leukemia.     

Cell-Free Urinary MicroRNA-99a and MicroRNA-125b Are Diagnostic Markers for the Non-Invasive Screening of Bladder Cancer

Background

Evidence implicated the diagnostic significance of microRNAs in whole urine/urine sediments in urothelial carcinoma of the bladder (UCB). However, the contaminated blood cells in patients with haematouria significantly altered the expression profiles of urinary microRNA, influencing the test accuracy.

Methods

MicroRNA profiles of the urine supernatants of UCB patients and controls without any malignancy and profiles of malignant and corresponding normal mucosa tissues from the patients were determined by microRNA microarray and compared to identify differentially expressed microRNAs. The differential expression was verified in the tissues of an independent patient cohort by RT-qPCR. The diagnostic significance of selected microRNAs as biomarkers in the urine supernatant was investigated in the expanded cohorts.

Results

MicroRNA-99a and microRNA-125b were down-regulated in the urine supernatants of UCB patients. The degree of down-regulation was associated with the tumor grade. A diagnostic model was developed using a combined index of the levels of microRNA-99a and microRNA-125b in the urine supernatant with a sensitivity of 86.7%, a specificity of 81.1% and a positive predicted value (PPV) of 91.8%. Discriminating between high- and low-grade UCB, the model using the level of microRNA-125b alone exhibited a sensitivity of 81.4%, a specificity of 87.0% and a PPV of 93.4%.

Conclusions

The results revealed a unique microRNA expression signature in the urine supernatants of UCB patients for the development of molecular diagnostic tests. An effective cell-free urinary microRNA-based model was developed using a combined index of the levels of microRNA-99a and microRNA-125b to detect UCB with good discriminating power, high sensitivity and high specificity.     

Prognostic Role of Common MicroRNA Polymorphisms in Cancers: Evidence from a Meta-Analysis

Background

The morbidity and mortality of cancer increase remarkably every year. It''s a heavy burden for family and society. The detection of prognostic biomarkers can help to improve the theraputic effect and prolong the lifetime of patients. microRNAs have an influential role in cancer prognosis. The results of articles discussing the relationship between microRNA polymorphisms and cancer prognosis are inconsistent.

Methods

We conduct a meta-analysis of 19 publications concerning the association of four common polymorphisms, mir-146a rs2910164, mir-149 rs2292832, mir-196a2 rs11614913 and mir-499 rs3746444, with cancer prognosis. Pooled Hazard Ratios with 95% Confidence Intervals for the relationship between four genetic polymorphisms and Overall Survival, Recurrence-free Survival, Disease-free survival, recurrence are calculated. Subgroup analysis by population and type of tumor are conducted.

Results

GG genotype of mir-146a may be the protective factor for overall survival, especially in Caucasian population. C-containing genotypes of mir-196a2 act as a risk role for overall survival. The same result exists in Asian population, in Non-Small Cell Lung Cancer and digestive cancer. The patients with C allele of mir-149 have a better overall survival, especially in Non-Small Cell Lung Cancer. No significant results are obtained for mir-499 polymorphisms.

Conclusions

Genetic polymorphisms in mir-146a, mir-196a2 and mir-149 may be associated with overall survival. This effect varies with different types of cancer. Genetic polymorphism in mir-499 may have nothing to do with cancer prognosis.     

Equivalence of conventionally-derived and parthenote-derived human embryonic stem cells

Background

As human embryonic stem cell (hESC) lines can be derived via multiple means, it is important to determine particular characteristics of individual lines that may dictate the applications to which they are best suited. The objective of this work was to determine points of equivalence and differences between conventionally-derived hESC and parthenote-derived hESC lines (phESC) in the undifferentiated state and during neural differentiation.

Methodology/Principal Findings

hESC and phESC were exposed to the same expansion conditions and subsequent neural and retinal pigmented epithelium (RPE) differentiation protocols. Growth rates and gross morphology were recorded during expansion. RTPCR for developmentally relevant genes and global DNA methylation profiling were used to compare gene expression and epigenetic characteristics. Parthenote lines proliferated more slowly than conventional hESC lines and yielded lower quantities of less mature differentiated cells in a neural progenitor cell (NPC) differentiation protocol. However, the cell lines performed similarly in a RPE differentiation protocol. The DNA methylation analysis showed similar general profiles, but the two cell types differed in methylation of imprinted genes. There were no major differences in gene expression between the lines before differentiation, but when differentiated into NPCs, the two cell types differed in expression of extracellular matrix (ECM) genes.

Conclusions/Significance

These data show that hESC and phESC are similar in the undifferentiated state, and both cell types are capable of differentiation along neural lineages. The differences between the cell types, in proliferation and extent of differentiation, may be linked, in part, to the observed differences in ECM synthesis and methylation of imprinted genes.     

MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R

Background

Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder.

Methodology/Principal Findings

Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca⁺⁺, miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a.

Conclusions/Significance

We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.     

Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates

Background

Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered.

Methodology/Principal Findings

Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm².

Conclusions/Significance

The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.