Analysis Of microRNA Expression Research Articles

Selection of reference genes for expression analysis of plant-derived microRNAs in Plutella xylostella using qRT-PCR and ddPCR.

The establishment of an expression quantification system that can be easily applied for the comparison of microRNAs (miRNAs) from biological samples is an important step toward understanding functional mechanisms in organisms. However, there is lack of attention on the selection of reference genes for miRNA expression profiling in insect herbivores. Here, we explored the candidate reference genes in a notorious pest of cruciferous crops, Plutella xylostella, for normalization of miRNA expression in developmental stages and tissues and in response to a change of food source from artificial diet to host plant Arabidopsis thaliana. We first compared the expression levels and stability of eight small RNAs using qRT-PCR, and found that miR11 was the most suitable reference gene for expression quantification of the miRNAs. We then confirmed this finding using digital droplet PCR and further validated with a well-studied cross-kingdom miRNA derived from A. thaliana (ath-miR159a). However, none of the reference genes was applicable for all experimental conditions, and multiple reference genes were sometimes required within the same experiment. Our work provides a method for the selection of reference genes for quantification of plant-derived miRNAs, which paves the way for unveiling their roles in the insect-plant coevolution.

MicroRNA Sequencing Reveals the Effect of Different Levels of Non-Fibrous Carbohydrate/Neutral Detergent Fiber on Rumen Development in Calves.

Simple SummaryBy histological sectioning and staining of rumen tissues from calves fed with a high or low ratio of non-fibrous carbohydrate/neutral detergent fiber diet, we found that the length and width of papillae were significantly affected by the ratio. From microRNA expression analysis we found cell proliferation, differentiation, physical and nutrient stimuli processes participate in the development of the rumen. In addition, bta-miR-128 was found to affect rumen development by negatively regulating PPARG and SLC16A1. Our findings provided an important resource for the continuing study of rumen development and absorption.Rumen development in calves is affected by many factors, including dietary composition. MicroRNAs (miRNAs) are known to function in the development of the rumen in cattle, what is not known is how these miRNAs function in rumen development of calves fed with high and low ratios of non-fibrous carbohydrate (NFC)/neutral detergent fiber (NDF). A total of six healthy Charolais hybrids bull calves of similar weight were divided into two groups; three calves were fed a mixed diet with NFC/NDF = 1.35 (H group), and three were fed a mixed diet with NFC/NDF = 0.80 (L group). After 105 days on the diet, calves were sacrificed and rumen tissues were collected. Tissues were subjected to histological observation and miRNA expression analysis. Functional enrichment analysis was conducted on the target genes of the miRNAs. Targeting and regulatory relationships were verified by luciferase reporter assay and quantitative PCR (qPCR). We found that the length of rumen papilla in the L group was significantly greater than that in the H group, while the width of rumen papilla in H group was significantly greater than that that in L group. We identified 896 miRNAs; 540 known miRNAs, and 356 novel predicted miRNAs. After statistical testing, we identified 24 differentially expressed miRNAs (DEmiRNAs). miRNA-mRNA-cluster network analysis and literature reviews revealed that cell proliferation, differentiation, physical and nutrient stimuli processes participate in rumen development under different NFC/NDF levels. The regulatory relationships between three DEmiRNAs and five target genes were verified by examining the levels of expression. The binding sites on bta-miR-128 for the peroxisome proliferator activated receptor gamma (PPARG) and solute carrier family 16 member 1 (SLC16A1) genes were investigated using a dual luciferase assay. The results of this study provide insight into the role of miRNAs in rumen development in calves under different NFC/NDF levels.

MicroRNA Profiling During Neural Differentiation of Induced Pluripotent Stem Cells.

MicroRNAs (miRNA) play an essential role in the regulation of gene expression and influence signaling networks responsible for several cellular processes like differentiation of pluripotent stem cells. Despite several studies on the neurogenesis process, no global analysis of microRNA expression during differentiation of induced pluripotent stem cells (iPSC) to neuronal stem cells (NSC) has been done. Therefore, we compared the profile of microRNA expression in iPSC lines and in NSC lines derived from them, using microarray-based analysis. Two different protocols for NSC formation were used: Direct and two-step via neural rosette formation. We confirmed the new associations of previously described miRNAs in regulation of NSC differentiation from iPSC. We discovered upregulation of miR-10 family, miR-30 family and miR-9 family and downregulation of miR-302 and miR-515 family expression. Moreover, we showed that miR-10 family play a crucial role in the negative regulation of genes expression belonging to signaling pathways involved in neural differentiation: WNT signaling pathway, focal adhesion, and signaling pathways regulating pluripotency of stem cells.

Ocular-Component-Specific miRNA Expression in a Murine Model of Lens-Induced Myopia.

To identify tissues and molecules involved in refractive myopic shift and axial length elongation in a murine lens-induced myopia model, we performed a comprehensive analysis of microRNA (miRNA) expression. Three weeks after negative 30 diopter lens fixation on three-week-old C57BL/6J mice, total RNA was extracted from individual ocular components including cornea, iris, lens, retina, retinal pigment epithelium (RPE)/choroid, and sclera tissue. The miRNA expression analysis was pooled from three samples and carried out using Agilent Mouse miRNA Microarray (8 × 60 K) miRBase21.0. The expression ratio was calculated, and differentially expressed miRNAs were extracted, using GeneSpring GX 14.5. Myopic induction showed a significant myopic refractive change, axial elongation, and choroidal thinning. Through the comprehensive miRNA analysis, several upregulated miRNAs (56 in cornea tissue, 13 in iris tissue, 6 in lens tissue, 0 in retina tissue, 29 in RPE/choroid tissue, and 30 in sclera tissue) and downregulated miRNAs (7 in cornea tissue, 28 in iris tissue, 17 in lens tissue, 9 in retina tissue, 7 in RPE/choroid tissue, and 40 in sclera tissue) were observed. Overlapping expression changes in miRNAs were also found in different ocular components. Some of this miRNA dysregulation may be functionally involved in refractive myopia shift and axial length elongation.

Abstract 1794: A comparative microRNA expression analysis in breast cancer and melanoma tissues

Abstract Recently studies have revealed that a subset of microRNAs (miRNAs) is aberrantly expressed in the development and progression of a variety of cancers including breast cancer and melanoma. Several miRNAs have been implicated in tumorigenesis and progression of human cancer. However, the underlying mechanisms in different types of cancers remain largely unknown. The aim of the study was to compare the expression profiled of miRNA between breast cancer and melanoma patient’s tissues. Methods: Expression levels of 7 miRNAs (miR-15b, miR-142-3p, miR-142-5p, miR-142a, miR-199a, miR-221, miR-424, ) were determine in 17 cases of breast cancer and 25 melanoma tissue samples. MiRNA isolation from tissue samples were performed using Trizol reagent ( Sigma, USA). RNA quality and quantity were assessed using a BioMate TM 3 Series Spectrophotometers (Thermo, Madison, WI). Synthesis of cDNA with reverse transcriptase was performed by TaqManTM microRNA Reversed Transcription Kits, For analysis of miRNA expression, real-time q-PCR analyses were using TaqMan MicroRNA Assays. All real-time q-RT-PCR were performed on a 7300 real-Time PCR system (Applied Biosystems, USA). MiR-24 was used as the internal control for normalization. The relative miRNA expression levels were calculated using the 2 DΔCt methods. Values were presented as means ± standard deviation (SD). The comparison of miRNA levels in breast cancer and melanoma tissues were performed using Student’s t-test. A p value less than 0.05 was considered statistically significant. The expression of miRNAs in different breast cancer subtypes (infiltrating and Invasive ductal cancer) and melanoma (primary and metastatic) was also performed. Results: Relative miRNA expression levels (miR-15b, miR-142-3p, miR-142-5p, miR-146a, miR-199a, miR-221 and miR-424) were found to be differentially expressed in breast cancer and melanoma tissues. Significantly higher relative miRNA expression levels were detected in melanoma tissues compared with breast cancer tissues in all miRNAs evaluated except for miR-424. Four miRNAs (miR-15b, miR-146a, miR-221, and miR-143-3p) had relative expressions over 1.5 compared with only 1 miRNA (miR-15b) in breast cancer tissue. DΔCt values are plotted following comparison with endogenous levels of miR-24 assessed in each sample. Conclusions: Our study demonstrated that miRNA is not only a potential biomarker, but also a valuable therapeutic target for breast cancer and melanoma. Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. A comparative microRNA expression analysis in breast cancer and melanoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1794.

MicroRNA-1246 regulates the radio-sensitizing effect of curcumin in bladder cancer cells via activating P53.

Radiotherapy is the primary option for bladder cancer patients, but it does not have obvious curative effects. This study was to investigate how to increase radiosensitivity in bladder cancer. The curcumin and irradiation treated T24 cells were used for analysis of microRNA expression (miRNA microarray), cell viability (Cell Proliferation Assay Kit), colony formation, apoptosis (Annexin V-FITC/7-AAD flow cytometry), miR-1246 and p53 mRNA (real-time PCR) and protein (Western blot) expression. Microarray assay identified 17 differentially expressed miRNAs (twofold change) in curcumin treated cells compared to control cells. Among them, miR-1246 was the miRNA with the largest change in expression after curcumin treatment. Curcumin significantly decreased T24 cell viability and colony formation in a concentration-dependent manner compared to control cells. miR-1246 expression was significantly higher in T24 cells than in SV-HUC-1 cells and the higher concentrations (10 or 20μM) of curcumin significantly down-regulated miR-1246 expression in T24 and HT-1376 cells. The combination of 10µM curcumin and irradiation was more effective in decreasing miR-1246 expression, cell viability and colony formation than curcumin or irradiation alone. Inhibition of miR-1246 significantly decreased cell viability and colony formation in T24 and HT-1376 cells. Transfection with antagomiR-1246 significantly increased the G0/G1-phase of T24 cells and induced apoptosis compared to cells transfected with antagomiR-NC. Luciferase reporter assay showed that the overexpression of miR-1246 suppressed the luciferase activity of the P53 3'-UTR reporter genes. miR-1246 is involved in the anti-cancer effects of curcumin and irradiation through targeting the inhibition of p53 gene translation in bladder cancer cells.

Prediction of Titanium Implant Success by Analysis of microRNA Expression in Peri-Implant Tissue. A 5-Year Follow-Up Study.

The aim of the present study is to evaluate the expression of microRNA (miRNA) in peri-implant soft tissue and to correlate epigenetic information with the clinical outcomes of the implants up to the five-year follow-up. Seven patients have been rehabilitated with fixed screw-retained bridges each supported by implants. Peri-implant bone resorption and soft tissue health parameters have been recorded over time with a five-year follow-up. Mini-invasive samples of soft peri-implant tissue have been taken three months after implant insertion. miRNA have been extracted from cells of the soft tissue samples to evaluate gene-expression at the implant sites by microarray analysis. The epigenomic data obtained by microarray technology has been statistically analyzed by dedicated software and compared with measured clinical parameters. Specific miRNA expression profiles predictive of specific clinical outcomes were found. In particular, some specific miRNA signatures appeared to be “protective” from bone resorption despite the presence of plaque accumulation. miRNA may be predictors of dental implant clinical outcomes and may be used as biomarkers for diagnostic and prognostic purposes in the field of implant dentistry.

Protocols for the Analysis of microRNA Expression, Biogenesis, and Function in Immune Cells.

MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures known as pre-miRNAs. Pre-miRNAs are translocated to the cytoplasm and cleaved by Dicer to form the mature miRNAs, which mediate mRNA degradation through their loading to the RNA-induced silencing complex (RISC) and binding to complementary sequences within target mRNAs to repress their translation by mRNA degradation and/or translation inhibition. Because ∼1900 miRNA genes are reported in the human genome, many associated with disease, appropriate methods to study miRNA expression and regulation under physiological and pathological conditions have become increasingly important to the study of many aspects of human biology, including immune regulation. As with small interfering RNA (siRNA), the mechanism of miRNA-mediated targeting has been used to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze the expression of pri-mRNAs, pre-miRNAs, and mature miRNAs and characterize their targets both in vitro and in vivo. Such studies will facilitate future novel drug design and development. This unit provides six basic protocols for miRNA analysis, covering next-generation sequencing, quantitative real-time PCR (qRT-PCR), and digoxigenin-based expression analysis of pri-mRNAs, pre-miRNAs, and mature miRNAs; mapping of pri-miRNA and their cleavage sites by rapid amplification of cDNA ends (RACE); electrophoretic mobility shift assays (EMSAs) or biotin-based nonradioactive detection of miRNA-protein complexes (miRNPs); and functional analysis of miRNAs using miRNA mimics and inhibitors. © 2019 by John Wiley & Sons, Inc.

Interplay between proinflammatory cytokines, miRNA, and tissue lesions in Anisakis-infected Sprague-Dawley rats.

BackgroundAnisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases.Methodology/Principal findingsThe aim of this study was to characterize the early cellular (6–72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM); ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, iNOS+, and S100A8/A9+ cells. The greatest number of iNOS+ and S100A8/A9+ cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls.Conclusions/SignificanceAnisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans.

Integrative microRNA and gene expression analysis identifies new drug repurposing candidates for fetal hemoglobin induction in β-hemoglobinopathies

Therapeutic induction of fetal hemoglobin (HbF) is one of the most promising approaches to ameliorate the severity of hemoglobinopathies like β-thalassemia and sickle cell anemia. Although several pharmacological agents have been investigated for HbF induction in adults, the majority of these are associated with significant side-effects. While drug repurposing is known to open new doors for the use of approved drugs in unexplored clinical conditions, the primary challenge lies in identifying such candidates. In this study, we aimed to identify repurposing candidates for HbF induction using a novel in silico approach utilizing microRNA-pathway-drug relationships. A computational drug repurposing strategy identified several unique candidates for HbF induction; among which Curcumin, Ginsenoside, Valproate, and Vorinostat were found to be most suitable for future trials. This study identified new drug repurposing candidates for HbF induction and demonstrates an easily adaptable methodology that can be used for other pathophysiological conditions.

Integrative Analysis of Long Noncoding RNA (lncRNA), microRNA (miRNA) and mRNA Expression and Construction of a Competing Endogenous RNA (ceRNA) Network in Metastatic Melanoma.

Worldwide, metastatic melanoma of the skin has an aggressive course with high morbidity and mortality. Therefore, an increased understanding of the pathogenesis of metastatic melanoma has gained increasing attention, including the role of epigenetic modification and competing endogenous RNA (ceRNA). This study aimed to used bioinformatics data to undertake an integrative analysis of long noncoding RNA (lncRNA), microRNA (miRNA) and mRNA expression to construct a ceRNA network in metastatic melanoma.Data from the Cancer Genome Atlas (TCGA), the Gene Ontology (GO) database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were analyzed. There were 471 cases that included 103 primary solid tumors and 368 cases of metastatic melanoma that included transcriptome sequencing data (including lncRNA and mRNA); 452 cases had miRNA sequencing data.Analysis of chip data identified 85 6 mRNAs, 67 miRNAs, and 250 lncRNAs that were differentially expressed in cases of metastatic melanoma, of which 25 miRNAs, 18 lncRNAs, and 18 mRNAs participated in the formation of ceRNAs. Survival analysis identified seven differentially expressed mRNAs, five differentially expressed miRNAs (miRNA-29c, miRNA-100, miR-142-3p, miR-150, miR-516a-2), and six differentially expressed lncRNAs (AC068594.1, C7orf71, FAM41C, GPC5-AS1, MUC19, LINC00402) that were correlated with survival time in patients with metastatic melanoma.Bioinformatics data and integrative analysis identified lncRNA, miRNA, and mRNA expression to construct a ceRNA and patient survival network in metastatic melanoma. These findings support the need for further studies on the mechanisms involved in the regulation of metastatic melanoma by ceRNAs.

Analysis of microRNA expression in brush cytology specimens improves the diagnosis of pancreatobiliary cancer

Background/ObjectivesMalignant pancreatobiliary strictures are in many cases clinically indistinguishable and present a major problem to endoscopy specialists. Intraductal sampling procedures such as brush cytology are commonly used for diagnosis with a sensitivity that is low for a diagnostic test used in daily clinical practice. MicroRNA (miR) alterations detected in many cancers are disease-specific, which can be utilized in clinical applications. The aim of the present study was to analyze whether determination of miR expression levels in intraductal brush cytology specimens is a feasible approach to improve the diagnosis of pancreatobiliary cancer. MethodsBrush cytology specimens have been collected during endoscopic retrograde cholangio-pancreatography (ERCP) and analyzed by routine cytology and ancillary miR assays. Total RNA was extracted using the miRNeasy Mini Kit and the expression of miRs frequently dysregulated in pancreatobiliary cancer (miR-16, miR-21, miR-196a, miR-221) were analyzed by quantitative real-time PCR using RNU6B as internal control. ResultsRoutine cytology resulted in no false positive diagnoses, however, the combined sensitivity remained at 53.8%. Expression (ΔCt values) of miR-16 (p = 0.0039), miR-196a (p = 0.0003) and miR-221 (p = 0.0049) showed a clear statistical significance between malignant and benign pancreatobiliary specimens (n = 35). Malignancy could be detected combining routine cytology and the miR-196a single marker expression levels with a sensitivity of 84.6% (92.9% in biliary strictures) with no false positives. ConclusionsThe results offer the first direct demonstration that microRNAs are readily detectable in brush cytology specimens obtained during ERCP, and have the potential to help the cytological diagnosis of pancreatobiliary malignancy.

Bioinformatics analysis of microRNA expression between patients with and without latent tuberculosis infections.

Tuberculosis (TB) is a globally prevalent infectious disease. The mechanisms of latent TB infection (LTBI) remain to be fully elucidated and may provide novel approaches for diagnosis. As therapeutic targets and molecular diagnostic markers, microRNAs (miRs) have been studied and utilized in various diseases. In the present study, the differentially expressed miRs (DEMs) in LTBI were screened and analyzed to determine the underlying mechanisms and identify potential biomarkers, thereby contributing to the diagnosis of LTBI. The GSE25435 and GSE29190 datasets from Gene Expression Omnibus were selected for analysis. The 2 datasets were analyzed individually using the Bioconductor package to screen the DEMs with specific cut-off criteria [P<0.01 and |log (fold change)|≥1]. Target gene prediction and interaction network construction were performed using Targetscan, the Search Tool for the Retrieval of Interacting Genes and Proteins and Cytoscape individually, and were merged using the latter tool. The hub genes were finally selected based on their degree of connectivity (DC). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the KEGG and GENCLIP. A total of 144 DEMs were identified from the 2 datasets. By exploring the overlapping miRs in the two datasets, Homo sapiens (hsa)-miR-29a and hsa-miR-15b were identified to be decreased, while hsa-miR-576-5p, hsa-miR-500 and hsa-miR-155 were identified to be upregulated. hsa-miR-500a-3p and hsa-miR-29a-3p, as well as 4 genes, namely cell division cycle (CDC)42, actin α1, skeletal muscle (ACTA1), phosphatase and tensin homolog (PTEN) and fos proto-oncogene (FOS), were selected as the key factors in this regulatory network. A total of 9 signaling pathways, including phosphoinositide-3 kinase (PI3K)/AKT and 11 biological processes, were identified to be associated with LTBI. In conclusion, the present analysis identified hsa-miR-500a-3p and hsa-miR-29a-3p, as well as CDC42, ACTA1, PTEN and FOS, as the most promising biomarkers and therapeutic candidates for LTBI. The PI3K/AKT signaling pathway is the key signaling pathway implicated in LTBI, and an in-depth investigation of the efficiency of PI3K/AKT signaling inhibitors may be used to prevent a chronic state of infection in LTBI.

Comparative expression analysis of let-7 microRNAs during ovary development in Megalobrama amblycephala.

As a critical regulator of gene expression, let-7 family miRNAs have been reported to be involved in multiple physiological processes. In this study, in order to elucidate the putative regulatory effect of let-7 miRNAs during fish gonadal development and to identify which member is crucial for this regulation, the expression of ten members including let-7a/b/c/d/e/f/g/h/i/j were quantified in ovary, pituitary, and brain tissues during the different ovarian developmental stages of blunt snout bream Megalobrama amblycephala. According to the data from analysis of expression patterns, let-7a showed the highest expression value in almost all the tested ovaries, pituitaries, and brains, with let-7b and let-7d moderately expressed, following by other let-7 miRNAs. In terms of the differential expression levels of ten let-7 miRNAs at each developmental stage, the results showed that let-7a/b/d/f/h expression gradually increased during the ovary development from stage I to V and dropped significantly at the phase VI in ovary tissues. However, the expression of let-7a/b/e/f in pituitary increased during the ovary development from stage I to IV and declined at stage V. Among all the let-7 miRNAs, let-7a/b/d had the highest expression and their expression patterns were consistent with the gonad development of M. amblycephala. Furthermore, the mostly predicted target genes of let-7 miRNAs are significantly enriched in signaling pathways closely related to gonadal development through KEGG enrichment analysis. These results indicate that let-7 miRNA members, especially let-7a/b/d, may play important roles in the regulation of ovary development in M. amblycephala through negatively regulating expression of their target genes.

MicroRNAs as biomarkers in Pompe disease

PurposeWe studied microRNAs as potential biomarkers for Pompe disease. MethodsWe analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients. ResultsIn the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement. ConclusionCirculating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.

MicroRNA-127-3p controls murine hematopoietic stem cell maintenance by limiting differentiation.

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

Analysis of microRNA expression in endometrial tissue in external genital endometriosis

Analysis of microRNA expression in endometrial tissue in external genital endometriosis

Function of berberine on porcine in vitro fertilization embryo development and differential expression analysis of microRNAs.

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU-23 embryonic culture medium was used for a control group, while NCSU-23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4- and 8-cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2-, 4-, 8-cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p<0.01). The differentially expressed miRNAs in the 8-cell versus the 4-cell stage in control group as well as in the 8-cell Ber group versus the 8-cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber-regulated miRNAs at the 8-cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8-cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.

MicroRNA expression analysis of human skin fibroblasts treated with high-fluence light-emitting diode-red light.

Skin fibrosis is a chronic debilitating feature of several skin diseases that lead to characteristic increases in dermal fibroblast proliferation and collagen deposition through upregulation in components of the transforming growth factor beta (TGF-B)/SMAD pathway. In contrast to ultraviolet phototherapy, high-fluence light-emitting diode-generated red light (HF-LED-RL, 633 ± 15 nm) is a safe, economic and non-invasive therapy with in vitro evidence that supports modulation of the key cellular characteristics involved in the pathogenesis of skin fibrosis. Limited data exists pertaining to the effects of HF-LED-RL on human skin fibroblast microRNA (miRNA). Herein, we explored the effects of HF-LED-RL on fibroblast miRNA levels using RNA-seq and miRNA expression analysis. Using RNA-seq analysis we found that HF-LED-RL at 320 and 640 J/cm2 increased transcription of key miRNA that are involved in skin fibrosis including miRNA-29, miRNA-196a and Let-7a, and decreased transcription of miRNA-21, miRNA-23b and miRNA-31. These microRNA findings provide insight into the molecular underpinnings of HF-LED-RL and highlight potential therapeutic targets of interest for the treatment of skin fibrosis. Additional research on the specific molecular mechanisms underlying HF-LED-RL effects on fibroblasts may provide further mechanistic insight into this therapy and may reveal additional future therapeutic targets for skin fibrosis.

Epigenetic Regulation of iASPP-p63 Feedback Loop in Cutaneous Squamous Cell Carcinoma

Keratinocyte skin cancer, comprising cutaneous squamous (cSCC) and basal cell carcinoma, is the most common malignancy in the United Kingdom. P53 is frequently mutated in cSCC. iASPP is a key inhibitor of p53 and NF-κB signaling pathways and has been documented as highly expressed in several types of human cancer. We have previously identified an autoregulatory feedback loop between iASPP and p63, which is critical in epidermal homeostasis. We hypothesized a potential role for dysregulation of this axis in the pathogenesis of keratinocyte malignancies. Immunostaining of 116 cSCC clinical samples revealed increased iASPP and ΔNp63 expression, but also highlighted a significant alteration of iASPP cellular localization, with consequent deregulation of its function. Expression patterns, functionality, and gene and microRNA expression analysis were further investigated in 10 cSCC cell lines. Our data suggest that while direct effects of iASPP and p63 upon each other's expression are maintained in cSCC, epigenetic dysregulation of the feedback loop occurs at the microRNA level by a previously unreported mechanism controlling p63 expression. We demonstrate that this autoregulatory feedback loop controls cell migration in cSCC by blocking epithelial-mesenchymal transition and promoting proliferation, and provides future directions for clinical biomarker and therapeutic target discovery in cutaneous SCC.