Low oxygen tension positively influences cardiomyocyte progenitor cell function

Previously we observed that cardiomyocyte progenitor cells (hCMPCs) isolated from the human heart differentiate spontaneously into cardiomyocytes and vascular cells when transplanted after myocardial infarction (MI) in the ischemic heart. After MI, deprivation of oxygen is the first major change in the cardiac environment. How cells handle hypoxia is highly cell type dependent. The effect of hypoxia on cardiac stem or progenitor cells remains to be elucidated. Here, we show for the first time that short- and long-term hypoxia have different effects on hCMPCs. Short-term hypoxia increased the migratory and invasive capacities of hCMPCs likely via mesenchymal transformation. Although long-term exposure to low oxygen levels did not induce differentiation of hCMPCs into mature cardiomyocytes or endothelial cells, it did increase their proliferation, stimulated the secretome of the cells which was shifted to a more anti-inflammatory profile and dampened the migration by altering matrix metalloproteinase (MMP) modulators. Interestingly, hypoxia greatly induced the expression of the extracellular matrix modulator thrombospondin-2 (TSP-2). Knockdown of TSP-2 resulted in increased proliferation, migration and MMP activity. In conclusion, short exposure to hypoxia increases migratory and invasive capacities of hCMPCs and prolonged exposure induces proliferation, an angiogenic secretion profile and dampens migration, likely controlled by TSP-2.     

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated “Focal adhesion” and “ECM-receptor interaction” as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren’t significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.     

MicroRNA‐26a is a novel regulator of vascular smooth muscle cell function

Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA‐26a for additional studies. Inhibition of miRNA‐26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA‐26a blunted differentiation. As a potential mechanism, we investigated whether miRNA‐26a influences TGF‐β‐pathway signaling. Dual‐luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA‐26a inhibition, and the opposite effect with miRNA‐26a overexpression in transfected human cells. Furthermore, inhibition of miRNA‐26a increased gene expression of SMAD‐1 and SMAD‐4, while overexpression inhibited SMAD‐1. MicroRNA‐26a was also found to be downregulated in two mouse models of AAA formation (2.5‐ to 3.8‐fold decrease, P < 0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA‐26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF‐β pathway signaling. MicroRNA‐26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease. J. Cell. Physiol. 226: 1035–1043, 2011. © 2010 Wiley‐Liss, Inc.     

Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis

Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre‐miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir‐15a and mir‐188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir‐15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir‐188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre‐mir‐10a, mir‐105, and mir‐182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.     

MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA‐155 (miR‐155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre‐miR‐155, anti‐miR‐155 or control‐miR (ctrl‐miR), followed by scratch‐ and transwell‐ assays. These functional assays displayed that miR‐155 over‐expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR‐155 transfected cells was collected and zymography analysis showed a significant decrease in MMP‐2 and MMP‐9 activities. The predicted 3′‐UTR of MMP‐16, an activator of MMP‐2 and ‐9, was cloned into the pMIR‐REPORT vector and luciferase assays were performed. Introduction of miR‐155 significantly reduced luciferase activity which could be abolished by cotransfection with anti‐miR‐155 or target site mutagenesis. By using MMP‐16 siRNA to reduce MMP‐16 levels or by using an MMP‐16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP‐16, miR‐155 efficiently inhibits cell migration via a reduction in MMP‐2 and ‐9 activities. Our study shows that miR‐155 might be used to improve local retention of hCMPCs after intramyocardial delivery.     

Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy

Background. In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart. Methods. hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine. Results. hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes. Conclusion. The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16: 163-9.)     

Identification of micro‐RNA networks in end‐stage heart failure because of dilated cardiomyopathy

Micro‐RNAs regulate gene expression by directly binding to the target mRNAs. The goal of the study was to examine the expression profiling of miRNAs in human failing hearts and identify the key miRNAs that regulate molecular signalling networks and thus contribute to this pathological process. The levels of miRNAs and expressed genes were analysed in myocardial biopsy samples from patients with end‐stage heart failure (n = 14) and those from normal heart samples (n = 8). Four networks were built including the Gene regulatory network, Signal‐Network, miRNA‐GO‐Network and miRNA‐Gene‐Network. According to the fold change in the network and probability values in the microarray cohort, RT‐PCR was performed to measure the expression of five of the 72 differentially regulated miRNAs. miR‐340 achieved statistically significant. miR‐340 was identified for the first time in cardiac pathophysiological condition. We overexpressed miR‐340 in cultured neonatal rat cardiomyocytes to identify whether miR‐340 plays a determining role in the progression of heart failure. ANP, BNP and caspase‐3 were significantly elevated in the miR‐340 transfected cells compared with controls (P < 0.05). The cross‐sectional area of overexpressing miR‐340 cardiomyocytes (1952.22 ± 106.59) was greater (P < 0.0001) than controls (1059.99 ± 45.59) documented by Laser Confocal Microscopy. The changes of cellular structure and the volume were statistical significance. Our study provided a comprehensive miRNA expression profiling in the end‐stage heart failure and identified miR‐340 as a key miRNA contributing to the occurrence and progression of heart failure. Our discoveries provide novel therapeutic targets for patients with heart failure.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

SLIT2/ROBO1‐miR‐218‐1‐RET/PLAG1: a new disease pathway involved in Hirschsprung's disease

Hirschsprung's disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. We investigated changes in expression of microRNAs (miRNAs) and the genes they regulate in tissues of patients with HSCR. Quantitative real‐time PCR and immunoblot analyses were used to measure levels of miRNA, mRNAs, and proteins in colon tissues from 69 patients with HSCR and 49 individuals without HSCR (controls). Direct interactions between miRNAs and specific mRNAs were indentified in vitro, while the function role of miR‐218‐1 was investigated by using miR‐218 transgenic mice. An increased level of miR‐218‐1 correlated with increased levels of SLIT2 and decreased levels of RET and PLAG1 mRNA and protein. The reductions in RET and PLAG1 by miR‐218‐1 reduced proliferation and migration of SH‐SY5Y cells. Overexpression of the secreted form of SLIT2 inhibited cell migration via binding to its receptor ROBO1. Bowel tissues from miR‐218‐1 transgenic mice had nerve fibre hyperplasia and reduced numbers of gangliocytes, compared with wild‐type mice. Altered miR‐218‐1 regulation of SLIT2, RET and PLAG1 might be involved in the pathogenesis of HSCR.     

Folate deficiency disturbs hsa‐let‐7 g level through methylation regulation in neural tube defects

Folic acid deficiency during pregnancy is believed to be a high‐risk factor for neural tube defects (NTDs). Disturbed epigenetic modifications, including miRNA regulation, have been linked to the pathogenesis of NTDs in those with folate deficiency. However, the mechanism by which folic acid‐regulated miRNA influences this pathogenesis remains unclear. It is believed that DNA methylation is associated with dysregulated miRNA expression. To clarify this issue, here we measured the methylation changes of 22 miRNAs in 57 human NTD cases to explore whether such changes are involved in miRNA regulation in NTD cases through folate metabolism. In total, eight of the 22 miRNAs tested reduced their methylation modifications in NTD cases, which provide direct evidence of the roles of interactions between DNA methylation and miRNA level in these defects. Among the findings, there was a significant association between folic acid concentration and hsa‐let‐7 g methylation level in NTD cases. Hypomethylation of hsa‐let‐7 g increased its own expression level in both NTD cases and cell models, which indicated that hsa‐let‐7 g methylation directly regulates its own expression. Overexpression of hsa‐let‐7 g, along with its target genes, disturbed the migration and proliferation of SK‐N‐SH cells, implying that hsa‐let‐7 g plays important roles in the prevention of NTDs by folic acid. In summary, our data suggest a relationship between aberrant methylation of hsa‐let‐7 g and disturbed folate metabolism in NTDs, implying that improvements in nutrition during early pregnancy may prevent such defects, possibly via the donation of methyl groups for miRNAs.     

Induced miR‐99a expression represses Mtor cooperatively with miR‐150 to promote regulatory T‐cell differentiation

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4⁺ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T‐cell‐expressed miRNAs in naive mouse CD4⁺ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR‐100, miR‐99a and miR‐10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR‐99a cooperated with miR‐150 to repress the expression of the Th17‐promoting factor mTOR. The comparably low expression of miR‐99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR‐150 could only repress Mtor in the presence of miR‐99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.     

Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.     

TGF-β1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro

The adult mammalian heart has limited regenerative capacity and was generally considered to contain no dividing cells. Recently, however, a resident population of progenitor cells has been identified, which could represent a new source of cardiomyocytes. Here, we describe the efficient isolation and propagation of human cardiomyocyte progenitor cells (hCMPCs) from fetal heart and patient biopsies. Establishment of hCMPC cultures was remarkably reproducible, with over 70% of adult atrial biopsies resulting in robustly expanding cell populations. Following the addition of transforming growth factor β, almost all cells differentiated into spontaneously beating myocytes with characteristic cross striations. hCMPC-derived cardiomyocytes showed gap-junctional communication and action potentials of maturing cardiomyocytes. These are the first cells isolated from human heart that proliferate and form functional cardiomyocytes without requiring coculture with neonatal myocytes. Their scalability and homogeneity are unique and provide an excellent basis for developing physiological, pharmacological, and toxicological assays on human heart cells in vitro.     

Cellular and extracellular miRNAs are blood‐compartment‐specific diagnostic targets in sepsis

Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment are of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High‐throughput technologies assessing circulating microRNAs represent an important tool for biomarker identification, but the blood‐compartment specificity of these miRNAs has not yet been investigated. We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients by next‐generation sequencing and RT‐qPCR (n = 3 × 22) and established differences in miRNA expression between blood compartments. In silico analysis was used to identify compartment‐specific signalling functions of differentially regulated miRNAs in sepsis‐relevant pathways. In septic shock, a total of 77 and 103 miRNAs were down‐ and up‐regulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes and 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment‐specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR‐199b‐5p was identified as a potential early indicator for sepsis and septic shock. miR‐125b‐5p and miR‐26b‐5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR‐27b‐3p) was present in all three compartments. The expression of sepsis‐associated miRNAs is compartment‐specific. Exosome‐derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly identified targets for the development of novel sepsis biomarkers.     

Differential expression of microRNAs in TM3 Leydig cells of mice treated with brain‐derived neurotrophic factor

Brain‐derived neurotrophic factor (BDNF) is a neurotrophin that can promote the development and proliferation of neurons. BDNF has been found to be involved in male reproduction. Leydig cells in testicular interstitial tissues can secrete testosterone in a luteinizing hormone‐dependent manner. We showed that BDNF and its receptor TrkB were expressed in mice TM3 Leydig cells in the present study. Furthermore, BDNF can promote proliferation of mouse TM3 Leydig cells in vitro. Results of microRNA (miRNA) deep sequencing showed that BDNF can alter the expression profile of miRNAs in TM3 Leydig cells. Eighty‐three miRNAs were significantly different in the BDNF‐treated and control groups (fold change of >2.0 or <0.5, P < 0.05) wherein 40 were upregulated and 43 were downregulated. The expression levels of miR‐125a‐5p, miR‐22‐5p, miR‐342‐59, miR‐451a, miR‐148a‐5p, miR‐29b‐3p, miR‐199b‐5p, and miR‐145a‐5p were further confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs participate in the pathways involved in signal transduction, cancer, metabolism, endocrine system, immune system, and nerve system. This study indicated that miRNAs might be involved in the BDNF‐regulated cellular functions of Leydig cells.