MicroRNAs (miRNAs) have recently come into focus as key posttranscriptional modulators of gene expression. In this work, we addressed whether in vitro angiogenesis is an miRNA-regulated process. We ...performed large-scale analysis of miRNA expression in human umbilical vein endothelial cells (HUVECs) and found that 15 highly expressed miRNAs have the receptors of angiogenic factors as putative targets. In particular, we demonstrated that miR-221 and miR-222 affect c-Kit expression and, as a consequence, the angiogenic properties of its ligand stem cell factor. Interaction between miR-222 and c-Kit is likely to be part of a complex circuit that controls the ability of endothelial cells to form new capillaries.
Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain ...significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in
increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.
To dissect the
TBX5
regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type
, ...CRE, Tbx5
lox/
+
and Tbx5
del/
+
mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt–Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between
TBX5
, arrhythmia and heart development.
tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to ...gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt-Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) ...cells affecting cell proliferation/survival, as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing, we collected the complete repertoire of miR-28-5p targets, obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22, we found that 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with an important role in PCa. We validated miR-28-5p/SREBF2 interaction, demonstrating that SREBF2 inhibition affects almost all the tumor processes altered by miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development, and potential new targets for anti-tumor therapy.
The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma ...cells.
Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos).
Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as
,
,
,
and
mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes-due to increased maturation and/or trafficking-confers resistance to vem.
We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells.
Ventricular assist device (VAD) implant represents a therapeutic option for pediatric patients with end-stage heart failure (HF). Heart unloading by VAD can modify several molecular pathways ...underlying cardiac function in HF. Among them, the potential role of microRNA (miRNAs) in response to VAD implant is emerging. This study was aimed at investigating in HF pediatric patients the effect of VAD-modified miRNAs on the adiponectin (ADPN) system, known to exert cardioprotective actions.
ADPN was measured in plasma samples obtained from HF children, before and 1 month after VAD implant, and from healthy control children. miRNA profile and molecules belonging to ADPN system were determined in cardiac biopsies collected at the time of VAD implantation (pre-VAD) and at the moment of heart transplant (post-VAD). An in vitro study using HL-1 cell line was performed to verify the regulatory role of the VAD-modified miRNA on the ADPN system.
VAD implant did not affect circulating and cardiac levels of ADPN, but increased the cardiac mRNA expression of ADPN receptors, including AdipoR1, AdipoR2, and T-cad. AdipoR2 and T-cad were inversely related to the VAD-modified miRNA levels. The in vitro study confirmed the regulatory role of miR-1246 and miR-199b-5p on AdipoR2, and of miR-199b-5p on T-cad.
These data suggest that VAD treatment could regulate the expression of the cardioprotective ADPN system by epigenetic mediators, suggesting that miRNAs have a potential role as therapeutic targets to improve cardiac function in HF pediatric patients.
The LncRNA my-heart (Mhrt) and the chromatin remodeler Brg1 inhibit each other to respectively prevent or favor the maladaptive α-myosin-heavy-chain (Myh6) to β-myosin-heavy-chain (Myh7) switch, so ...their balance crucially guides the outcome of cardiac remodeling under stress conditions. Even though triiodothyronine (T3) has long been recognized as a critical regulator of the cardiac Myh isoform composition, its role as a modulator of the Mhrt/Brg1 axis is still unexplored. Here the effect of T3 on the Mhrt/Brg1 regulatory circuit has been analyzed in relation with chromatin remodeling and previously identified T3-dependent miRNAs. The expression levels of Mhrt, Brg1 and Myh6/Myh7 have been assessed in rat models of hyperthyroidism or acute myocardial ischemia/reperfusion (IR) treated with T3 replacement therapy. To gain mechanistic insights, in silico analyses and site-directed mutagenesis have been adopted in combination with gene reporter assays and loss or gain of function strategies in cultured cardiomyocytes. Our results indicate a pivotal role of Mhrt over-expression in the T3-dependent regulation of Myh switch. Mechanistically, T3 activates the Mhrt promoter at two putative thyroid hormone responsive elements (TRE) located in a crucial region that is necessary for both Mhrt activation and Brg1-dependent Mhrt repression. This newly identified T3 mode of action requires DNA chromatinization and is critically involved in mitigating the repressive function of the Brg1 protein on Mhrt promoter. In addition, T3 is also able to prevent the Brg1 over-expression observed in the post-IR setting through a pathway that might entail the T3-mediated up-regulation of miR-208a. Taken together, our data evidence a novel T3-responsive network of cross-talking epigenetic factors that dictates the cardiac Myh composition and could be of great translational relevance.
On the grounds that miRNAs present in the blood of prostate cancer (PCa) patients are released in the growth medium by PCa cells, it is conceivable that PCa cells resistant to docetaxel (DCT) (DCT
) ...will release miRNAs that may be found in PCa patients under DCT therapy if resistant PCa cells appear. We isolated DCT
clones respectively from 22Rv1 and DU-145 PCa cell lines and performed through next-generation sequencing (NGS) the miRNAs profiles of the released miRNAs. The analysis of the NGS data identified 105 and 1 miRNAs which were differentially released in the growth medium of the 22Rv1/DCT
and DU-145/DCT
clones, respectively. Using additional filters, we selected 12 and 1 miRNA more released by all 22Rv1/DCT
and DU-145/DCT
clones, respectively. Moreover, we showed that 6 of them were more represented in the growth medium of the DCT
cells than the ones of DCT-treated cells. We speculated that they have the pre-requisite to be tested as predictive biomarkers of the DCT resistance in PCa patients under DCT therapy. We propose the utilization of clones resistant to a given drug as in vitro model to identify the differentially released miRNAs, which in perspective could be tested as predictive biomarkers of drug resistance in tumor patients under therapy.
Docetaxel (DCT) resistance is one of the main factors responsible for treatment failure in metastatic prostate cancer (PCa). Although several mechanisms of DCT resistance have been elucidated, the ...issue is still far from comprehensive. In this work we show that miR-96-5p, miR-183-5p and miR-210-3p (referred to as sDCT
-miRNAs) are specifically released by DCT resistant (DCT
) PCa clones and decrease the efficacy of DCT in PCa cells when overexpressed. Through bioinformatic analysis, we identified several potential targets of sDCT
-miRNAs' activity including FOXO1, IGFBP3, and PDCD4 known to exert a role in DCT resistance. Additionally, we found that PPP2CB and INSIG1 mediated the ability of sDCT
-miRNAs to reduce the efficacy of DCT. We explored whether secreted sDCT
-miRNAs could affect the phenotype of PCa cells. We found that exposure to exosomes derived from DCT
PCa clones (in which the content of sDCT
-miRNAs was higher than in exosomes from parental cells), as well as exposure to exosome loaded with sDCT
-miRNAs, reduced the cytotoxicity of DCT in PCa cells sensitive to the drug. Finally, we validated circulating miR-183-5p and miR-21-5p as potential predictive biomarkers of DCT resistance in PCa patients. Our study suggests a horizontal transfer mechanism mediated by exosomal miRNAs that contributes to reduce docetaxel sensitivity and highlights the relevance of cell-to-cell communication in drug resistance.
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