Circulating microRNA profile in response to remdesivir treatment in coronavirus disease 2019 (COVID-19) patients.

Coronavirus disease 2019 (COVID-19), a serious infectious disease caused by the recently discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a major global health crisis. Although no specific antiviral drugs have been proven to be fully effective against COVID-19, remdesivir (GS-5734), a nucleoside analogue prodrug, has shown beneficial effects when used to treat severe hospitalized COVID-19 cases. The molecular mechanism underlying this beneficial therapeutic effect is still vaguely understood. In this study, we assessed the effect of remdesivir treatment on the pattern of circulating miRNAs in the plasma of COVID-19 patients, which was analyzed using MiRCURY LNA miRNA miRNome qPCR Panels and confirmed by quantitative real-time RT-PCR (qRT-PCR). The results revealed that remdesivir treatment can restore the levels of miRNAs that are upregulated in COVID-19 patients to the range observed in healthy subjects. Bioinformatics analysis revealed that these miRNAs are involved in diverse biological processes, including the transforming growth factor beta (TGF-ß), hippo, P53, mucin-type O-glycan biosynthesis, and glycosaminoglycan biosynthesis signaling pathways. On the other hand, three miRNAs (hsa-miR-7-5p, hsa-miR-10b-5p, and hsa-miR-130b-3p) were found to be upregulated in patients receiving remdesivir treatment and in patients who experienced natural remission. These upregulated miRNAs could serve as biomarkers of COVID-19 remission. This study highlights that the therapeutic potential of remdesivir involves alteration of certain miRNA-regulated biological processes. Targeting of these miRNAs should therefore be considered for future COVID-19 treatment strategies.

Effect of Ultraviolet Radiation and Benzo[a]pyrene Co-Exposure on Skin Biology: Autophagy as a Potential Target.

The skin is the outermost protective barrier of the human body. Its role is to protect against different physical, chemical, biological and environmental stressors. The vast majority of studies have focused on investigating the effects of single environmental stressors on skin homeostasis and the induction of several skin disorders, such as cancer or ageing. On the other hand, much fewer studies have explored the consequences of the co-exposure of skin cells to two or more stressors simultaneously, which is much more realistic. In the present study, we investigated, using mass-spectrometry-based proteomic analysis, the dysregulated biological functions in skin explants after their co-exposure to ultraviolet radiation (UV) and benzo[a]pyrene (BaP). We observed that several biological processes were dysregulated, among which autophagy appeared to be significantly downregulated. Furthermore, immunohistochemistry analysis was carried out to validate the downregulation of the autophagy process further. Altogether, the output of this study provides an insight into the biological responses of skin to combined exposure to UV + BaP and highlights autophagy as a potential target that might be considered in the future as a novel candidate for pharmacological intervention under such stress conditions.

Glutamine transport as a possible regulator of nitrogen catabolite repression in Saccharomyces cerevisiae.

Nitrogen catabolite repression (NCR) is a major transcriptional control pathway governing nitrogen use in yeast, with several hundred of target genes identified to date. Early and extensive studies on NCR led to the identification of the 4 GATA zinc finger transcription factors, but the primary mechanism initiating NCR is still unclear up till now. To identify novel players of NCR, we have undertaken a genetic screen in an NCR-relieved gdh1Δ mutant, which led to the identification of four genes directly linked to protein ubiquitylation. Ubiquitylation is an important way of regulating amino acid transporters and our observations being specifically observed in glutamine-containing media, we hypothesized that glutamine transport could be involved in establishing NCR. Stabilization of Gap1 at the plasma membrane restored NCR in gdh1Δ cells and AGP1 (but not GAP1) deletion could relieve repression in the ubiquitylation mutants isolated during the screen. Altogether, our results suggest that deregulated glutamine transporter function in all three weak nitrogen derepressed (wnd) mutants restores the repression of NCR-sensitive genes consecutive to GDH1 deletion.

Comparing the osteogenic potential of schneiderian membrane and dental pulp mesenchymal stem cells: an in vitro study.

Mesenchymal stem cells, being characterized by high self-renewal capacity and multi-lineage differentiation potential, are widely used in regenerative medicine especially for repair of bone defects in patients with poor bone regenerative capacity. In this study, we aimed to compare the osteogenic potential of human maxillary schneiderian sinus membrane (hMSSM)-derived stem cells versus permanent teeth dental pulp stem cells (DPSCs). Both cells types were cultivated in osteogenic and non-osteogenic inductive media. Alkaline phosphatase (ALP) activity assay and quantitative real-time PCR analysis were carried out to assess osteogenic differentiation. We showed that ALP activity and osteoblastic markers transcription levels were more striking in hMSSM-derived stem cells than DPSCs. Our results highlight hMSSM-derived stem cells as a recommended stem cell type for usage during bone tissue regenerative therapy.

Isoconazole and Clemizole Hydrochloride Partially Reverse the Xeroderma Pigmentosum C Phenotype.

Xeroderma Pigmentosum protein C (XPC) is involved in recognition and repair of bulky DNA damage such as lesions induced by Ultra Violet (UV) radiation. XPC-mutated cells are, therefore, photosensitive and accumulate UVB-induced pyrimidine dimers leading to increased cancer incidence. Here, we performed a high-throughput screen to identify chemicals capable of normalizing the XP-C phenotype (hyper-photosensitivity and accumulation of photoproducts). Fibroblasts from XP-C patients were treated with a library of approved chemical drugs. Out of 1280 tested chemicals, 16 showed ≥25% photo-resistance with RZscore above 2.6 and two drugs were able to favor repair of 6-4 pyrimidine pyrimidone photoproducts (6-4PP). Among these two compounds, Isoconazole could partially inhibit apoptosis of the irradiated cells especially when cells were post-treated directly after UV irradiation while Clemizole Hydrochloride-mediated increase in viability was dependent on both pre and post treatment. No synergistic effect was recorded following combined drug treatment and the compounds exerted no effect on the proliferative capacity of the cells post UV exposure. Amelioration of XP-C phenotype is a pave way towards understanding the accelerated skin cancer initiation in XP-C patients. Further examination is required to decipher the molecular mechanisms targeted by these two chemicals.

An update on human periapical cyst-mesenchymal stem cells and their potential applications in regenerative medicine.

The broad clinical applications of Mesenchymal Stem Cells (MSCs) in the regenerative medicine field is attributed to their ability to self-renew and differentiate into multiple cellular lineages. Nowadays, MSCs can be derived from a variety of adult and fetal tissues including bone marrow, adipose tissue, umbilical cord and placenta. The difficulties associated with the isolation of MSCs from certain tissues such as bone marrow promoted the search for alternative tissues which are easily accessible. Oral derived MSCs include dental pulp stem cells (DPSCs), dental follicle progenitor cells (DFPC), and periodontal ligament stem cells (PDLSC). Being abundant and easily accessible, oral derived MSCs represent an interesting alternative MSC type to be employed in regenerative medicine. Human periapical cyst-mesenchymal stem cells (hPCy-MSCs) correspond to a newly discovered and characterized MSC subtype. Interestingly, hPCy-MSCs are collected from periapical cysts, which are a biological waste, without any influence on the other healthy tissues in oral cavity. hPCy-MSCs exhibit cell surface marker profile similar to that of other oral derived MSCs, show high proliferative potency, and possess the potential to differentiate into different cell types such as osteoblasts, adipocytes and neurons-like cells. hPCy-MSCs, therefore, represent a novel promising MSCs type to be applied in regenerative medicine domain. In this review, we will compare the different types of dental derived MSCs, we will highlight the isolation technique, the characteristics, and the therapeutic potential of hPCy-MSCs.

MiR302c, Sp1, and NFATc2 regulate interleukin-21 expression in human CD4+CD45RO+ T lymphocytes.

Interleukin-21 (IL-21) is a cytokine with potent regulatory effects on different immune cells. Recently, IL-21 has been contemplated for use in the treatment of cancers. However, the molecular mechanisms regulating human IL-21 gene expression has not yet been described. In this study, we initially studied the promoter region and identified the transcription start site. We thereafter described the essential region upstream of the transcription start site and showed the in vivo binding of NFATc2 and SP1 transcription factors to this region, in addition to their positive role in IL-21 expression. We also studied the role of microRNAs (miRNAs) in regulating IL-21 expression. We, thus, established the miRNA profile of CD4+CD45RO+ versus CD4+CD45RA+ isolated from healthy volunteers and identified a signature composed of 12 differentially expressed miRNAs. We showed that miR-302c is able to negatively regulate IL-21 expression by binding directly to its target site in the 3'-untranslated region. Moreover, after using fresh human CD4-positive T cells, we observed the high acetylation level of histone H4, an observation well in line with the already described high expression of IL-21 in CD4+CD45RO+ versus CD4+CD45RA+ T cells. Altogether, our data identified different molecular mechanisms regulating IL-21 expression.

Human CD8+ CD25 + CD127 low regulatory T cells: microRNA signature and impact on TGF-ß and IL-10 expression.

Regulatory T cells (Tregs) are central for maintaining immune balance and their dysfunction drives the expansion of critical immunologic disorders. During the past decade, microRNAs (miRNAs) have emerged as potent regulators of gene expression among which immune-related genes and their immunomodulatory properties have been associated with different immune-based diseases. The miRNA signature of human peripheral blood (PB) CD8+ CD25 + CD127 low Tregs has not been described yet. We thus identified, using TaqMan low-density array (TLDA) technique followed by individual quantitative real-time polymerase chain reaction (qRT-PCR) confirmation, 14 miRNAs, among which 12 were downregulated whereas two were upregulated in CD8 + CD25 + CD127 low Tregs in comparison to CD8 + CD25 - T cells. In the next step, microRNA Data Integration Portal (mirDIP) was used to identify potential miRNA target sites in the 3'-untranslated region (3'-UTR) of key Treg cell-immunomodulatory genes with a special focus on interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Having identified potential miR target sites in the 3'-UTR of IL-10 (miR-27b-3p and miR-340-5p) and TGF-ß (miR-330-3p), we showed through transfection and transduction assays that the overexpression of two underexpressed miRNAs, miR-27b-3p and miR-340-5p, downregulated IL-10 expression upon targeting its 3'-UTR. Similarly, overexpression of miR-330-3p negatively regulated TGF-ß expression. These results highlighted an important impact of the CD8 + Treg mirnome on the expression of genes with significant implication on immunosuppression. These observations could help in better understanding the mechanism(s) orchestrating Treg immunosuppressive function toward unraveling new targets for treating autoimmune pathologies and cancer.

Immunomodulatory effects of foreskin mesenchymal stromal cells on natural killer cells.

Foreskin-mesenchymal stromal cells (FSK-MSCs) are immune-privileged thus making them valuable immunotherapeutic cell product. Characterization of the relationship between FSK-MSCs and natural killer (NK) cells is essential to improve cell-based therapy. In the present study, we studied for the first time FSK-MSCs-NK interaction and showed that the result of such cross talk was robustly dependent on the type of cytokines (IL-2, IL-12, IL-15, and IL-21) employed to activate NK cells. Distinctly activated-NK cells showed uneven cytotoxicity against FSK-MSCs, triggering their death in fine. The expression of different cell-surface ligands (CD112, CD155, ULPB-3) and receptors (LAIR, KIRs) ensuring such interaction was altered following co-culture of both populations. Despite their partial negative effect on NK cell proliferation, FSK-MSCs boosted the capacity of activated NK-cells to secrete IFN-γ and TNF-α. Moreover, FSK-MSCs enhanced degranulation of NK cells, reinforced secretion of perforin and granzymes, while only modestly increased ROS production. On the other hand, FSK-MSCs-mediated expression of C1 and B9 serpins was significantly lowered in the presence of activated NK cells. Altogether, our results highlight major immunological changes following FSK-MSCs-NK interaction. Understanding these outcomes will therefore enhance the value of the therapeutic strategy.

Characterization and assessment of potential microRNAs involved in phosphate-induced aortic calcification.

Medial artery calcification, a hallmark of type 2 diabetes mellitus and chronic kidney disease (CKD), is known as an independent risk factor for cardiovascular mortality and morbidity. Hyperphosphatemia associated with CKD is a strong stimulator of vascular calcification but the molecular mechanisms regulating this process remain not fully understood. We showed that calcification was induced after exposing Sprague-Dawley rat aortic explants to high inorganic phosphate level (Pi , 6 mM) as examined by Alizarin red and Von Kossa staining. This calcification was associated with high Tissue-Nonspecific Alkaline Phosphatase (TNAP) activity, vascular smooth muscle cells de-differentiation, manifested by downregulation of smooth muscle 22 alpha (SM22α) protein expression which was assessed by immunoblot analysis, immunofluorescence, and trans-differentiation into osteo-chondrocyte-like cells revealed by upregulation of Runt related transcription factor 2 (Runx2), TNAP, osteocalcin, and osteopontin mRNA levels which were determined by quantitative real-time PCR. To unravel the possible mechanism(s) involved in this process, microRNA (miR) expression profile, which was assessed using TLDA technique and thereafter confirmed by individual qRT-PCR, revealed differential expression 10 miRs, five at day 3 and 5 at day 6 post Pi treatment versus control untreated aortas. At day 3, miR-200c, -155, 322 were upregulated and miR-708 and 331 were downregulated. After 6 days of treatment, miR-328, -546, -301a were upregulated while miR-409 and miR-542 were downregulated. Our results indicate that high Pi levels trigger aortic calcification and modulation of certain miRs. These observations suggest that mechanisms regulating aortic calcification might involve miRs, which warrant further investigations in future studies.

First-line treatment of Helicobacter pylori in Lebanon: Comparison of bismuth-containing quadruple therapy versus 14-days sequential therapy.

Helicobacter Pylori (H. Pylori) is the most common cause of peptic ulcer disease (PUD) and represents a strong risk factor for gastric cancer. Treatment of H. Pylori is, therefore, a persistent need to avoid serious medical complications. Resistance to antibiotics remains to be the major challenge for H. Pylori eradication. In this study, we determined the prevalence of H. pylori infection and evaluated H. pylori eradication efficacy of bismuth-containing quadruple therapy (Pylera) versus 14-days sequential therapy in treatment naïve-Lebanese patients. 1030 patients, showing symptoms of peptic ulcer (PU) and gastritis, underwent 14C-Urea Breath Test and esophagogastroduodenoscopy to examine H. Pylori infection and gastrointestinal disorders. Among the H. Pylori-positive patients 60 individuals were randomly selected, separated into two groups (each consisting of 30 patients) and treated with either bismuth-containing quadruple therapy or 14-days sequential therapy. We show that of the 1050 patients tested: 46.2% were H. pylori-positive, 55% had gastritis, 46.2% had both gastritis and H. pylori infection, 8.8% had gastritis but no H. pylori infection, 44.9% had neither gastritis nor H. pylori infection. Following the 14-days sequential therapy, the eradication rate was significantly higher than that obtained upon using bismuth-containing quadruple therapy [80% (24/30) versus 50% (15/30), χ2 = 5.93, P = 0.015]. In conclusion, we determined H. pylori and gastritis prevalence among Lebanese PU-patients and showed that 14-days sequential therapy is more efficient than bismuth-containing quadruple therapy in terms of H. Pylori-eradication.

Immunological impact of Wharton's Jelly mesenchymal stromal cells and natural killer cell co-culture.

Due to their easier isolation, multilineage potential, and immunomodulatory capacity, Wharton's Jelly-derived mesenchymal stromal cells (WJ-MSCs) exhibit promising efficacy in the field of regenerative medicine and immunotherapy. Characterization of WJ-MSCs-natural killer (NK) cells crosstalk is required for ameliorating the medicinal value of WJ-MSCs. Here, we revealed that the outcome of WJ-MSCs-NK cells crosstalk varied according to the type of cytokines (IL-2, IL-12, IL-15 and IL-21) utilized to activate NK cells. Differently activated NK cells exerted distinct cytotoxicities against WJ-MSCs causing their probable death. Cell surface ligands (CD112, CD155, ULPB-3) and receptors (LAIR, CD226, CD314, CD335, CD336 and CD337) governing the interaction between NK cells and their targets, exhibited altered expression profiles following the co-culture with WJ-MSCs. Although partly inhibited NK cell proliferation, WJ-MSCs enhanced activated NK-cell-mediated secretion of IFN-γ and TNF-α. Moreover, WJ-MSCs reinforced NK cells' degranulation as well as secretion of perforin and granzymes. On the other hand, WJ-MSCs displayed only slight increase in ROS generation but significant decrease in A1 and C1 serpins expression following co-culture with activated NK cells. Altogether, our results highlight that WJ-MSCs-NK cells interaction may affect both cell type features and, therefore, their therapeutic properties.

The micronome of mesenchymal stromal cells is partially responsive to inflammation.

Mesenchymal stromal cells (MSCs) display a special immunological profile that allows their potential use as immunotherapeutic cells. Nowadays, foreskin (FSK) represents a valuable reservoir of MSCs with International Society for Cellular Therapy (ISCT) compliant criteria and relevant functional properties. However, their mode of action is poorly understood and needs to be more elucidated to optimize their therapeutic use. Because microRNAs (miRNAs) act as key regulators in a wide variety of biological processes, we decided to establish the micronome of FSK-MSCs, the influence of inflammation and the predicted target pathways. Here, we provide the full list of unchanged and additional four differentially expressed miRNAs, miR-199b, -296-3p and -589-5p being downregulated whilst miR-146-3p being upregulated, in MSCs following their exposure to a cocktail of proinflammatory cytokines. MicroRNA target prediction in addition to Pathway enrichment analysis performed using miRNet, showed that miR-296-3p is linked to antigen processing and presentation pathway. Collectively, our data indicate that the micronome of FSK-MSCs is partially responsive to inflammation. Differentially expressed miRNAs are subsequently modulated by inflammation and seem to be involved in regulating the immunological fate of FSK-MSCs. These miRNAs deserve more attention in order to optimize MSC-based therapy and achieve the appropriate therapeutic effect.

Yeast nitrogen catabolite repression is sustained by signals distinct from glutamine and glutamate reservoirs.

Nitrogen catabolite repression (NCR) is a wide transcriptional regulation program enabling baker's yeast to downregulate genes involved in the utilization of poor nitrogen sources when preferred ones are available. Nowadays, glutamine and glutamate, the major nitrogen donors for biosyntheses, are assumed to be key metabolic signals regulating NCR. NCR is controlled by the conserved TORC1 complex, which integrates nitrogen signals among others to regulate cell growth. However, accumulating evidence indicate that the TORC1-mediated control of NCR is only partial, arguing for the existence of supplementary regulatory processes to be discovered. In this work, we developed a genetic screen to search for new players involved in NCR signaling. Our data reveal that the NADP-glutamate dehydrogenase activity of Gdh1 negatively regulates NCR-sensitive gene transcription. By determining the total, cytoplasmic and vacuolar pools of amino acids, we show that there is no positive correlation between glutamine/glutamate reservoirs and the extent of NCR. While our data indicate that glutamine could serve as initial trigger of NCR, they show that it is not a sufficient signal to sustain repression and point to the existence of yet unknown signals. Providing additional evidence uncoupling TORC1 activity and NCR, our work revisits the dogmas underlying NCR regulation.

Study of the microRNA expression profile of foreskin derived mesenchymal stromal cells following inflammation priming.

BACKGROUND: Due to their self-renewal capacity, multi-lineage potential, and immunomodulatory properties, mesenchymal stromal cells (MSCs) are an attractive tool for different therapeutic strategies. Foreskin (FSK), considered as a biological waste material, has already been shown to be a valuable source of MSCs. Besides their typical fibroblast like morphology and International Society for cellular Therapy compliant phenotype, foreskin-MSCs (FSK-MSCs) are clonogenic, and highly proliferative cells with multi-lineage and strong immunomodulatory capacities. Of importance, FSK-MSCs properly adjust their fate following exposure to inflammatory signals. Being potent regulators of gene expression, miRNAs are involved in modulating nearly all cellular processes and in orchestrating the roles of different immune cells. In this study, we characterized the miRNome of FSK-MSCs by determining the expression profile of 380 different miRNAs in inflammation primed vs. control non-primed cells. METHODS: TaqMan low density array (TLDA) was performed to identify dysregulated miRNAs after exposing FSK-MSCs to inflammatory signals. Quantitative real-time RT-PCR was carried out to validate the observations. DIANA-miRPath analysis web server was used to identify potential pathways that could be targeted by the dysregulated miRNAs. RESULTS: Sixteen miRNAs were differentially expressed in inflammation-primed vs. non-primed FSK-MSCs. The expression level of miR-27a, -145, -149, -194, -199a, -221, -328, -345, -423-5p, -485-3p, -485-5p, -615-5p and -758 was downregulated whilst that of miR-155, -363 and -886-3p was upregulated. Target pathway prediction of those differentially expressed miRNAs identified different inflammation linked pathways. CONCLUSIONS: After determining their miRNome, we identified a striking effect of inflammatory signals on the miRNAs' expression levels in FSK-MSCs. Our results highlight a potential role of miRNAs in modulating the transcription programs of FSK-MSCs in response to inflammatory signals. Further, we propose that specific miRNAs could serve as interesting targets to manipulate some functions of FSK-MSCs, thus ameliorating their therapeutic potential.

Characterization of circulating leptin-receptor levels following acute sleep restriction: A pilot study on healthy adult females.

BACKGROUND: Insufficient sleep adversely affects energy homeostasis by decreasing leptin levels. The underlying physiological mechanisms; however, remain unclear. Circulating leptin is well described to be regulated by its soluble receptor (sOB-R). Intriguingly, the impact of short sleep duration on sOB-R levels has never been characterized. AIM: In this study, we investigated, for the first time, the variation of sOB-R levels and its temporal relationship with circulating leptin upon acute sleep restriction. METHODS: Five adult females were maintained on an 8-hour sleep schedule (bedtime at 00:00) for 1 week before restricting their sleep to 4.5 h (bedtime at 03:30) on 2 consecutive nights. Balanced meals were scheduled to specific hours and sleep was objectively measured. Four-hour blood samples were regularly collected during waking hours between 08:00 and 00:00. RESULTS: Sleep restriction resulted in lower leptin (20.9 ± 1.7 vs 25.7 ± 1.7 ng/ml) and higher sOB-R concentrations (24.4 ± 1.2 vs 19.8 ± 1.6 ng/ml). Neither the discordant temporal relationship nor the pattern of leptin and sOB-R were altered in response to sleep restriction. CONCLUSION: Our results suggest that sleep restriction may modulate circulating leptin levels and possibly metabolism via upregulating its soluble receptor. This observation may have valuable therapeutic implications when considering sOB-R as a potential target during the management of metabolic disturbances.

Proteomic Methods to Study Autophagy in Skin Exposed to Pollutants.

Autophagy refers to the natural cellular process by which cells degrade and recycle their own damaged or dysfunctional cellular components. It is an essential mechanism for maintaining cellular homeostasis removing toxic substances and providing energy during times of stress or nutrient deprivation. When autophagy is dysregulated or impaired, it can have detrimental effects on cell function and overall health. Studying autophagy in skin exposed to pollutants can provide valuable insights into the cellular mechanisms underlying pollutant-induced skin damage. Proteomic methods, which involve the large-scale analysis of proteins, can be employed to investigate the changes in protein expression associated with biological processes including autophagy. Here, we thus describe a method where LC-MS/MS was applied to identify the deregulated proteins in pollutant exposed-skin. Using bioinformatics and statistical analysis, we extracted the qualitative and quantitative information for proteins involved in autophagy. These deregulated proteins were then validated by immunohistochemistry (IHC). These methods help to understand how the pollutants affect the autophagy process.

Lipoic acid alters the microRNA signature in breast cancer cells.

BACKGROUND: Breast cancer, the deadliest disease affecting women globally, exhibits heterogeneity with distinct molecular subtypes. Despite advances in cancer therapy, the persistence of high mortality rates due to chemotherapy resistance remains a major challenge. Lipoic acid (LA), a natural antioxidant, has proven potent anticancer properties. Yet, the impact of LA on microRNA (miRNA) expression profile in breast cancer remains unexplored. AIM: The aim of this study was to unravel the effect of LA on miRNA expression profiles in different breast cancer cell lines. METHODS: The MiRCURY LNA miRNA miRNome qPCR Panel was used to compare the miRNA signature in MDA-MB-231 and MCF-7 cells treated or not with LA. RESULTS: We identified six upregulated and six downregulated miRNAs in LA-treated MDA-MB-231 cells and 14 upregulated and four downregulated miRNAs in LA-treated MCF-7 cells compared to control cells. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that the deregulated miRNAs could alter different signaling cascades including FoxO, P53 and Hippo pathways. CONCLUSION: The outcome of this study provides further insights into the molecular mechanisms underlying the therapeutic benefit of LA. This in turn could assist the amelioration of LA-based anticancer therapies.

MicroRNA-138 inhibits hypoxia-inducible factor 1α expression in breast cancer cells.

BACKGROUND: Hypoxia, a critical feature during cancer development, leads to the stabilization and activation of the hypoxia-inducible factor 1-alpha (HIF-1α) to drive the expression of many target genes which in turn can promote many aspects of breast cancer biology, mainly metastasis and resistance to therapy. MicroRNAs are known to modulate the expression of many genes involved in breast cancer tumorigenesis. In this study, we examined the regulatory effect of miRNAs on HIF1α expression. METHODS: MCF-7 and MDA-MB-231 were cultivated under normoxia or hypoxia conditions. TaqMan-Low Density Array (TLDA) was used to characterize the miRNA signatures. Wild-Type (WT) or mutated fragments of HIF-1α 3'UTR containing the miR-138 potential target site were cloned downstream of the Renilla luciferase gene in the psiCHECK-1 plasmid. Luciferase assays were then carried out. A lentiviral vector containing copGFP as a reporter gene was prepared and transduced into MCF-7 and MDA-MB-231 cells to assess the effect of identified deregulated miRNAs on HIF-1α expression. RESULTS: Under hypoxic conditions, MCF-7 cells showed deregulated expression for 12 miRNAs. In the case of MDA-MB-231 cells, 16 miRNAs were deregulated in response to hypoxia. Interestingly, miR-138 that was downregulated in both MCF-7 and MDA-MB-231 cells cultivated under hypoxic conditions appeared to have a binding site in 3'UTR of HIF-1α. Moreover, our results indicated that miR-138 could down regulate HIF-1α expression, upon binding directly to its 3'UTR. CONCLUSIONS: Interestingly, our data highlights miR-138 as a potential therapeutic target to reduce HIF-1α expression and subsequently restrain breast cancer invasion and metastasis.

MicroRNA profile of circulating CD4-positive regulatory T cells in human adults and impact of differentially expressed microRNAs on expression of two genes essential to their function.

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.