3,5-dicaffeoylquinic acid lowers 3T3-L1 mitotic clonal expansion and adipocyte differentiation by enhancing heme oxygenase-1 expression

Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment

Upregulation of miR-34a-5p, miR-20a-3p and miR-29a-3p by onconase in A375 melanoma cells correlates with the downregulation of specific onco-proteins

Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity

Regulation of microRNAs in satellite cell renewal, muscle function, sarcopenia and the role of exercise

Sarcopenia refers to a condition of progressive loss of skeletal muscle mass and function associated with a higher risk of falls and fractures in older adults. Musculoskeletal aging leads to reduced muscle mass and strength, affecting the quality of life in elderly people. In recent years, several studies contributed to improve the knowledge of the pathophysiological alterations that lead to skeletal muscle dysfunction; however, the molecular mechanisms underlying sarcopenia are still not fully understood. Muscle development and homeostasis require a fine gene expression modulation by mechanisms in which microRNAs (miRNAs) play a crucial role. miRNAs modulate key steps of skeletal myogenesis including satellite cells renewal, skeletal muscle plasticity, and regeneration. Here, we provide an overview of the general aspects of muscle regeneration and miRNAs role in skeletal mass homeostasis and plasticity with a special interest in their expression in sarcopenia and skeletal muscle adaptation to exercise in the elderly

Human melanoma cells differentially express RNASEL/RNase-L and miR-146a-5p under sex hormonal stimulation

Polymorphisms in the ribonuclease L (RNASEL) coding gene and hsa-miR-146a-5p (miR-146a) have been associated with melanoma in a sex-specific manner. We hypothesized that RNASEL and miR-146a expression could be influenced by sex hormones playing a role in the female advantages observed in melanoma incidence and survival. Thus, we explored the effects of testosterone and 17β-estradiol on RNASEL and miR-146a expression in LM-20 and A375 melanoma cell lines. Direct targeting of miR-146a to the 3' untranslated region (3'UTR) of RNASEL was examined using a luciferase reporter system. Our results indicate that RNASEL is a direct target of miR-146a in both melanoma cell lines. Trough qPCR and western blot analyses, we explored the effect of miR-146a mimic transfection in the presence of each hormone either on RNASEL mRNA level or on protein expression of RNase-L, the enzyme codified by RNASEL gene. In the presence of testosterone or 17β-estradiol, miR-146a overexpression did not influence RNASEL transcript level in LM-20 cell line, but it slightly induced RNASEL mRNA level in A375 cells. Remarkably, miR-146a overexpression was able to repress the protein level of RNase-L in both LM-20 and A375 cells in the presence of each hormone, as well as to elicit high expression levels of the activated form of the extracellular signal-regulated kinases (ERK)1/2, hence confirming the pro-tumorigenic role of miR-146a overexpression in melanoma. Thereafter, we assessed if the administration of each hormone could affect the endogenous expression of RNASEL and miR-146a genes in LM-20 and A375 cell lines. Testosterone exerted no significant effect on RNASEL gene expression in both cell lines, while 17β-estradiol enhanced RNASEL transcript level at least in LM-20 melanoma cells. Conversely, miR-146a transcript augmented only in the presence of testosterone in either melanoma cell line. Importantly, each hormone acted quite the opposite regarding the RNase-L protein expression, i.e., testosterone significantly decreased RNase-L expression, whereas 17β-estradiol increased it. Overall, the data show that, in melanoma cells treated with 17β-estradiol, RNase-L expression increased likely by transcriptional induction of its gene. Testosterone, instead, decreased RNase-L expression in melanoma cell lines with a post-transcriptional mechanism in which miR-146a could play a role. In conclusion, the pro-tumor activity of androgen hormone in melanoma cells could be exacerbated by both miR-146a increase and RNase-L downregulation. These events may contribute to the worse outcome in male melanoma patients

Expression of FBXW11 in normal and disease-associated osteogenic cells

The ubiquitin-proteasome system (UPS) plays an important role in maintaining cellular homeostasis by degrading a multitude of key regulatory proteins. FBXW11, also known as b-TrCP2, belongs to the F-box family, which targets the proteins to be degraded by UPS. Transcription factors or proteins associated with cell cycle can be modulated by FBXW11, which may stimulate or inhibit cellular proliferation. Although FBXW11 has been investigated in embryogenesis and cancer, its expression has not been evaluated in osteogenic cells. With the aim to explore FBXW11gene expression modulation in the osteogenic lineage we performed molecular investigations in mesenchymal stem cells (MSCs) and osteogenic cells in normal and pathological conditions. In vitro experiments as well as ex vivo investigations have been performed. In particular, we explored the FBXW11 expression in normal osteogenic cells as well as in cells of cleidocranial dysplasia (CCD) patients or osteosarcoma cells. Our data showed that FBXW11 expression is modulated during osteogenesis and overexpressed in circulating MSCs and in osteogenically stimulated cells of CCD patients. In addition, FBXW11 is post-transcriptionally regulated in osteosarcoma cells leading to increased levels of beta-catenin. In conclusion, our findings show the modulation of FBXW11 in osteogenic lineage and its dysregulation in impaired osteogenic cells

miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators

BACKGROUND: Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance.METHODS: The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy.RESULTS: miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor.CONCLUSIONS: Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Video Abstract

Biopsychosocial model of resilience in young adults with multiple sclerosis (BPS-ARMS): an observational study protocol exploring psychological reactions early after diagnosis

INTRODUCTION: Multiple sclerosis (MS), the most common neurological disease causing disability in young adults, is widely recognised as a major stress factor. Studies have shown that the first years after the diagnosis are distressing in terms of adjustment to the disease and that MS negatively affects patients' psychological well-being, quality of life (QoL) and social functioning. However, the links between disease-specific variables at diagnosis, resilience and psychological adjustment of patients with MS remain largely unexplored, especially in adolescents and young adults. This observational study aims to fill the gap of knowledge on biopsychosocial characteristics and resilience of young adults with MS to evaluate the relationship among these variables and to develop a biopsychosocial model of resilience. METHODS AND ANALYSIS: Biological and clinical characteristics of young adults newly diagnosed with MS will be investigated by collecting clinical information, performing neurological examinations, MRI and analysing cerebrospinal fluid and blood biomarkers (eg, measures of inflammation), body composition, gut microbiota and movement/perceptual markers. Psychosocial characteristics (eg, psychological distress, coping strategies), QoL, psychological well-being and resilience will be assessed by self-report questionnaires. Comparative statistics (ie, analysis of variance or unpaired samples t-test, correlation and regression analyses) will be applied to evaluate the relationship among biological, psychological and social factors. The results are expected to allow a comprehensive understanding of the determinants of resilience in young patients with MS and to inform resilience interventions, tailored to young patients' specific needs, aiming to reduce the risk of maladaptive reactions to the disease and to improve psychological well-being and QoL. ETHICS AND DISSEMINATION: The study has been approved by the Verona University Hospital Ethics Committee (approval number: 2029CESC). The findings will be disseminated through scientific publications in peer-reviewed journals, conference presentations, social media and specific websites. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03825055)

Rehabilitation and biomarkers of stroke recovery: study protocol for a randomized controlled trial

Background: Stroke is a leading cause of disability. Nonetheless, the care pathway for stroke rehabilitation takes partially into account the needs of chronic patients. This is due in part to the lack of evidence about the mechanisms of recovery after stroke, together with the poor knowledge of related and influencing factors. Here we report on the study protocol \u201cRehabilitation and Biomarkers of Stroke Recovery,\u201d which consists of 7 work-packages and mainly aim to investigate the effects of long-term neurorehabilitation on stroke patients and to define a related profile of (clinical-biological, imaging, neurophysiological, and genetic-molecular) biomarkers of long-term recovery after stroke. The work-package 1 will represent the main part of this protocol and aims to compare the long-term effects of intensive self-rehabilitation vs. usual (rehabilitation) care for stroke. Methods: We planned to include a total of 134 adult subacute stroke patients (no more than 3 months since onset) suffering from multidomain disability as a consequence of first-ever unilateral ischemic stroke. Eligible participants will be randomly assigned to one of the following groups: intensive self-rehabilitation (based on the principles of \u201cGuided Self-Rehabilitation Contract\u201d) vs. usual care (routine practice). Treatment will last 1 year, and patients will be evaluated every 3 months according to their clinical presentation. The following outcomes will be considered in the main work-package: Fugl-Meyer assessment, Cognitive Oxford Screen Barthel Index, structural and functional neuroimaging, cortical excitability, and motor and somatosensory evoked potentials. Discussion: This trial will deal with the effects of an intensive self-management rehabilitation protocol and a related set of biomarkers. It will also investigate the role of training intensity on long-term recovery after stroke. In addition, it will define a set of biomarkers related to post-stroke recovery and neurorehabilitation outcome in order to detect patients with greater potential and define long-term individualized rehabilitation programs. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT04323501

Polymorphism analysis in cox-2 gene regulatory regions in non-melanoma skin cancer after transplantation

Una sovra-espressione di ciclossigenasi 2, \ue8 stata osservata nei cheratinociti cutanei dopo danno da raggi ultravioletti, nel carcinoma squamocellulare e negli stadi precoci della cancerogenesi di vari tessuti. Questa alterazione dell\u2019espressione di COX2 potrebbe essere dovuta a cambiamenti funzionali di elementi regolatori nel promotore o nella regione del 3\u2019 UTR (UnTraslated Region) del gene. Due polimorfismi comuni (-1195A>G e -765G>C) nella regione del promotore e un polimorfismo comune nella regione non tradotta del 3\u2019 (3\u2019UTR) del gene sono stati descritti e riportati associati a vari tipi di tumori. In questo lavoro \ue8 stato studiato COX2 (ciclossigenasi 2) come gene candidato ad avere un ruolo nella predisposizione al tumore cutaneo non melanoma (NMSC) dopo trapianto d\u2019organo. Sono stati reclutati 107 individui riceventi trapianto d\u2019organo (OTR) che presentavano NMSC (casi) e 133 individui OTR che non presentavano tale tumore (controlli) e sono stati genotipizzati per tre polimorfismi nelle regioni regolatrici del gene (-1195A>G, \u2013765G>C e 8473T>C). L\u2019analisi ha dimostrato che l\u2019allele \u2013765C rappresenta un fattore protettivo verso lo sviluppo di carcinoma basocellulare (BCC) in individui che sono stati sottoposti a trapianto prima dei 50 anni (CC+CG vs GG test di Fisher p=0.003). La ricerca di nuovi polimorfismi nelle regioni regolatrici al 5\u2019 e al 3\u2019 UTR del gene ha identificato una nuova sostituzione nucleotidica (-62C>G) con una frequenza dell\u2019allele minore \u201362G di 0.019 e un polimorfismo noto nella regione 3\u2019 UTR (8293G>C) con frequenza 0.02. Studi funzionali non hanno dimostrato alcun effetto di quest\u2019ultimo polimorfismo, nel del polimorfismo comune (8473T>C) sulla regolazione post trascrizionale del gene. L\u2019analisi degli aplotipi ha mostrato che l\u2019aplotipo contenente tutti gli alleli pi\uf9 frequenti rappresenta un fattore protettivo verso il carcinoma squamocellulare (SCC) nei pazienti che sono stati sottoposti a trapianto dopo i 50 anni [p=0.009; OR=0.37 (0.18-0.79)] e che la variante \u20131195G pu\uf2 rappresentare un fattore di rischio in questo sottogruppo di pazienti [p=0.01; OR=4.77 (1.47-16.41)]. La variante 8473T>C non ha mostrato associazione con il rischio di NMSC dopo trapiant. In conclusione i polimorfismi \u2013765G>C e \u20131195A>G appaiono associati al rischio di NMSC, sebbene in differenti modi in SCC e BCC e in diversi gruppi di et\ue0, indicando che fattori di rischio genetico possono giocare diversi ruoli nell\u2019insorgenza dei due fenotipi.Over expression of cyclooxygenase-2 (COX-2), resulting in excessive prostaglandin production, has been observed in human epidermal keratinocytes after ultraviolet B (UVB) injury. The dysregulation of COX-2 expression can in part be due to functional changes affecting regulatory elements of the gene. Two common polymorphisms (-1195A>G and -765 G>C) in the promoter region of the COX-2 gene, and one common polymorphism in 3\u2019UTR (8473T>C) have been described, and associated to various malignancies. This study was designed to determine if common known polymorphisms in the regulatory region of the COX-2 gene can be associated to non melanoma skin cancer (NMSC) predisposition after transplantation, and to identify possible new genetic polymorphisms in the proximal regulatory regions of the gene associated with disease. Frequency of the three known polymorphisms was determined in two hundred fourty North Italian transplant recipient patients (107 cases and 133 controls) by PCR-RFLP, and the proximal regulatory regions of the gene were screened by heteroduplex analysis to identify new variants. Allele \u2013765C represented a protective factor in basal cell cancer (BCC) patients undergoing transplantation before 50 years of age (CC+CG vs GG Fisher exact test P=0.003). One novel polymorphism, -62C>G, was detected in the 5\u2019flanking region; allele frequency was 0.019. Screening of the 3\u2019 UTR region revealed the presence of one known polymorphism -8293G>, with a frequency of 0.02. Functional studies showed no effect of this variant, or of the common polymorphism 8473T>C, upon post-transcriptional regulation of gene expression. Haplotype analysis showed that the haplotype containing all major alleles represents a protective factor in squamous cell cancer (SCC) patients undergoing transplantation after 50 years of age [P=0.008; OR= 0.37 (0.18-0.79)] and that haplotype containing variant -1195G may represent a risk factor in this subgroup of patients [P=0.01; OR=3.92 (1.22- 12.46)]. Haplotype analysis in basal cell cancer (BCC) patients revealed that the haplotype containing variant \u2013765C might be a protective factor in patients undergoing transplantation before 50 years of age (P=0.003). Conclusion: COX-2 common variants \u20131195A>G and \u2013765G>C appear to be associated to SCC or BCC, respectively, indicating that the gene may be a risk factor for the development of NMSC after transplantation