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Background DNA-Damaged Binding protein 2 (DDB2) is a protein involved in the early step of Nucleotide Excision Repair. Recently, it has been reported that DDB2 is involved in epithelial-to-mesenchymal transition (EMT), key process in tumour invasiveness and metastasis formation. However, its role is not completely known. Methods Boyden chamber and cell adhesion assays, and ICELLigence analysis were performed to detect HEK293 adhesion and invasion. Western blotting and gelatine zymography techniques were employed to assess the EMT protein levels and MMP enzymatic activity. Immunofluorescence analysis and pull-down assays facilitated the detection of NF-kB sub-cellular localization and interaction. Results We have previously demonstrated that the loss of DDB2-PCNA binding favours genome instability, and increases cell proliferation and motility. Here, we have investigated the phenotypic and molecular EMT-like changes after UV DNA damage, in HEK293 clones stably expressing DDB2 Wt protein or a mutant form unable to interact with PCNA (DDB2 PCNA− ), as well as in HeLa cells transiently expressing the same DDB2 constructs. Cells expressing DDB2 PCNA− showed morphological modifications along with a reduced expression of E-cadherin, an increased activity of MMP-9 and an improved ability to migrate, in concomitance with a significant upregulation of EMT-associated Transcription Factors (TFs), whose expression has been reported to favour tumour invasion. We observed a higher expression of c-Myc oncogene, NF-kB, both regulating cell proliferation and metastatic process, as well as ZEB1, a TF significantly associated with tumorigenic potential and cell migratory ability. Interestingly, a novel interaction of DDB2 with NF-kB was detected and found to be increased in cells expressing the DDB2 PCNA− , suggesting a direct modulation of NF-kB by DDB2. Conclusion These results highlight the role of DDB2-PCNA interaction in counteracting EMT since DDB2 PCNA− protein induces in HEK293 transformed cells a gain of function contributing to the acquisition of a more aggressive phenotype.

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

Background The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2 PCNA- ), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro. Methods In this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2 Wt or DDB2 PCNA- , were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy. Results The results have shown that DDB2 Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2 PCNA- , as also confirmed by the different extent of co-localization of DDB2 Wt and some NER proteins (such as XPG), vs the DDB2 mutant form. Conclusion The HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.

The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A is known to induce cell cycle arrest by inhibiting CDK activity and by interfering with DNA replication through binding to proliferating cell nuclear antigen. Although the molecular mechanisms have been elucidated, the temporal dynamics, as well as the intracellular sites of the activity of p21 bound to cyclin/CDK complexes during cell cycle arrest, have not been fully investigated. In this study we have induced the expression of p21CDKN1A fused to green fluorescent protein (GFP) in HeLa cells, in order to visualize the intracellular localization of the inhibitor during the cell cycle arrest. We show that p21-GFP is preferentially expressed in association with cyclin E in cells arrested in G1 phase, and with cyclin A more than with cyclin B1 in cells arrested in the G2/M compartment. In addition, we show for the first time that p21-GFP colocalizes with cyclin E in the nucleolus of HeLa cells during the G1 phase arrest.

Background Osteoarthritis (OA) is the most prevalent joint disease, and to date, no options for effective tissue repair and restoration are available. With the aim of developing new therapies, the impact of mesenchymal stem cells (MSCs) has been explored, and the efficacy of MSCs started to be deciphered. A strong paracrine capacity relying on both secreted and vesicle-embedded (EVs) protein or nucleic acid-based factors has been proposed as the principal mechanism that contributes to tissue repair. This work investigated the mechanism of internalization of extracellular vesicles (EVs) released by adipose-derived MSCs (ASCs) and the role of shuttled miRNAs in the restoration of homeostasis in an in vitro model of human fibroblast-like synoviocytes (FLSs) from OA patients. Methods ASC-EVs were isolated by differential centrifugation and validated by flow cytometry and nanoparticle tracking analysis. ASC-EVs with increased hyaluronan (HA) receptor CD44 levels were obtained culturing ASCs on HA-coated plastic surfaces. OA FLSs with intact or digested HA matrix were co-cultured with fluorescent ASC-EVs, and incorporation scored by flow cytometry and ELISA. ASC-EV complete miRNome was deciphered by high-throughput screening. In inflamed OA FLSs, genes and pathways potentially regulated by ASC-EV miRNA were predicted by bioinformatics. OA FLSs stimulated with IL-1β at physiological levels (25 pg/mL) were treated with ASC-EVs, and expression of inflammation and OA-related genes was measured by qRT-PCR over a 10-day time frame with modulated candidates verified by ELISA. Results The data showed that HA is involved in ASC-EV internalization in FLSs. Indeed, both removal of HA matrix presence on FLSs and modulation of CD44 levels on EVs affected their recruitment. Bioinformatics analysis of EV-embedded miRNAs showed their ability to potentially regulate the main pathways strictly associated with synovial inflammation in OA. In this frame, ASC-EVs reduced the expression of pro-inflammatory cytokines and chemokines in a chronic model of FLS inflammation. Conclusions Given their ability to affect FLS behavior in a model of chronic inflammation through direct interaction with HA matrix and miRNA release, ASC-EVs confirm their role as a novel therapeutic option for osteoarthritic joints.

Background Mesenchymal stromal cell (MSC)-enriched products showed positive clinical outcomes in regenerative medicine, where tissue restoration and inflammation control are needed. GMP-expanded MSCs displayed an even higher potential due to exclusive secretion of therapeutic factors, both free and conveyed within extracellular vesicles (EVs), collectively termed secretome. Moreover, priming with biochemical cues may influence the portfolio and biological activities of MSC-derived factors. For these reasons, the use of naive or primed secretome gained attention as a cell-free therapeutic option. Albeit, at present, a homogenous and comprehensive secretome fingerprint is still missing. Therefore, the aim of this work was to deeply characterize adipose-derived MSC (ASC)-secreted factors and EV-miRNAs, and their modulation after IFNγ preconditioning. The crucial influence of the target pathology or cell type was also scored in osteoarthritis to evaluate disease-driven potency. Methods ASCs were isolated from four donors and cultured with and without IFNγ. Two-hundred secreted factors were assayed by ELISA. ASC-EVs were isolated by ultracentrifugation and validated by flow cytometry, transmission electron microscopy, and nanoparticle tracking analysis. miRNome was deciphered by high-throughput screening. Bioinformatics was used to predict the modulatory effect of secreted molecules on pathologic cartilage and synovial macrophages based on public datasets. Models of inflammation for both macrophages and chondrocytes were used to test by flow cytometry the secretome anti-inflammatory potency. Results Data showed that more than 60 cytokines/chemokines could be identified at varying levels of intensity in all samples. The vast majority of factors are involved in extracellular matrix remodeling, and chemotaxis or motility of inflammatory cells. IFNγ is able to further increase the capacity of the secretome to stimulate cell migration signals. Moreover, more than 240 miRNAs were found in ASC-EVs. Sixty miRNAs accounted for > 95% of the genetic message that resulted to be chondro-protective and M2 macrophage polarizing. Inflammation tipped the balance towards a more pronounced tissue regenerative and anti-inflammatory phenotype. In silico data were confirmed on inflamed macrophages and chondrocytes, with secretome being able to increase M2 phenotype marker CD163 and reduce the chondrocyte inflammation marker VCAM1, respectively. IFNγ priming further enhanced secretome anti-inflammatory potency. Conclusions Given the portfolio of soluble factors and EV-miRNAs, ASC secretome showed a marked capacity to stimulate cell motility and modulate inflammatory and degenerative processes. Preconditioning is able to increase this ability, suggesting inflammatory priming as an effective strategy to obtain a more potent clinical product which use should always be driven by the molecular mark of the target pathology.

Mesenchymal stem cells (MSCs) derived from adipose tissue and used either as expanded cells or minimally manipulated cell preparations showed positive clinical outcomes in regenerative medicine approaches based on tissue restoration and inflammation control, like in osteoarthritis (OA). Recently, MSCs’ healing capacity has been ascribed to the large array of soluble factors, including soluble cytokines/chemokines and miRNAs conveyed within extracellular vesicles (EVs). Therefore, in this study, 200 secreted cytokines, chemokines and growth factors via ELISA, together with EV-embedded miRNAs via high-throughput techniques, were scored in adipose-derived MSCs (ASCs) cultivated under inflammatory conditions, mimicking OA synovial fluid. Both factors (through most abundantly expressed TIMP1, TIMP2, PLG and CTSS) and miRNAs (miR-24-3p, miR-222-3p and miR-193b-3p) suggested a strong capacity for ASCs to reduce matrix degradation activities, as those activated in OA cartilage, and switch synovial macrophages, often characterized by an M1 inflammatory polarization, towards an M2 phenotype. Moreover, the crucial importance of selecting the target tissue is discussed, showing how a focused search may greatly improve potency prediction and explain clinical outcomes. In conclusion, herein presented data shed light about the way ASCs regulate cell homeostasis and regenerative pathways in an OA-resembling environment, therefore suggesting a rationale for the use of MSC-enriched clinical products, such as stromal vascular fraction and microfragmented adipose tissue, in joint pathologies.

Bone-marrow-mesenchymal-stromal-cells (BMSCs)- and platelet-rich-plasma (PRP)-based therapies have shown potential for treating osteoarthritis (OA). Recently, the combination of these two approaches was proposed, with results that overcame those observed with the separate treatments, indicating a possible role of PRP in ameliorating BMSCs’ regenerative properties. Since a molecular fingerprint of BMSCs cultivated in the presence of PRP is missing, the aim of this study was to characterize the secretome in terms of soluble factors and extracellular-vesicle (EV)-embedded miRNAs from the perspective of tissues, pathways, and molecules which frame OA pathology. One hundred and five soluble factors and one hundred eighty-four EV-miRNAs were identified in the PRP-treated BMSCs’ secretome, respectively. Several soluble factors were related to the migration of OA-related immune cells, suggesting the capacity of BMSCs to attract lympho-, mono-, and granulocytes and modulate their inflammatory status. Accordingly, several EV-miRNAs had an immunomodulating role at both the single-factor and cell level, together with the ability to target OA-characterizing extracellular-matrix-degrading enzymes and cartilage destruction pathways. Overall, anti-inflammatory and protective signals far exceeded inflammation and destruction cues for cartilage, macrophages, and T cells. This study demonstrates that BMSCs cultivated in the presence of PRP release therapeutic molecules and give molecular ground for the use of this combined and innovative therapy for OA treatment.

Osteoarthritis (OA) leads to chronic pain and disability, and traditional conservative treatments are not effective in the long term. The intra-articular injection of mesenchymal stem cells (MSCs) is considered a novel therapy for OA whose efficacy mainly relies on the adaptive release of paracrine molecules which are either soluble or extracellular vesicles (EVs) embedded. The correct quantification of EV-miRNAs using reliable reference genes (RGs) is a crucial step in optimizing this future therapeutic cell-free approach. The purpose of this study is to rate the stabilities of literature-selected proposed RGs for EV-miRNAs in adipose derived-MSCs (ASCs). EVs were isolated by ultracentrifugation from ASCs cultured with or without inflammatory priming mimicking OA synovial fluid condition. Expression of putative RGs (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA) was scored by using the algorithms geNorm, NormFinder, BestKeeper and ΔCt method. miR-16a-5p/miR-23a-3p yielded the most stable RGs, whereas let-7a-5p/miR-425-5p performed poorly. Outcomes were validated by qRT-PCR on miR-146a-5p, reported to be ASC-EVs enriched and involved in OA. Incorrect RG selection affected the evaluation of miR-146a-5p abundance and modulation by inflammation, with both values resulting strongly donor-dependent. Our findings demonstrated that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for ASC-EVs miRNAs evaluation. A correct approach would increase the accuracy of embedded molecule assessments aimed to develop therapeutic strategies for the treatment of OA based on EVs.