Molecular markers in human pathologies

Our aim is to search for clinically applicable biomarkers for human diseases. We analyse circulating Extracellular Vesicles (EVs) isolated from preclinical models as well as from patients’ biofluids (serum, plasma) for the presence of disease biomarkers.  Two examples of the studies performed in the lab are described herein. The first regards detecting biomarkers for early liver fibrosis in cholestatic mouse models. Cholestatic diseases, resulting from impaired bile flow from the liver to the duodenum, account for 10% of all liver diseases and exert a significant burden on healthcare systems. Liver fibrosis preludes complications such as cirrhosis and hepatobiliary cancers. Liver fibrosis is often reversible in the early phases but remains under-diagnosed due its asymptomatic presentation in the initial stage, until complications occur. There is thus an urgent need for reliable biomarkers in the clinic to intervene early with therapy. The evolution and resolution of liver fibrosis are influenced by the underlying cause, and general biomarkers may not be sufficient to detect fibrotic development in all liver diseases. There is increasingly strong evidence demonstrating that the molecular cargo of EVs, which represents the phenotypic state of donor cells, is a critical determinant of EV action in the progression of fibrosis in various liver diseases. Our study was designed to lay the foundation for translational research in humans by identifying potential biomolecules indicative of cholestasis-induced liver fibrosis in mouse models. Whole transcriptome and small RNA sequencing analyses of circulating EVs revealed enrichment of RNA species, such as hepatic mRNAs, like Albumin and Haptoglobin and microRNAs like miR192-5p, miR194-5p, miR22-3p, and miR29a-3p, in cholestatic mice versus controls. This panel of mRNAs and miRNAs contained in circulating EVs indicates hepatic damage and fibrosis in mice, hence offering promising biomarkers for human severe cholestasis-induced liver fibrosis. Another recent study performed in our lab aimed at clarifying, using a transcriptomic approach, the relationship between serum Vitamin D levels, genomic response to Vitamin D treatment (analysis of serum EVs) and SARS-CoV-2 positivity during the follow-up of Inflammatory Bowel Disease (IBD) patients recently affected by COVID-19. In this study, EV-enclosed miRNAs were analysed by RNA sequencing, which evidenced a statistically significant increase in miR30d-5p, miR150-5p, Let-7f-5p, and Let-7a-5p in the anti-SARS-CoV-2-positive and low 25(OH)D and Vit.D supplemented groups with respect to the non-Vit.D supplemented group, indicating their responsiveness to Vit.D treatment. These biomolecules are promising as biomarkers for Vit.D responsivity in these patients. 

Under normal conditions, physiological quantities of EVs containing liver-derived biomolecules are released. Upon cholestasis, the damage that occurs in the liver induces massive release of EVs that are enriched with bioactive molecules such as miRNAs and mRNAs, which provide a molecular fingerprint of the underlying pathological events in the liver and can thus be used as biomarkers (taken from Antioxid Redox Signal. 2022 Mar;36(7- 9):480-504)

The secretome of stem cells, especially extracellular vesicles (EVs), have recently attracted much attention due to their small size, biomolecule cargo, and low immunogenicity compared to the parent cells in novel, cell-free therapeutic strategies for human diseases. We assessed the potential of EVs derived from bone marrow mesenchymal stem cells (BMSC-EVs), applied in a bio-adhesive and lubricant material that enables prolonged effects in vivo and reduces the frequency of daily applications, in promoting corneal repair. Our study showed that BMSC-EV treatment mitigated corneal damage while controlling inflammation and neo-angiogenesis, crucial for repairing the avascularized cornea. Importantly, we observed no side effects despite the non-autologous nature of the EVs, as they were not internalized in the non-damaged corneas, indicating that as the damage resolves, these EVs no longer penetrate the corneal epithelium. Thus, BMSC-EVs are promising as a new cell-free approach for intervening on burn wounds in the avascularized region of the eye and deserve further investigation.

RNA binding proteins are well recognized as critical regulators of tumorigenic processes through their capacity to modulate RNA biogenesis, including alternative splicing, RNA stability and mRNA translation. We have previously identified a new role for the RNA-binding protein Epithelial Splicing Regulatory Protein 1, ESRP1, as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. In particular, ESRP1 exerted its action by modulating the polysomal loading of some core pluripotency factors, such as Oct4 and Sox2, thus controlling their expression and the responsiveness of Embryonic Stem (ES) cells to differentiate stimuli. In fact, ESRP1-silenced ES cells showed enhanced self-renewal and defective differentiation capacity. This initial study sparked further research in the field of colorectal cancer (CRC), considering that certain embryogenesis-related pathways are reactivated during tumorigenesis. ESRP1 is mostly known for its tumor suppressive role through the control of alternative splicing events. However, our work regarding the role of ESRP1 in CRC indicated, for the first time, that ESRP1 played a role in anchorage-independent growth of CRC cells, hence acting as pro-oncogene. ESRP1 also promoted the ability of CRC cells to generate macrometastases in mice livers. High ESRP1 expression may thus stimulate growth of cancer epithelial cells in the colon as well as at distant sites, and promote colorectal cancer progression. Our data also evidenced that ESRP1 acted, at least partially, through RAC1b in its tumor-promoting activities in CRC cells, and interacts and is co-modulated with a group of proteins that are strongly associated with a pro-mitotic and pro-proliferative activity. Thus, ESRP1 can suppress or promote tumorigenesis depending on the cell type and disease context and is a promising therapeutic target in a subgroup of CRC patients. 

We also recently performed a bioinformatics search for RBPs differentially modulated in Primary Sclerosing Cholangitis (PSC) and cholangiocarcinoma (CCA) patients and found several RBP transcripts that exhibit differential expression in liver and gall bladder, as well as in body fluids, and are promising as biomarkers for the early diagnosis of PSC and for predicting its progression to CCA.