Biogem

Molecular oncology and precision oncology

Director Prof. Michele Caraglia

Research areas

The Molecular Oncology and Precision Oncology laboratory, coordinated by Prof. Michele Caraglia, focuses on the characterization of circulating miRNA signatures, with the aim of suggesting new diagnostic and therapeutic approaches. The main goal is to determine a circulating miRNA signature with innovative nanosensors, for the diagnostic and prognostic determination of squamous cell carcinoma of the larynx. This new technology will be applicable for any other neoplasia of which there are known circulating miRNA signatures to be determined in real time in the patients' blood. The biological role of these miRNAs in human cancers is also studied in order to identify new therapeutic strategies, based on the regulation of miRNA expression in cancer cells, through the use of nanotechnological approaches. The laboratory is also able to perform genotypic characterization in next generation sequencing (NGS) of human cancers, such as colorectal and breast cancer and renal clear cell cancer. These molecular features are useful for understanding the biological mechanisms that guide their development and determine their response to treatments.

Technologies used / developed

The Molecular Oncology and Precision Oncology laboratory is equipped with equipment for molecular and cell biology. In particular, it has clean rooms for cell cultures with safety devices for the manipulation of viral strains, a confocal microscope in white light and fluorescence, equipped with a CCD camera, FACS, equipment for gene sequencing of Next Generation Sequencing (PGM, Ion Torrent and Ion Proton), QuantStudio 7 Flex Real-Time; RT-PCR equipment (Pyromax and Therascreen).

Services

The laboratory aims to offer the genotypic characterization of colorectal and breast carcinomas of samples from patients affected by these diseases, so as to develop a collaborative network between the main universities and hospitals in Campania. Thanks to a bioinformatics tool that optimizes and simplifies the annotation of sequence data, the analysis obtained by evaluating quality score and frequency parameters, would allow for the development of a personalized medicine. In this way, therapeutic treatments could be developed based on the characteristics of each patient, with the right combination of drugs (based on their genetic profile and that of the disease) in the appropriate dose and at the right time.

Research

Internal seminar

MARCO BOCCHETTI PhD, miR423-5p/Malat loop in HCC

DNA-RNA-Protein-Function is the founding of biology but today we suppose that there is something more. Some RNAs, such as micro-RNAs and long non-coding RNAs, do not code for proteins but still modulate their expression having powerful effects on cell biology and function of both normal and cancer cells. We recently found that micro-RNA (miR)423-5p is overexpressed in serum of hepatocellular cancer (HCC) patients responding to sorafenib inducing also autophagy in HCC cells. Bioinformatic analysis and luciferase reporter assay defined Malat-1, a long non-coding RNA associated with the aggressive phenotype of many different cancer types, as a target of miR423-5p. On these bases, we generated stable miR423-5p or Malat-1-overexpressing Hep-3B and SNU-387 HCC cell clones using a custom-made lentiviral vector. miR423-5p overexpressing cell lines had reduced levels of Malat-1 and were struggling to proliferate, migrate and invade compared to parental cells while Malat-1 had opposite effects. Mass Spectrometry experiments and other observations suggested a reduced level of mitochondrial proteins in cells transfected with miR-423-5p pushing us to investigate the mitochondrial activity. MiR423-5p-transfected cell lines had a reduced number of mitochondria with smaller size if compared to parental cells as shown after Mitotracker staining at confocal microscopy while opposite effect was recorded in Malat-1 overexpressing cells. The transfection of HCC cells with MitoRed Plasmid confirmed these results. To investigate the mitochondrial genome modifications, we isolated mitochondria and extracted their specific RNA analysing MT-ATP, MT-CO and MT-NT genes families and assessed Cox4i2, Cox6a2, Ndufb7, Ndufv1, and Ndufs4 gene expression from total cellular RNA. Interestingly, we found most of those genes, directly correlated to cell energetic and metabolic activity, downregulated in miR423-5p- overexpressing cell lines and upregulated or unmodified in Malat-1 overexpressing cell lines. Finally, we performed NGS Analysis on Long Non-Coding RNAs and in vivo experiments for xenografts growth evaluation, with interesting results. The above findings demonstrate how strong is the impact of these two non-coding RNAs on cell phenotype, offering an interesting starting point to be further investigated for novel therapeutic approaches in cancer research.