Ceni, E.

Galli, A.

Materozzi, M.

Mello, T.

Polvani, S.

Simeone, I.

Tarocchi, M.

English

Florence Research

Abstracts / Digestive and Liver Disease 51 (2019) e18–e42 e39T-43A new regulator of mitochondrial OXPHOS inhepatocytes and HCC cell lines: the AAA+ ATPaseRuvBL1I. Simeone 1,2, M. Materozzi 1,2, E. Ceni 1,S. Polvani 1, M. Tarocchi 1, A. Galli 1, T. Mello 11 Department of Experimental and ClinicalBiomedical Sciences “Mario Serio”, University ofFlorence, Florence, Italy2 GenOMeC Doctorate, University of Siena, Siena,ItalyHepatocellular carcinoma (HCC) is currently a major challengein medicine for its poor prognosis and lack of effective therapeuticoptions. The AAA+ ATPase RuvBL1 associates with Hsp90 in sev-eral multiprotein complexes regulating key cellular pathways suchas cell proliferation, gene expression, chromatin remodeling andtelomere maintenance. In several human cancer, including HCC,RuvBL1 overexpression correlates with a poor prognosis. Despitethe several functions potentially regulated by RuvBL1, its role in theonset and progression of HCC is still unknown. We had previouslygenerated a RuvBL1 hepatocyte-specific knock-out mouse modeland observed by TEM major structural alterations in mitochon-dria of hepatocytes. Moreover, by a 2D-proteomic screening, wefound that several key mitochondrial proteins and Hsp90 weresignificantly reduced in RuvBL1-silenced hepatoma cells in vitro.Therefore, we aimed to dissect the role of RuvBL1 as a potentialregulator of mitochondria functionality in HCC cells.First, we found that RuvBL1 targeting by RNAi significantlyimpairs mitochondrial respiration (OXPHOS) in normal hepa-tocytes (AML-12) and hepatoma cell lines (Hepa1-6, HepG2,Huh7, Hep3B). Live-cell imaging experiments showed a signifi-cant increase of mitochondrial ROS production and mitochondrialdepolarization in RuvBL1-silenced cells. We next explored theRuvBL1-Hsp90 axis in mitochondrial functionality. We found thatRuvBL1 is a positive regulator of HSP90AB1 promoter and mRNAlevels, both in hepatoma and normal hepatocyte cell lines. Interest-ingly, Hsp90 inhibitors (17-AAG, VER-50589) effectively reducedmitochondrial OXPHOS paralleling RuvBL1 silencing. Analyzing theLIHC-TCGA database, we found a significant correlation betweenRUVBL1 and HSP90AB1 expression in HCC patients, consistentlywith our in vitro data. Finally, Gene Ontology analysis performedon genes differentially expressed in high- vs low-RUVBL1 HCC sam-ples in the TGCA database, highlighted a significant enrichment ofmitochondria-related terms.In conclusion, our data uncover a novel role of RuvBL1, possiblythrough its interaction with Hsp90, in mitochondria integrity andfunction.https://doi.org/10.1016/j.dld.2018.11.109T-44Surface enhanced Raman spectroscopy as adiagnostic tool for circulating miRNA detectionin HCC: the SERMI4cancer projectD. Pascut 1, A. Bonifacio 2, A. Visintin 3,4,A. Di Silvestre 2, E. Mitri 2, L.S. Crocè 1,3,4,C. Tiribelli 11 Fondazione Italiana Fegato - ONLUS, Centre forLiver Studies, Area Science Park Basovizza, Trieste,Italy2 Department of Architecture and Engineering,University of Trieste, Trieste, Italy3 Departmento of Medical Sciences, University ofTrieste, Trieste, Italy4 Clinica Patologie Fegato, Azienda SanitariaUniversitaria Integrata di Trieste (ASUITS), Trieste,ItalyIntroduction: Hepatocellular carcinoma (HCC) is one of themost common cancers worldwide with more than 800,000 newlydiagnosed cases every year. The late diagnosis, that limits the treat-ments options, makes this tumor the fifth cause of cancer-relateddeath. The existing diagnostic procedures are expensive and lackof adequate sensitivity and the specificity. The growing interestin the use of circulating miRNAs as reliable biomarkers for cancerdiagnosis opens new perspectives in clinical oncology.Aim: The main objective of the SERMI4Cancer project is todevelop a portable, user friendly, and cost-effective Point Of Care(POC) system integrated with the innovative Surface EnhancedRaman Spectroscopy (SERS) technology for circulating miRNAdetection in HCC patients.Materials andmethods results: The system allows a label-freedirect detection of the analyte making this technology suitablefor a POC system for multiple applications. The SERMI4Cancerproject includes the development of three components: The miRNAseparation and binding support (polyfunctional matrix); the detec-tion system consisting of a Portable Raman Spectrometer andnanostructured SERS Sensor; the data integration system. Molec-ular beacons targeting miRNAs are immobilized onto silver coatedsilicon nanopillars spotted on a nitrocellulose membrane andincubated with patients’ plasma. Quantifications are performedwith the Raman Renishaw spectrometer, a simple, rapid andpowerful tool yielding a unique “chemical fingerprint” of thechemical composition of the sample. SERS technology allow acost-effective detection of very low concentrations of miRNAs(attomolar) increasing the accuracy of the diagnostic biomarkercandidate at early stages of the disease.Conclusion: These results will open new perspectives on HCCdiagnosis that could be useful for the development of a point of caresystem with the innovative SERS technology for miRNAs detectionblood samples.Funding: Supported by the project surface-enhanced RamanmicroRNA for cancer(SERMI4CANCER) PORFESR 2014-2020 FVG,decree no.3028 05/05/2017 and no.4526 16/06/2017.https://doi.org/10.1016/j.dld.2018.11.110

A new regulator of mitochondrial OXPHOS in hepatocytes and HCC cell lines: the AAA plus ATPase RuvBL1

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A new regulator of mitochondrial OXPHOS in hepatocytes and HCC cell lines: the AAA plus ATPase RuvBL1

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