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Baculovirus-based gene silencing of Humanin for the treatment of pituitary tumors

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Abstract

Pituitary tumors are the most common primary intracranial neoplasms. Humanin (HN) and Rattin (HNr), a rat homolog of HN, are short peptides with a cytoprotective action. In the present study, we aimed to evaluate whether endogenous HNr plays an antiapoptotic role in pituitary tumor cells. Thus, we used RNA interference based on short-hairpin RNA (shRNA) targeted to HNr (shHNr). A plasmid including the coding sequences for shHNr and dTomato fluorescent reporter gene was developed (pUC-shHNr). Transfection of somatolactotrope GH3 cells with pUC-shHNr increased apoptosis, suggesting that endogenous HNr plays a cytoprotective role in pituitary tumor cells. In order to evaluate the effect of blockade of endogenous HNr expression in vivo, we constructed a recombinant baculovirus (BV) encoding shHNr (BV-shHNr). In vitro, BV-shRNA was capable of transducing more than 80% of GH3 cells and decreased HNr mRNA. Also, BV-shHNr increased apoptosis in transduced GH3 cells. Intratumor injection of BV-shHNr to nude mice bearing s.c. GH3 tumors increased the number of apoptotic cells, delayed tumor growth and enhanced survival rate, suggesting that endogenous HNr may be involved in pituitary tumor progression. These preclinical data suggests that the silencing of HN expression could have a therapeutic impact on the treatment of pituitary tumors.

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Acknowledgements

This work was supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas, National Research Council (PIP 11420110100353 to M.C.; PIP 11220120100261 to A.S, doctoral fellowships to MFG, MP, ASA), Agencia Nacional de Promoción Científica y Tecnológica (PICT 2013-0310, PICT-2015-3309 to M.C.; PICT 2014-0334 to A.S.; PICT 2014-1827 to V.R., doctoral fellowship to CZ), the University of Buenos Aires (20020130100020 to A.S.) and the National University of La Plata (X703 to V.R.).

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Author notes

  1. María Florencia Gottardo and Matías L. Pidre contributed equally to this work.

Authors and Affiliations

  1. Departamento de Biología Celular e Histología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

    María Florencia Gottardo, Gabriela Jaita & Adriana Seilicovich

  2. Instituto de Investigaciones Biomédicas (INBIOMED), Facultad de Medicina, CONICET- Universidad de Buenos Aires, Paraguay 2155, piso 10, C1121ABG, Buenos Aires, Argentina

    María Florencia Gottardo, Camila Zuccato, Antonela S. Asad, Mercedes Imsen, Gabriela Jaita, Marianela Candolfi & Adriana Seilicovich

  3. Instituto de Biotecnología y Biología Molecular (IBBM), Universidad Nacional de La Plata - CONICET, La Plata, Argentina

    Matías L. Pidre & Víctor Romanowski

Authors
  1. María Florencia Gottardo
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  2. Matías L. Pidre
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Correspondence to Adriana Seilicovich.

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Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the Ethics Committee of the School of Medicine, University of Buenos Aires.

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Supplementary Fig. 1

Generation of recombinant baculovirus for delivery of shRNAs in mammalian cells. Representative images of Sf9 insect cells co-transfected with bAcGoza bacmid and the plasmid pBacPAK9-shHNr; observation made at 96 h post-transfection. Left panel: DAPI; medium panel: cells with recombinant baculovirus expressing dTomato protein; right panel: cells infected with recombinant baculovirus BV-shHNr observed under phase contrast microscopy (TIF 124 KB)

Supplementary Fig. 2

Transfection efficiency of the plasmid with HNr interfering RNA. GH3 cells were transfected with 1 μg of plasmid DNA pUC-shHNr, incubated for 16, 24 or 48 h and processed for the detection of HNr by immunofluorescence. (a) Representative microphotographs of transfected cells. Arrows indicate transfected cells showing dTomato and HNr expression. Nuclei were counterstained with DAPI. Left panels: DAPI; medium panels: HNr; right panels: dTomato. (b) Percentage of transfected (dTomato-positive) cells. Each column represents the percentage ± CL of dTomato-positive GH3 cells (n ≥ 1000 cells/group) (TIF 143 KB)

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Gottardo, M.F., Pidre, M.L., Zuccato, C. et al. Baculovirus-based gene silencing of Humanin for the treatment of pituitary tumors. Apoptosis 23, 143–151 (2018). https://doi.org/10.1007/s10495-018-1444-0

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