Biopolym. Cell. 2010; 26(2):153-159.
http://dx.doi.org/10.7124/bc.000152
Genomics, Transcriptomics and Proteomics
RNA interference of genes of ubiquitn and proteasome β7 subunit in cardiomyocytes culture
1Kyrychenko V. O., 1Nagibin V. S., 1Tumanovska L. V., 2Rybalchenko V. K., 1Dosenko V. E., 1Moibenko O. O.
  1. Bogomoletz Institute of Physiology, NAS of Ukraine
    4, Bogomolets Str., Kyiv, Ukraine, 01024
  2. Taras Shevchenko National University of Kyiv
    64, Volodymyrska Str., Kyiv, Ukraine, 01601

Abstract

Aim. Ubiquitin (UBB) and proteasome 7 subunit (PSMB7) genes silencing is interesting due to the crucial role of ubiquitin-dependent proteasomal system (UPS) in turnover of functional proteins, which controls program cell death (apoptosis, auophagy). Methods. We used methods of RNA interference (for UBB and PSMB7 genes silencing), fluorescence microscopy and real-time PCR. Results. It was shown that small interference RNA injection in cell culture decreased ubiquitin expression in 2,4 (P < < 0.05), PSMB7 – in 1,6 times (P > 0.05). At the same time, there was augmented level of necrotic cells with no change in number of apoptotic cells. Cells with signs of autophagy were much augmented as consequence of UPS impairment and intracellular proteins accumulation, which degrade in lysosomes. Conclusions. The data obtained testify that UBB and PSMB7 genes silencing induces cell necrosis and autophagy without changing number of apoptotic cells.
Keywords: ubiquitin, proteasome, RNA-interference, cardiomyocytes
Full text: (PDF, in Ukrainian)

References

[1] Staub O., Rotin D. Role of ubiquitylation in cellular mem brane transport Physiol Rev 2006 86, N 2 P. 669–707.
[2] Ikeda F., Dikic I. Atypical ubiquitin chains: new molecular signals EMBO Reps 2008 9, N 6 P. 536–542.
[3] Piper R. C., Luzio J. P. Ubiquitin-dependent sorting of integral membrane proteins for degradation in lysosomes Curr. Opin. Cell Biol 2007; 19(4):459–465.
[4] Bulteau A. L., Lundberg K. C., Humphries K. M., Sadek H. A., Szweda P. A., Friguet B., Szweda L. I. Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion J. Biol. Chem 2001 276, N 32 P. 30057– 30063.
[5] Dosenko V. E., Nagibin V. S., Tumanovskaya L. V., Zagoriy V. Y., Moibenko A. A., Vaage J. Proteasomal proteolysis in anoxia-reoxygenation, preconditioning and postconditioning of isolated cardiomyocytes Pathophysiology 2006 13, N 2 P. 119–125.
[6] Mearini G., Schlossarek S., Willis M. S., Carrier L. The ubiquitin–proteasome system in cardiac dysfunction Biochim. Biophys. Acta 2008 1782, N 12 P. 749–763.
[7] Luss H., Schmitz W., Neumann J. A proteasome inhibitor confers cardioprotection Cardiovasc. Res 2002 54, N 1 P. 140–151.
[8] Sledz C. A. Williams B. R. RNA interference in biology and disease Blood 2005 106, N 3:787–794.
[9] Siomi H., Siomi M. C. On the road to reading the RNA-interference code Nature 2009 457, N 7228:396–404.
[10] Reinecke H., Zhang M., Bartosek T. Murry C. E. Survival, integration, and differentiation of cardiomyocyte grafts Circulation 1999 100, N 2:193–202.
[11] Franzen R., Pfeilschifter J., Huwiler A. Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures FEBS Lett 2002 532, N 3:441–444.
[12] Evdonin A. L., Tsupkina N. V., Nikol'skii N. N., Medvedeva N. D. Effect of EGF on ubiquitination and proteasomedependent degradation of phospholipase C gamma1 in A431 cells. Tsitologiia 2003 45, N 10:1013–1018.
[13] Marquis H., Goldfine H., Portnoy D. A. Proteolytic pathways of activation and degradation of a bacterial phospholipase C during intracellular infection by Listeria monocytogenes J. Cell Biol 1997 137, N 6:1381–1392.
[14] Perez-Alvarez S., Solesio M. E., Manzanares J., Jordan J., Galindo M. F. Lactacystin requires reactive oxygen species and Bax redistribution to induce mitochondria-mediated cell death Br. J. Pharmacol 2009 158, N 4:1121–1130.
[15] Ling Y. H., Liebes L., Zou Y., Perez-Soler R. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells J. Biol. Chem 2003 278, N 36:33714–33723.
[16] Dallaporta B., Pablo M., Maisse C., Daugas E., Loeffler M., Zamzami N., Kroemer G. Proteasome activation as a critical event of thymocyte apoptosis Cell. Death Differ 2000 7, N 4:368–373.
[17] Fan X. M., Wong B. C., Wang W. P., Zhou X. M., Cho C.H., Yuen S. T., Leung S. Y., Lin M. C., Kung H. F., Lam S. K. Inhibition of proteasome function induced apoptosis in gastric cancer Int. J. Cancer 2001 93, N 4:481–488.
[18] Surova O. V., Nagibin V. S., Tumanovskaya L. V., Dosenko V. E., Moibenko A. A. Effect of a low dose of proteasome inhibitor on cell death and gene expression in neonatal rat cardiomyocyte cultures exposed to anoxia-reoxygenation Exp Clin. Cardiol 2009 14, N 2:57–61.
[19] Wojcik C. Proteasomes in apoptosis: villains or guardians? Cell Mol. Life Sci 1999 56, N 11–12:908–917.