Bìol. Tvarin, 2017, Volume 19, Issue 2, pp. 70–78

ShRNA-MEDIATED KNOCKDOWN OF PITUITARY TUMOR TRANSFORMING GENE 1 (PTTG1): OPTIMIZATION OF THE EFFECT ON EARLY OSTEOBLAST DIFFERENTIATION

Kh. V. Malysheva1,2,3, O. K. Pavlenko4, R. S. Stoika1,4, O. G. Korchynskyi1,3

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1Institute of Cell Biology NAS of Ukraine,
14/16 Drahomanov str., Lviv 79005, Ukraine

2Insitute of Animal Biology NAAS,
38 V. Stus str., Lviv 79034, Ukraine

3Centre for Innovative Research in Medical and Natural Sciences,
Rzeszow University and Medical Faculty,
1A Warshawna str., Rzeszow 35-310, Poland

4Ivan Franko National University of Lviv,
4 Grushevsky str., Lviv 79005, Ukraine

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by massive joint destruction. Bone erosion belongs to the most painful consequences of RA that are tightly associated with disease severity and poor functional outcome. It is well known that bone morphogenetic protein (BMP) and Wnt regulatory pathways are involved in cartilage and bone formation and maintenance. We hypothesize that pituitary tumor transforming gene 1 (PTTG1)/(PTTG)-binding factor 1 (PBF1) axis serves as a new negative regulator of bone homeostasis with involvement into RA progression and pathogenesis.

The aim of this study was to investigate the effect of small hairpin (sh) RNA-mediated knockdown of PTTG1 mRNA expression on the early stages of BMP-induced osteoblast differentiation. Analysis of the experiment results was performed by spectrophotometric measurement of alkaline phosphatase activity, which is widely used as a marker of early osteogenesis.

We have found that shRNA-mediated knockdown of PTTG1 mRNA expression potentiated early stages of osteoblast differentiaion in stable multi-clonal cultures of C2C12 and KS483 cell lines. The most pronounced effect was found at the action of anti-PTTG1_shRNA-1 whose stable expression stimulated osteoblast differentiation of C2C12 and KS483 cells (2.1 fold and 2.7 fold, respectively), whereas anti-PTTG1_shRNA-3 stable expression did not show any significant effect on the osteoblast differentiation of these cells. Thus, we demonstrated that PTTG1 is an important repressor of early stages of osteogenesis and can serve as an inhibitor of bone remodelling, in particular, during RA progression.

Keywords: PITUITARY TUMOR TRANSFORMING GENE 1, SMALL HAIRPIN RNAs, OSTEOBLAST DIFFERENTIATION, MURINE MESENCHYMAL STEM CELLS, BONE MORPHOGENETIC PROTEINS, RHEUMATOID ARTHRITIS

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