The study on lentivirus mediated Aggrecanase-1 shRNA transfection in vitro cultured chondrocytes of rheumatoid arthritis patients

Abstract

Objective The major change found in rheumatoid arthritis (RA) is the rapid destruction of cartilage, Aggrecanase-1 plays an important role in Aggrecan degradation in RA patients which is the main components of cartilage matrix. This study aims to identify an effective way to inhibit Aggrecanase-1 expres-sion, to explore if the RNAi mediated by lentivirus in cartilage is feasible, and determine whether the Aggrecan in chondrocyte can be protected by inhibiting the expression of Aggrecanase-1. Methods The lentivirus vectors expressing shRNA targeting Aggrecanase-1 was constructed and transfected into cultured RA patients articular chondrocyte. Gene mRNA levels of Aggrecanase and Aggrecan in different time points after transfec-tion with different MOI were analyzed by RT-PCR. Aggrecan concentration was measured by immuno-histoche-mistry, the results were processed by SPSS 19.0 software, with the method of paired t test and ANOVA. Results The specific inhibition of Aggrecanase-1 by RNAi mediated by lentivirus had no negative effect on the morphology and growth velocity of chondrocytes. With the mRNA of Aggrecanase-1 decreased significantly, the Aggrecan expression increased accordingly both at mRNA and protein level, especially the change of shRNA4 group was significant, in which the Aggrecanase-1 mRNA decreased from the baseline level 1.083±0.133 to 0.074±0.004. Aggrecan mRNA increased from the baseline 1.182±0.160 to 2.922±0.169; at the same time, Aggrecan proteoglycan increased from the baseline 507±265 to 2 270±334 (P<0.05). Conclusion The results suggest that lentivirus mediated inhibition of Aggrecanase-1 by RNAi can retard Aggrecan degra-dation, without interfering with chondrocytic phenotype and viability. RNAi technology can be a useful tool to mitigate cartilage degeneration. Key words: Chondrocyte; RNA interference; Aggrecanase-1