Function of matrix metalloprotenase-2 by RNA interference / 中国实验血液学杂志

ABSTRACT

The study was aimed to reveal the effects of matrix metalloproteinase-2 (MMP-2) on cell proliferation, migration, invasion, angiogenesis and cell cycle. The small interfering RNA (siRNA) of MMP-2 transfected into endothelial cells EAhy926, the transformation efficiency at protein and gene levels was evaluated by using flow cytometry and RT-PCR respectively. MTT method was used to detect the proliferation ability of EAhy926. The migration and invasion abilities of EAhy926 induced by two kinds of factors, type IV collagen (COL IV) and fibronectin (Fn) were assayed with Rose Bengal dying method. The changes of angiogenesis were determined by three-dimension culture. The changes of cell cycle and related gene expression were assayed by using flow cytometry and RT-PCR respectively. The results indicated that the proliferation ability of EAhy926 had no obvious difference between interference or not. The migration ability of EAhy926 induced by two kinds of factors, type IV collagen (COL IV) and fibronectin (Fn) was inhibited after interfered by siRNA for 48 hours, and was stronger inhibition to COL IV than Fn (Fn control 0.581+/-0.012 vs 0.261+/-0.002; COL IV control 0.467+/-0.009 vs 0.110+/-0.010, p<0.01). The invasion test had the similar result as migration test. (Fn vs control 0.365+/-0.012 vs 0.101+/-0.002; COL IV vs control 0.317+/-0.009 vs 0.102+/-0.010, p<0.01). The angiogenesis ability of endothelial cells dropped to 58.9% of control after interference with siRNA for 48 hours. In the cell cycle experiments, after RNAi for 48 hours and 72 hours, the cell ratio of G1 rose from [(65.9+/-2.53)%; (63.2+/-1.89)%] to [(83.9+/-2.53)%, (89.2+/-1.24)%]% (p<0.01); the cell ratio of S and G2 dropped from [(32.7+/-1.91)%, (37.1+/-2.65)%] to [(18.1+/-1.49)%, (10.2+/-0.85)%] (p<0.01). The results analyzed by semi-quantitative RT-PCR showed that the expression levels of Rb, cyclin D1 PCNA genes dropped to 35%, 51% and 22% of normal control respectively. It is concluded that the expression of MMP-2 does not distinctly correlate with the proliferation of endothelial cells, but plays a very important role in affecting endothelial cell migration, invasion and angiogenesis, and participates in regulating cell cycle through Rb, cyclinD1 and PCNA.