Heparin Impairs Angiogenesis through Inhibition of MicroRNA-10b

Xiaokun Shen,Jianping Fang,Xiaofen Lv,Zhicao Pei,Ying Wang,Songshan Jiang,Kan Ding

doi:10.1074/jbc.m111.224212

Abstract

Heparin, which has been used as an anticoagulant drug for decades, inhibits angiogenesis, whereas thrombin promotes tumor-associated angiogenesis. However, the mechanisms underlying the regulation of angiogenesis by heparin and thrombin are not well understood. Here, we show that microRNA-10b (miR-10b) is down-regulated by heparin and up-regulated by thrombin in human microvascular endothelial cells (HMEC-1). Overexpression of miR-10b induces HMEC-1 cell migration, tube formation, and angiogenesis, and down-regulates homeobox D10 (HoxD10) expression via direct binding of miR-10b to the putative 3' UTR of HoxD10. In addition, HMEC-1 cell migration and tube formation are induced by HoxD10 knockdown, whereas angiogenesis is arrested when HoxD10 expression is increased after anti-miR-10b or heparin treatments. Furthermore, expression of miR-10b and its transcription factor Twist are up-regulated by thrombin, whereas HoxD10 expression is impaired by thrombin. Using quartz crystal microbalance analysis, we show that heparin binds to thrombin, thereby inhibiting thrombin-induced expression of Twist and miR-10b. However, the expression of miR-10b is not attenuated by heparin any more after thrombin expression is silenced by its siRNA. Interestingly, we find that heparin attenuates miR-10b expression and induces HoxD10 expression in vivo to inhibit angiogenesis and impair the growth of MDA-MB-231 tumor xenografts. These results provide insight into the molecular mechanism by which heparin and thrombin regulate angiogenesis.

Highlights

Bin and thrombosis through the clotting cascade or platelet activation [4]
Our results argue that heparin and thrombin play conflicting roles in angiogenesis through their regulation of Twist, a transcription factor of miR-10b to further influence the expression of miR-10b, whereas miR-10b induces angiogenesis by decreasing the levels of a functional target, Hoxd10, which impairs angiogenesis
We find that thrombin treatment increases Twist expression levels and leads to increased expression of miR-10b and decreased expression of homeobox D10 (HoxD10) in HMEC-1 cells

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—HMEC-1 were cultured in MCDB131 (Invitrogen) medium containing 15% fetal bovine serum Semi-quantitative RTPCR was performed to evaluate HoxD10 and Twist mRNA levels as previously described [14]. The membranes were blocked with Tris-buffered saline (TBS) plus 5% nonfat dry milk and 0.1% Tween 20 before incubation with antibody against ␤-actin (Sigma), HoxD10 (Santa Cruz), Twist (Santa Cruz), and thrombin (Boster Biological Technology, Ltd., Wuhan, China) at 4 °C overnight. Cells were grown to ϳ70% confluence in 48-well plates and co-transfected with psiCHECK2–3ЈUTR plus pll3.7-miR-10b or empty vector as described above and previously [28]. HMEC-1 cells were transfected with miR-10b vector or antimiR-10b siRNA. 1 ϫ 106 HMEC-1 cells (as control) or miR-10b-transfected HMEC-1 cells (100 ␮l) were mixed with 400 ␮l Matrigel, and subcutaneously injected into the midventral abdominal region of 4 – 6-week-old nude mice. Statistical analysis was performed using Student’s t test and p Ͻ 0.05 was considered statistically significant (*, p Ͻ 0.05; **, p Ͻ 0.01)

RESULTS

DISCUSSION

Kan Ding