Figure 7.

Nucleolin binded to VEGF‐A and MMP‐9 mRNA and up‐regulated the expressions of VEGF‐A and MMP‐9 mRNA through enhancing their stability. (A) and (B) IP‐RT‐PCR analysis demonstrated that nucleolin binded to VEGF‐A and MMP‐9 mRNA. Nucleolin in the HUVECs was precipitated by nucleolin antibody and magnetic A/G magnetic beads, Western blotting was performed to identify the precipitation efficiency of nucleolin, and the mRNA expressions of MMP‐9 and VEGF‐A were detected in the extracted from the magnetic beads, the representative images (A) and the analysis of results (B) (n = 3 per group; *P < .05 vs IgG). (C) and (D) Western blotting demonstrated the effect of transfection with pCMV‐NCL (C) or siRNA‐NCL (D) on the protein expression of nucleolin in the HUVECs. E and F, RT‐PCR analysis demonstrated the effect of transfection with pCMV‐NCL (E) or siRNA‐NCL (F) on the expressions of VEGF‐A and MMP‐9 mRNA (n = 3 per group; *P < .05 vs pCMV‐GFP (pCMV) or siRNA‐scramble (siRNA‐Scr). (G) and (H) Effect of up‐regulation and down‐regulation of nucleolin expression on the stability of VEGF‐A (G) and MMP‐9 (H) mRNA. HUVECs were transfected with pCMV, pCMV‐NCL, siRNA‐Scr or siRNA‐NCL and then incubated with actinomycin D (5 mg/mL) for various time (0, 1, 3, 6 hours), and the mRNA levels of VEGF‐A and MMP‐9 were determined by real‐time PCR, respectively (n = 3 per group; *P < .05 vs pCMV; # P < .05 vs siRNA‐Scr). I–K, IP‐RT‐PCR assay demonstrated the interaction between nucleolin and VEGF‐A, nucleolin and MMP‐9 in the myocardial tissue of AMI mice and HSYA‐treated AMI mice. Nucleolin in the myocardial tissue was precipitated by nucleolin antibody and magnetic A/G beads, Western blotting was performed to identify the precipitation efficiency of nucleolin, the mRNA expressions of VEGF‐A and MMP‐9 were detected in the extracted from magnetic beads by RT‐PCR, and the representative images (I) and analysis of VEGF‐A (J) and MMP‐9 (K) mRNA were shown (n = 3 per group; *P < .05, #P < .05 vs AMI + NS)