Down-regulation Of ROCK-Ⅱ And The Migration Of Vascular Smooth Muscle Cell

ObjectivesThe incidence of atherosclerosis (AS) has increased in developed countries, even in China. Proliferation and migration of vascular smooth muscle cells play an important role in the development of AS. Platelet-Derived Growth Factor (PDGF) is one of the numerous growth factors, and it plays an important role in cell proliferation,cell migration and atherosclerosis. In addition, it can induce VSMC migration. PDGF-BB, as one type of PDGE, is known as the strongest chemotatic agent. RhoA is a small GTPase protein. It acts upon known an effector protein: ROCK (Rho-associated coiled-coil containing protein kinase). ROCK has two types: ROCK-â… and ROCK-â…¡. Recently, accumulating evidences have been suggested that activation of the Rho/ROCK signaling pathway is substantially involved in a wide range of cellular functions, such as cystoskeleton reorganization, VSMC contraction, proliferation, adhesion, migration and inflammatory reaction. Rho/ROCK mediated signaling in physiology and pathophysiology. Accordingly, Rho/ROCK pathway appears to play a key role in the regulation of several arterial diseases.The main focus of this article was to verify the effect of ROCK-â…¡in cell migration of VSMC stimulated by PDGF-BB. Applying to the RNA interference to knock down the expression of ROCK-â…¡gene, and then to observe whether there are some changes in the cell migration of VSMC. From our study, it may provide novel viewpoints for new medicines to prevent AS and Cardiovascular disease.Methods1. Sequences of short interfering RNA (siRNA) were designed and then chemically synthesized according to the gene sequence of ROCK-â…¡(GenBank number NM₀13022). In addition, siRNA(sc-36433) from Santa Cruz Biotechnology incorporation was also used into our experiments.2. The siRNA were transfected into A7r5 VSMC by lipofectamine TM 2000, during the transfection of A7r5, control siRNA (Fluorescein Conjugate) transfection efficiency was detected and calculated by fluorescent microscopy. The best transfection dosage was determined in the toxicity assay of lipofectamine TM 2000.3. Total RNA was extracted from A7r5 cells with RNAiso reagent for reverse transcription and proliferation of cDNA. Data were analyzed by software and the results were in optical density ratio of ROCK-â…¡gene to GAPDH.4. Total protein from cell lysis was electrophoresised followed by membrane transfer. After incubation with primary and secondary antibodies, the protein bands were detected by enhanced chemiluminescence(ECL). Data were analyzed by computer and the results were in optical density ratio of ROCK-â…¡protein toβ-actin.5. Cell migration was monitored using a Boyden chamber. Applying the most efficient siRNA to interfere ROCK-â…¡gene in rat VSMC, the A7r5 cell migration assay induced by PDGF-BB was examined by Boyden Chamber assay through a membrane with 8μm pore, which was applied to quantitate the effect of RNAi.Results1. Green fluorescein in the A7r5 cells was seen clearly in control siRNA (Fluorescein Conjugate) transfected group under the fluorescent microscope, the transfection efficiency of lipofectamineTM 2000 was approximation 92% after a 5-hour transfection.2. In the toxicity assay of lipofectamine TM 2000, compared to blank control group, the total of A7r5 cells had decreased by 46.9% in 1.7μl lipofectamine TM 2000 transfected group and the total numberof A7r5 cells had decreased by 66.2 % in 2.5μl lipofectamine TM 2000 transfected group after a 24-hour transfection.3. RT-PCR results indicate: (1) Compared to blank control groups, the mRNA expression of ROCK-â…¡in A7r5 cells could not be inhibited by siRNA (sc-36433)(P>0.05);(2) the mRNA expression of ROCK-â…¡in A7r5 cells was significantly inhibited by (CBR0915)(P<0.05). The suppression of ROCK-â…¡mRNA expression was 60.3%.4. Western blot indicate (1). Compared to blank control groups, the protein expression of ROCK-â…¡in A7r5 cells could not be inhibited by siRNA (sc-36433)(P>0.05);(2) the protein expression of ROCK-â…¡in A7r5 cells was significantly inhibited by siRNA(CBR0915). The suppression of ROCK-â…¡mRNA expression was 60.1%. It indicated that siRNA(CBR0915) could effectively inhibit ROCK-â…¡protein expression in A7r5 cells.5. (1) In the migration assay, according to the number of VSMC passing through the membrane, 5ng/ml PDGF-BB was selected as the optimal dosage to induce VSMC migration. (2)The number of VSMC passing through the membrane in the RNAi group was 70.2±8.81 (n=3) cells, in the blank control groups with 59.3±6.22 (n=3) cells and in the lipofectamine TM 2000 transfected groups with 60.1±4.11 (n=3)cells in the 34p mol siRNA transfected groups. There was no statistical difference among the three groups.Conclusions1. In A7r5 cells transfection, the transfection efficiency of lipofectamineTM 2000 was approximation 92% after a 5-hour transfection. The optimal dosage of lipofectamineTM 2000 was 1.7μl and the final concentration of siRNA was 17nM.2. It was indicated that siRNA(CBR0915) could effectively inhibit ROCK-â…¡in mRNA and protein expression. However, siRNA (sc-36433) could not effectively inhibt ROCK-â…¡in mRNA and protein expression.3. Down-regulation of ROCK-â…¡gene could not influence the number of VSMC passing through the membrane. There was no statistical difference among the three groups. The role of ROCK-â…¡in VSMC migration should still be elucidated in future.