Adeno-associated Virus-mediated RNA Interference Inhibits Expression Of Myocardin

Background Atherosclerosis (AS) is one of diseases which endanger the health of mankind. About its outbreak mechanism, traditionary hypothesizes think that the smooth muscle cells (SMC) in AS plaques come from the moving and increasing of SMCs in tunica media of the blood vessels. However, recently, a series of study enunciated that the SMCs in AS plaques not only come from tunica medias, also may come from bone marrow mesenchymal stem cells (BMSC) and hematopoietic stem cells. Myocardin is the most important factor promoting SMCs differentiation what have been found so far. It only expresses in embryo growth period,cardiac muscles and tissues riching in smooth muscles, just as adults` blood vessels etc. If adeno-associated virus-mediated RNA interference can inhibit expression of myocardin, the differentiation from MSCs to SMCs will be inhibited. It may provide new therapeutic strategies for the gene therapy of atherosclerosis.Objective To construct the recombinant adeno-associated virus vector encoding the specific small hairpin RNA (shRNA) for mouse myocardin gene and to observe its silencing effect on myocardin gene and its contribution during the differentiation of mouse bone marrow mesenchymal stem cells into smooth-muscle-like cells induced by PDGF-BB in vitro.Methods Using the technique of gene recombination, the rAAV expression plasmid pAAV-MCS-myocardin-siRNA-U6 was constructed with the specific myocardin-siRNA sequence. It was identified by DNA sequencing and restriction digestion. Then HEK293T cells were cotransfected with pAAV-MCS-myocardin-siRNA-U6 together with pAAV-RC plasmid and pHelper plasmid. Meanwhile, rAAV-hrGFP and rAAV-LacZ were made by the same method. The titer was indirectly measured by rAAV-LacZ. The recombination adeno- associated virus vector infected the mouse BMSCs induced by PDGF-BB. The expression of myocardin mRNA and SM22αmRNA,α-SM-actin mRNA was identified by RT-PCR.Results The recombinant adeno-associated virus vector carrying the myocardin-siRNA sequences was successfully constructed. The viral titer was about 1.5×1010/ml. And the recombinant virus vector inhibited the expression of the myocardin gene in mouse BMSCs. Meanwhile the expression of the SM22αandα-SM-actin was down-regulated.Conclusions The recombinant adeno-associated virus vector rAAV-myocardin-siRNA-U6 can interfere the expression of myocardin gene significantly. And it can also inhibit the differentiation from mouse BMSC into SMC-like. It illustrates that myocardin plays a key role in the differentiation of mouse bone marrow mesenchymal stem cells induced by PDGF-BB into smooth-muscle-like cells.