Effect Of FSCN1on The Migration Capacity Of Wuzhishan Mini Pigs’ Bone Marrow Mesenchymal Stem Cells

Wuzhishan mini pig (WZSP) is the unique well-qualified breed in our country, and its physiological index is close to that of human being, which makes it a ideal animal model for human being. Mesenchymal stem cell (MSC) is a kind of non-hematopoietic stem cell generated from mesoderm. It can survive in adherence in vitro, and it can also differentiate to mesenchyma cell (MC) in large amount under suitable condition,, and continuously create connective tissue cell such as bone, cartilage, fat, fibroblast, endothelial cell, etc. At present, cell migration is an important topic in cytobiology, and it has been involved in many physiological phenomena including cell foraging, wound healing, cancer metastasis and so on. As a result, it is very important for the understanding of the mechanism of the MSCs migration. As a cross-link protein, fascin-1is coded by FSCN1gene, crosses link to F-actin, which is involved in the formation of filopodia, lamellipodia and micro spine. These filopodia and spine has close relationship with the cell motion, invasion and metaptosis, which indicates that FSCN1is involved in the migration of MSCs. In this study, the effect of Fascin on the migration capacity of WZSPs’bone marrow MSC is explored thro μ gh knock-down the expression of FSCN1, which leads to gene silencing for FSCN1.In this experiment, four specific short hairpin RNA (shRNA) sequences for Fascin was designed, linked with vectors, and the bone marrow MSCs were transfected by above four vectors, which were mediated by liposome. The qRT-PCR assay was used to evaluate the interference effect, and the shRNA with best interference effect was selected. After the shRNA vectior was digested with the restriction endonuclease, and the bone marrow MSCs were electroporated, and then were transfected the linearized shRNA vector. The stabilized cell line which knockdown expression of FSCN1is obtained after removing the untransfected cells by supplemented with G418. The interference effect was detected thro μ gh qRT-PCR assay and Western blot analysis at mRNA level and protein level of the FSCN1. The result showed that the expression of FSCN1level was effectively cut down in the transferred cells transfected with the specific shRNA. The migration ability of the bone marrow MSCs for the interference cells and non-interference cells was detected through scratch assay and transwell migration assay. The results indicated that there was a lower migration ability for the interference cells, which illustrated that FSCN1gene was implicated in the regulation of migration ability for the bone marrow MSCs from WZSP.In this experiment, the effective interference shRNA was selected and the stable interference cell line for FSCN1and control with non-interference cell line for FSCN1had been obtained. The migration ability was tested through scratch assay and transwell migration assay. The results showed that as actin binding protein, Fascin was involved in regulating cytoskeleton function, which had established the foundation for farther study in FSCN1.