Effects Of HAIF-1 Recombinant Adenovirus On Proliferation And Migration Of A549 Cells

AIF-1 is a 17kDa Ca2+ bingding protein which locates in cytoplasm. It contains two EF-hand motifs, and has the ability to bind and polymerize F actin. AIF-1 is so little in nature that it is difficult to get a large number of natural products. It has been confirmed that AIF-1 could promote the proliferation and migration of different kinds of cells such as vascular smooth muscle cells, endothelial cells, macrophages, breast cancer cells and so on, and then affect the occurrence and development of related diseases. Meanwhile, there are few studies on the relationship between AIF-1 and cell differentiation. In this research, the human AIF-1 cDNA was cloned into prokaryotic expression vector, the expression of human AIF-1 was induced by IPTG, the protein of AIF-1 was purified. The influence of AIF-1 on the proliferation and migration was studied on the human lung adenocarcinoma cell line A549. The relationship between AIF-1 and the differentiation of murine skeletal muscle C2C12 cells was explored at the first time.Firstly, the primers were designed according to the human AIF-1 gene, the hAIF-1 cDNA was cloned into the plasmid pET-28a(+) for constructing the prokaryotic expression vector pET-28a(+)-hAIF-1. The plasmid pET-28a(+)-hAIF-1 was transformed into E.coli BL21 (DE3)cells. The expression was induced with IPTG, analyzed by SDS-PAGE and Western Blot. The solubility of recombinant hAIF-1 protein was identified by sonication and purification. The results showed that a clear recombinant protein about 20 kDa appears in the samples after induction of 1mM IPTG for 4 hours. The recombinant protein mainly existed in the form of soluble protein, and the high-purity AIF-1 protein was prepared by Ni-NTA.Next, the shuttle plasmid pDC-EGFP-hAIF-1 was constructed and co-transfected into 293 cells with adenovirus genomic plasmid pBHGloxdeltaE13Cre for packaging recombinant adenovirus. The titer of the virus was measured by TCID50. A549 cells were infected with the recombinant adenovirus, and then the expression of hAIF-1 was confirmed by PT-PCR and Western Blot. The effect of hAIF-1 on the proliferation and migration of A549 cells was examined by MTT, wound healing and Transwell assay. The results showed that the AdDC315-EGFP-hAIF-1 was successfully constructed and the titer of the virus was 2.5×108pfu/ml. Analysis of RT-PCR and Western Blot indicated that A549 cells could overexpress AIF-1 after infected with the recombinant adenovirus. The MTT assay showed that AIF-1 could increase the proliferative rate of A549 cells. The wound healing and Transwell assay proved that AIF-1 could promote the migration of A549 cells.Finally, C2C12 cells were induced to differentiate by the culture medium with 2% horse serum, and Myosin heavy chain (MHC) was used as the marker for the cell differentiation. The expression of AIF-1 was tested by Western Blot and Q-PCR. The results indicated that C2C12 cells started to express MHC protein, fused to form myotube after induced 3 days, then the number of myotube was increasing and the myotube was coarsening, the expression of MHC protein was also gradually increasing. The expression of AIF-1 was on the rise on protein level, but trended down on mRNA level. It could be said that the differentiation of C2C12 cells could affect the expression of AIF-1.