Study On The Roles Of Osteopontin In Regulating The Growth Of BRL-3A Cell Line

The liver is the only organ with extraordinary regenerative capabilities.However,as the largest digestive gland,the liver is vulnerable to a variety of injure caused by metabolites,toxin,microorganisms,resection and so on.Therefore,the mammalian liver has developed an unique capability to restore when the liver is damaged.Osteopontin(OPN)is a kind of secretory phosphorylation glucoprotein,and is widely distributed in a variety of tissues,cells and body fluids.Meanwhile,increasing studies have shown that liver regeneration is closely related to inflammation,cell activation and proliferation and so on.OPN,as acute phase protein and inflammation regulatory factor,is not only closely associated with liver diseases,may also participate in liver regeneration and repair of damaged liver cells.Our preliminary in vivo experiments showed that OPN had obvious effects on promoting liver regeneration,and hepatocytes are the main mobilized cell types during liver regeneration.However,it remains unclear whether OPN can effectively target hepatocytes,and then affect liver regeneration.Therefore,this study aplplied BRL-3A cells as our study subject,which belong to normal rat liver cell line,investigated the effects of OPN on growth of BRL-3A cells in vitro by changing the expression level of exogenous OPN,and then clarified the signaling pathway of OPN.Firstly,the over-expression of OPN in BRL-3A cells was achieved by lentivirus-mediated transgene,and the decreased expression of OPN was conducted by transfecting with short interfering RNA(siRNA).Then the effects of OPN on biological functions of BRL-3A cells were detected by MTT,BrdU incorporation,flow cytometry,qRT-PCR and Western blot.The results showed that the number of S phase and G2/M phase cells increased when OPN gene is over-expressed in BRL-3A cells,the expression levels of key proteins including ERK1/2,AKT,NFKB1 and JAK/STAT3 were increased,which resulted in the down-regulation of BAX and up-regulation of BCL-2 and CCND1.In contrast,cell viability,cell proliferation rate and the number of S phase and G2/M phase cells were all decreased after the expression of OPN in BRL-3A cells were redued by siRNA,and the expressions of key proteins MAPK,AKT1,STAT3 and NFKB1 were significantly reduced,which consequently led to the increasing of their target proteins BAX and the decreasing of BCL-2 and CCND1.In conclusion,OPN may regulate the expression of target genes BCL-2,BAX and CCND1 through JAK/STAT3,ERK1/2,PI3K/AKT and NF-?B signaling pathways,and then eventually promot hepatocyte proliferation.