The Molecular Mechanism Of SPINK3 Regulating Rat Hepatocyte Proliferation

Objective Serine peptidase inhibitor(SPINK3)is not only a trypsin inhibitor,but also a growth factor similar to epidermal growth factor(EGF),which could bind to epidermal growth factor receptor(EGFR),activating its downstream signaling pathway and then promoting cell proliferation.Previous studies in our laboratory have shown that the overexpression or interference of SPINK3 could influence the proliferation of rat normal hepatocytes,but its regulation mechanism is not yet clear.Therefore,in this paper we will do some further study on the mechanism of SPINK3 overexpression and interference on the proliferation of primary rat hepatocytes,and the regulation of SPINK3 recombinant protein on rat normal hepatocyte and hepatoma cell proliferation.Method In the study of gene level,the expression vector of SPINK3(p HBAd-MCMV-RFP-SPINK3)was constructed and infected primaty rat hepatocytes by adenoviral packaging,making SPINK3 overexpressed in primary rat hepatocytes.The SPINK3 siRNA fragment was designed and transfected into primary rat hepatocyte by liposome to reduce the expression of SPINK3.In the study of protein level,BRL-3A cell and RH-35 cell were treated with rat recombinant SPINK3(rrSPINK3)to exogenously increase SPINK3 content in both cells.MTT method was used to detect the effect of SPINK3 on cell activity,Brd U method was used to detect the effect of SPINK3 on cell proliferation,and flow cytometry was used to detect the change of cell cycle and cell apoptosis.Detection the expression of related genes and proteins in SPINK3 signaling pathway by qRT-PCR and Western blot.To analyze the mechanism of SPINK3 expression changes on primary rat hepatocytes,BRL-3A cells and RH-35 cells.Results The results of SPINK3 overexpression and interference on primary rat hepatocytes showed that overexpression of SPINK3 promoted the cell proliferation,but inhibited the cell apoptosis.Interference the expression of SPINK3 inhibited the cell proliferation,but promoted the cell apoptosis.In addition,the results of qRT-PCR and Western blot showed that SPINK3 may promote the cell ptoliferation by PI3K/AKT?JAK/STAT and SRC/P38 signaling pathways.The results of rrSPINK3 on BRL-3A cell and RH-35 cell showed that rrSPINK3 could promote BRL-3A and RH-35 cell proliferation,and significantly inhibited the apoptosis of RH-35,but had no obvious effect on the apoptosis of BRL-3A cell.The results of qRT-PCR and Western blot showed that rrSPINK3 might regulate BRL-3A cell proliferation through PI3K/AKT and SRC/P38 signaling pathways,while might ptomote RH-35 cell proliferation through JAK/STAT?hRAS/JNK1 and SRC/P38 singnaling pathways.Conclusions In summary,SPINK3 could promote the proliferation of primary rat hepatocytes,BRL-3A cells and RH-35 cells.