Research On The Molecular Mechanism Of SPINK3 Regulating The Proliferation Of Rat Normal Liver Cells BRL-3A

Studies have shown that serine peptidase inhibitor,Kazal type 3(SPINK3)is a trypsin inhibitor,and also a growth factor that has an identical structure to epidermal growth factor(EGF),which could combine with epidermal growth factor receptor(EGFR)to promote cell proliferation.Through the functional genomics research of rat liver regeneration(LR),we discovered that SPINK3 was significantly up-regulated during rat LR.Many researches have reported that SPINK3 was closely related to the proliferation of rat intestinal epithelial cell(IEC-6).However,the role and regulation mechanism of SPINK3 on liver cell proliferation and LR remains unclear.Therefore,this paper will further to study the molecular mechanism of SPINK3 on rat normal liver cells BRL-3A proliferation.To shed light on the role and regulation mechanism of SPINK3 regulating hepatic cell proliferation in rat liver regeneration(LR),Rat Genome 230 2.0 microarrays were used to detect the expression profiles of LR-related genes after partial hepatectomy(PH).The results showed that SPINK3 was significantly up-regulated at 2–24 h and 72–168 h after PH,qRT-PCR and immunoblotting were used to validate the reliability of assay results.The process of its promoting cell proliferation is basically consistent with rat liver regeneration progress.Ingenuity Pathway Analysis 9.0(IPA)software was used to analyze the the possible mechanism of SPINK3 signaling regulating LR,and the results showed that SPINK3 could combine with EGFR and regulate hepatocyte proliferation during rat liver regeneration via p38 MAPK,PKC,JAK-STAT and AKT pathways.Further analysis showed that the above four signaling pathways could accelerate cell proliferation.Thus,SPINK3 was likely to promote cells proliferation in rat LR through p38 MAPK,PKC,JAK-STAT and AKT pathways,and then promote liver regeneration.To check this theory,this paper studied the effect of SPINK3 on rat normal hepatic cell line BRL-3A via gene addition and gene interference.Firstly,we synthetized the expression vector of pCDH-CMV-MCS-EF1-copGFP-SPINK3,and then the SPINK3 overexpress lentivirus was acquired by lentivirus packaging,and infected the BRL-3A cells.Then the SPINK3 over-expressing lentivirus infected the BRL-3A cells.Monoclonal BRL-3A which could over-express SPINK3 was picked out.At the same time,two short interfering RNA(siRNA)fragments of SPINK3 were designed and synthesized;qRT-PCR was used to screen out the best interference fragment.Then the interference fragment was infected in BRL-3A to down-regulated the expression of SPINK3 with the method of liposome transfection.The influence of SPINK3 expression changes on the cell vitality of BRL-3A was detected by MTT assay,and the results showed that the BRL-3A which transfected with recombinant plasmid pCDH-copGFP-SPINK3 proliferated quickly than the control group at 24,48,72 and 96 h after transfection,the proliferation of BRL-3A which infected with siRNA1 was significantly inhibited at 48 and 72 h after transfection.BrdU assay was used to analysis the influence of down-regulating SPINK3 expression on cell proliferation.We found that siRNA1 inhibited cell proliferation obviously at 48 h after transfection.Flow cytometry was used to detect the influnce of SPINK3 expression changes on cell proliferation and cell apoptosis,we found that over-expression of SPINK3 promoted BRL-3A cell proliferation and inhibited cell apoptosis significantly(P<0.05);the down-expression of SPINK3 promoted cell apoptosis and inhibited cell proliferation significantly(P<0.05).Western Blot and qRT-PCR were used to detect the expression changes of SPINK3,EGFR and SPINK3 signaling pathways-associated genes and proteins,and then further to analysis the molecule mechanism of SPINK3 regulating hepatic cell proliferation during rat LR.The results indicated that over-expressing SPINK3 could up-regulated the expression of pathways-associated gene ERK1,AKT1,STAT3,transcription factor NF-?B1 and the downstream target gene CCND1.And knockdown of SPINK3 expression with siRNA1,the above genes and proteins were down-regulating expression.It was concluded that SPINK3 could activate ERK1/2,JAK-STAT and PI3K-AKT pathway through regulating the expression of ERK1,STAT3 and AKT1,then regulate the expression of downstream cell proliferation or apoptosis-related transcription factors,such as CCND1,NF-?B1,BAX and CASPASE3 et al,and eventually regulate the proliferation and apoptosis of rat normal hepatic cell BRL-3A in vitro.