Comparative Analysis Of The Molecular Mechanism Of SPINK1 Regulating The Proliferation Of Rat Normal Liver Cells BRL-3A And Rat Hepatoma Cells RH-35

Serine peptidase inhibitor,Kazal type 1(SPINK1)acts as a trypsin inhibitor,its spatial structure is similar to that of epidermal growth factor(EGF).Studies indicate that,the same function as EGF,SPINK1 can interact with epidermal growth factor receptor(EGFR)and promote various normal cells and cancer cells proliferation and differentiaion.In our laboratory,Rat Genome 230 2.0 microarray was used to detect the gene expression profiles of hepatocytes of rat regenerative liver(RL)after partial hepatectomy(PH),and the results indicate that Spink1 was significantly up-regulated at 6,24 and 72 h after PH,it was known that hepatocytes proliferation occurred during 6-72 h after PH,indicating that SPINK1 was possibly related with hepatocytes proliferation in LR.The gene expression profiles of hepatocytes in diethylnitrosamine(DENA)induced rat hepatic cancer(HC)was also detected by Rat Genome 230 2.0 microarray,and the results showed that Spink1 significantly up-regulated at 12-16 w after DENA treatment,as we knew,hepatocytes was rapidly proliferating at 12-16 w,indicating that Spink1 was possibly related with hepatoma cell proliferation.But the regulatory function of SPINK1 on hepatocytes and hepatoma cell proliferation and LR is not sure.Recent researches have shown that SPINK1 could regulate cell proliferation and migration via interacting with EGFR.In this study,immunoprecipitation(IP)assay was used to conform it,and the result indicated that SPINK1 could interact with EGFR.Previous studies suggested that activated EGFR could promote cell proliferation via p38 MAPK,ERK,JNK,PKC,JAK-STAT and AKT signaling pathways,SPINK1-EGFR signaling pathway was built by Ingenuity Pathway Analysis 9.0(IPA)software,and conformed by relevant literature information,and the results showed that SPINK1 could interact with EGFR and regulate cell proliferation via the above signaling pathways.To study the mechanism of SPINK1 regulating hepatocytes and hepatoma cell proliferation,recombinant plasmid of Spink1 was used to treat BRL-3A cell,recombinant protein of rat SPINK1(rrSPINK1)was used to treat BRL-3A cell and RH-35 cell respectively,MTT,EdU and flow cytometry were used to detected the effect of SPINK1 up-regulation on cell proliferation and cell cycle of BRL-3A and RH-35,qRT-PCR and western blot were used to detect the genes/proteins expression of signaling pathways induced by SPINK1 based on IPA software,the regulatory mechanism of SPINK1 on BRL-3A cell and RH-35 cell proliferation was speculated based on the results above all.The results showed that the up-regulation of Spink1 significantly increased cell viability and proliferation,the number of cells in S and G2/M phase,and the genes/proteins expression related to cell proliferation and anti-apoptosis in BRL-3A.Conversely,the number of cells in G1 phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased.The expression of p38,ERK and JNK signaling pathways-related genes were significantly changed,and were consistent with expected results.When the BRL-3A cells were treated with rrSPINK1,the similar results obtained.After treating with rrSPINK1,cell viability and proliferation,the number of cells in S and G2/M phase,and the genes/proteins expression related to cell proliferation and anti-apoptosis in RH-35 were significantly increased,the number of cells in G1 phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased.The expression of p38 and JAK-STAT signaling pathways-related genes were significantly changed,and were consistent with expected results.We also used MTT to analyze the effect of anti-pEGFR monoclonal antibody on the cell viability of BRL-3A cells that overexpressed Spink1,and the results indicated that anti-pEGFR antibody could inhibited the viability of BRL-3A cells that overexpressed Spink1,and the effect was enhanced by the increased dose of anti-pEGFR antibody.The above results further proved that SPINK1 promoted cell proliferation via connecting with EGFR.Above all,SPINK1 combine with EGFR to active the genes/proteins related p38,ERK1/2 and JNK signaling pathways,and promote BRL-3A cell proliferation via regulating cell proliferation-gens/proteins MYC and CCND1 and apoptosis-related genes/proteins BAX and CASPASE3;and to active the genes/proteins related p38,JAK-STAT3 signaling pathways,and promote RH-35 cell proliferation via regulating cell proliferation-gens/proteins MYC and CCND1 and apoptosis-related genes/proteins CASPASE3.