PLCG2 Promotes Hepatocyte Proliferation Via NF-κB And ERK Pathways During Rat Liver Regeneration

Phospholipase C(PLC) is an enzyme prevailed in eukaryotes, which is composed of a set of functionally similar protein, including PLC gamma 1 and PLC gamma 2, etc. Phospholipase Cγ2(PLCG2) has been implicated in the regulation of cell proliferation, transformation, and tumor growth. But its role in regulating liver regeneration is still unknown. To understand the mechanism underlying hepatocyte prolifertation during rat liver regeneration, we use the Rat Genome 230 2.0 Array to determine the expression changes of genes, then search the GO and NCBI databases for genes associated with cell proliferation, and QIAGEN and KEGG databases for the cell proliferation-related signaling pathways. We use expression profile function(Et) to calculate the activity level of the known cell proliferation signal pathways, and Ingenuity Pathway Analysis 9.0(IPA) to determine the interactions among these signaling pathways. The results showed that by the method of Rat Genome 230 2.0 Array, 606 rat liver regeneration related genes(RLR) were involed in signaling pathways regulating cell proliferation and the Et results indicated that the signaling pathways mediated by FGF, PDGF, INS, OSM, IL2 and HRH3 played key roles in regualating cell proliferation, where PLCG2 was in the centrol position among these pathways stated above. In conclusion, PLCG2 might play an important role in promoting cell proliferation during rat liver regeneration.In addition, recently inhibition of gene expression through RNAi is a tool increasingly used for the study of gene function in model systems. Thus, in this study, we investigated the mechanism of PLCG2 action using a short interference RNA(siRNA) method. The effects of PLCG2 on rat liver BRL-3A cells treated with siRNA are studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT assay), bromodeoxyuridine(BrdU) labeling assay, flow cytometry method(FCM), quantitative real time-polymerase chain reaction(qRT-PCR) and Western Blot. The results showed when PLCG2 was reduced, cell vitality and proliferation rate were significantly decreased(p<0.05). FCM analysis showed that the number of cell division phase(G2+M) was declined(p<0.05). RT-PCR and Western Blot revealed that the expression levels of NF-κB and ERK signaling related genes NF-κB2, FOS and JUN, target genes BCL2, CCNB1 and CCND1 were remarkably down-regulated in cells treated with PLCG2 siRNAs. Based on these results, we concluded that PLCG2 played an important role in rat liver cell proliferation via NF-κB and ERK pathways by regulating the expression of BCl2, MYC and CCND1.