Study On The Effects And Mechanism Of Reg?? On The Growth Of Rat Hepatocytes BRL-3A

Regenerating islet-derived 3 gamma(Reg??),as an immunity and inflammation regulatory cytokine,is expressed in inflammation,tumors and other diseases or damaged tissues.It mainly participates in promoting cell proliferation,inhibiting apoptosis and preventing wound infection.Previous studies have shown that Reg?? is closely related to the gastrointestinal tract digestive system diseases,such as pancreas and liver,can promote repair of pancreas,intestine and skin tissue injuries.Our previous research found that Reg?? may be involved in modulating liver injury and repair.However,there is so little scientific research on its receptors and the underlying mechanism during liver regeneration.Therefore,this study intends to explore the role and mechanism of Reg?? in the growth of BRL-3A in rat hepatocytes.In this study,the Reg??-overexpressed rat BRL-3A hepatocytes were obtained by the lentivirus mediated transfection,and effects of Reg?? on the activity and proliferation of BRL-3A were detected by MTT and EdU methods respectively,so as cell cycle and apoptosis by flow cytometry.The results showed that cell growth activity of BRL-3A was increased significantly,proliferative cell number increased obviously,and cell cycle process was promoted after overexpression of Reg??.However,the apoptosis of BRL-3A cells was not significantly affected.To further explore the regulatory mechanism of Reg?? on rat hepatocytes BRL-3A,IPA,GenMAPP and KEGG database and related literature were used to build the Reg?? signal pathway network diagram,then Western Blot was carried out to detect the downstream proteins in signal pathway,and the expressions of p-STAT3,p-PKC and cell proliferation-related proteins CCNA2,CCND1,c-MYC were significantly increased,while obvious expression changes of MAP3K12,p-JNK,P38 MAPK,MEK1,p-ERK1/2 and NF-?B(p-p65)were not observed compared to those in control group.The immunofluorescence result after transient transfection of pcDNA3.1(-)-Reg?? plasmid to BRL-3A cells suggested that Reg?? was transported into the nucleus from cytoplasm and finally located around the nuclear membrane and in the cytoplasm.Further detection by co-immunoprecipitation indicated that Reg?? could interact with itspotential receptors EGFR and EXTL3.Furthermore,the result of immunofluorescence indicated that Reg?? was co-located with EGFR or EXTL3 in BRL-3A hepatocytes at different time points after transfection.It can be concluded that Reg?? may activate JAK-STAT and PKC signaling pathways by interacting with EGFR or EXTL3,then regulate the expressions of cell proliferation/apoptosis related proteins,and ultimately promote the proliferation of BRL-3A hepatocytes.