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

Regenerating islet-derived 3 gamma(RegⅢγ),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 RegⅢγ 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 RegⅢγ 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 RegⅢγ in the growth of BRL-3A in rat hepatocytes.In this study,the RegⅢγ-overexpressed rat BRL-3A hepatocytes were obtained by the lentivirus mediated transfection,and effects of RegⅢγ 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 RegⅢγ.However,the apoptosis of BRL-3A cells was not significantly affected.To further explore the regulatory mechanism of RegⅢγ on rat hepatocytes BRL-3A,IPA,GenMAPP and KEGG database and related literature were used to build the RegⅢγ 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-κB(p-p65)were not observed compared to those in control group.The immunofluorescence result after transient transfection of pcDNA3.1(-)-RegⅢγ plasmid to BRL-3A cells suggested that RegⅢγ 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 RegⅢγ could interact with itspotential receptors EGFR and EXTL3.Furthermore,the result of immunofluorescence indicated that RegⅢγ was co-located with EGFR or EXTL3 in BRL-3A hepatocytes at different time points after transfection.It can be concluded that RegⅢγ 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.