Tmub1 Negatively Regulates Liver Regeneration Via Inhibiting STAT3 Phosphorylation

Background:All sorts of benign and malign disease of liver often causes liver Insufficiency at different levels.Liver failure is a major cause of death after surgery and one of the difficulties restricting the surgical treatment of patients with advanced liver cancer,especially in the surgical treatment of liver cancer.The common treatment methods for liver failure include non-biological artificial liver support therapy and liver transplantation,but these technologies still have many limitations and cannot solve the problems of postoperative liver failure well.Previous research found that the liver has a strong regeneration ability,the liver could restore their size and function in the short time after partial hepatectomy(PH).Therefore,the artificial regulation of liver regeneration which can restore the original function can be used as an effective method for the treatment of liver failure.For hepatology,if liver regeneration of hepatopathy patients can be enhanced,it can not only avoid liver failure after hepatectomy,but also broaden the indications of hepatectomy,providing strong support for the treatment of liver diseases.Therefore,further study on the mechanism of hepatocyte proliferation during liver regeneration and the role of liver regeneration regulatory network is essential.Transmembrane and ubiquitin like domain containing 1(Tmub1),also called hepatocyte odd protein shuttling(Hops),is a nucleocytoplasmic shuttling protein which contains a ubiquitin-like domain,a putative NES and a proline rich region.In the process of liver regeneration,Tmub1 rapidly shuttles out of the nucleus and is highly expressed,negatively regulating hepatocyte proliferation.Researchers found that Tmub1 starts to be expressed and transfers to the cytoplasm in 30 minutes after partial hepatectomy,and Tmub1 gradually moves back to the nucleus 12 hours after PH meanwhile the protein expression of Tmub1 reaches its peak within 48 hours.It has been reported that transcription factor C/EBP may positively regulate the expression of Tmub1 in hepatocyte proliferation by IL-6,and CAML may regulate the function of Tmub1 in IL-6-induced liver regeneration.Silencing of Tmub1 generates supernumerary centrosomes,multinucleated cells and multipolar spindle,suggesting that Tmub1 plays an important role in regulating the proliferation of hepatocyte by affecting the process of cell cycle.Tmub1 participates in the regulation network of liver regeneration,but the role of Tmub1 in liver regeneration and the effect of Tmub1 on cell proliferation have not been elucidated,and the related mechanisms need to be further studied.Objective:1.To investigate the influence of Tmub1 on hepatocyte proliferation during liver regeneration.2.To determine the effect of Tmub1 on hepatocyte proliferation by inhibiting STAT3 phosphorylation.3.To explore the interaction mechanism between Tmub1 and STAT3.Materials and methods:1.A regeneration model of 70% hepatectomy in mice was established,and the mice were transfected with Tmub1 overexpression plasmid via tail vein injection at the indicated time points(48 h before partial hepatectomy and 24 h after the surgery).No hepatectomy was performed in the negative control group.Liver regeneration of mice in each group after hepatectomy was evaluated by detecting liver weight/body weight and serum AST/ALT levels of mice 3 days after surgery.Proliferation-related proteins were detected by Western Blotting technology,and the regenerated liver tissues of mice in each group were analyzed by immunohistochemical technology,so as to determine the effect of Tmub1 on liver regeneration in mice and the expression of STAT3 pathway-related proteins..2.Human Tmub1 overexpression and shRNA plasmids were used to transfect human normal hepatocytes with L02,and the effect of Tmub1 ectopic expression on proliferation of L02 cells was detected by EdU.The effect of Tmub1 ectopic expression on STAT3 pathway-related protein expression were detected by Western Blotting technology.Using immunofluorescence and WB,the effect of Tmub1 ectopic expression on STAT3 and p-stat3 subcellular localization were detected.3.The reverse effect of STAT3 inhibitor stattic/ agonist OSM on tmub1-induced hepatocyte proliferation was observed by corresponding Rescue experiments.4.Bioinformatics analysis,chromatin immunoprecipitation(ChIP),and Luciferase reporter gene assay were used to investigate the induction effect of STAT3 on Tmub1 expression,and the interaction between Tmub1 and STAT3 protein was determined by immunoprecipitation.5.The data in this article were analyzed by SPSS19.0 software.The data are shown as the means ± S.E.M.The statistical analyses were performed using an unpaired Student's t-test,and P < 0.05(two tailed)was considered statistically significant.Results:1.Three days after hepatectomy,the liver weight/body weight ratio of mice with overexpression of Tmub1 was significantly lower than that of negative control group and blank group,suggesting that overexpression of Tmub1 delayed liver regeneration in mice.Serum aminotransferase test indicated that the serum ALT and AST levels in the overexpression Tmub1 group were significantly higher than those in the control group,suggesting poor recovery of liver function.Meanwhile,immunohistochemistry and HE staining showed that hepatocyte injury was more serious in the Tmub1 overexpression group,and the positive rates of ki-67 and p-h3 proliferation indicators were significantly lower than those in the negative control group and the blank group.Further Western Blotting results indicated that the phosphorylation level of STAT3 and the expression levels of MCM2 and Cyclin D1 were significantly decreased in overexpression of Tmub1 mice,suggesting that Tmub1 significantly inhibited the proliferation of liver cells during liver regeneration.2.Western Blotting showed that overexpression of Tmub1 significantly reduced the phosphorylation level of STAT3 and the expression levels of cyclin D1 and c-myc,but did not affect the total STAT3 protein level.On the contrary,after interfering with the expression of Tmub1,the phosphorylation level of STAT3 and the expression levels of cyclin D1 and c-myc were significantly increased,but the expression level of total STAT3 remained unchanged.Immunofluorescence indicated that Tmub1 did not affect the subcellular localization of STAT3.3.The EdU test suggested that L02 proliferation of human normal hepatocytes with overexpression of Tmub1 was significantly inhibited,but cell proliferation recovered after the addition of STAT3 agonist OSM.Interference with Tmub1 expression significantly promoted L02 cell proliferation,which was reverted by STAT3 inhibitor stattic.Western Blotting results also suggested that overexpression of Tmub1 caused a decrease in the phosphorylation level of STAT3 and the expression levels of cyclin D1 and c-myc,which could be recovered after the addition of OSM.And vice versa.4.Bioinformatics analysis showed that there was a STAT3 binding site in the promoter region of human Tmub1 gene.Chromatin immunoprecipitation(ChIP)and Luciferase reporter gene assay suggested that STAT3 could bind to the Tmub1 promoter,enhance the activity of the Tmub1 promoter and enhance the expression of Tmub1.5.Using co-ip,we found that Tmub1 and STAT3 can form protein complex.Conclusions:Tmub1 is a negative regulator of liver regeneration.STAT3 can induce the gene expression of Tmub1,and Tmub1 can inhibit the proliferation of liver cells by binding and inhibiting the phosphorylation and activation of STAT3.Tmub1 and STAT3 may form a negative feedback regulatory loop in the process of liver cell proliferation to regulate the process of liver regeneration.