| Background:As the end stage of various kinds of liver diseases,liver failure has extreme high mortality rate and low cure rate.Recently major breakthroughs have been made in artificial liver and histological engineering techniques,but due to the limited clinical effects,there are still limitations to apply these techniques in clinical practice.Liver transplantation has been the only effective way to treat liver failure,however,due to the lacking of donors,high costs and immunological rejection,it still cannot be applied widely.Liver is an organ with strong regeneration ability to recover from some severe damages,so properly stimulating the regeneration ability of liver can be regarded as an ideal treatment for liver failure.Partial hepatectomy is widely used to treat multiple kinds of liver diseases in the filed of hepatic surgery,but the resection size of human liver must be limited in prevention of postoperative liver failure.After partial hepatectomy,the mature hepatocytes can reenter the cell cycle to implement regeneration process.If the liver regeneration of hepatopath patients can be enhanced and properly regulated,the indication of hepatectomy can extended to treat liver failure more effectively.Therefore,the researches on the regulatory network of liver regeneration after partial hepatectomy is of great importance to understand the liver regeneration process and to prevent and treat liver failure.Tmub1(transmembrane and ubiquitin like domain containing 1)is a shuttling protein which contains one ubiquitin-like domain and three transmembrane domains.Researchers found that during the liver regeneration,Tmub1 rapidly exports from nuclear and negatively regulates hepatocyte proliferation.However,the mechanisms of Tmub1 regulation are not clear.Our previous studies found that securin may be one of the target proteins of Tmub1.Securin is the inhibitor of separase and plays important roles in the cell cycle regulation.Securin must be degraded via the ubiquitin-proteasome system before the anaphase of mitosis for the proper separation of sister chromatids.In the endoplasmic reticulum associated degradation(ERAD)pathway,Tmub1 can form a protein complex with gp78 and SPFH2 to regulate the ubiquitination and degradation of HMG-CoA reductase.Based on this study,we infer that Tmub1 may influence the hepatocyte proliferation by regulating the ubiquitination of securin,but the effects and mechanisms of Tmub1 still need further investigation.Furthermore,the relationship between Tmub1 and ubiquitination lacks reports,and the functions of ubiquitinlike proteins still need to be illustrated.Objective:1.To investigate the expression pattern and the relationship between Tmub1 and securin during liver regeneration after rat 70% hepatectomy.2.To further observe the effect of Tmub1 on cell proliferation,cell cycle and the mitosis of rat hepatocytes.3.To identify the influence of Tmub1 and its ubiquitin-like domain(UBL)on securin's ubiquitination.Materials and Methods:1.The 70% rat hepatectomy was performed to establish the animal model of liver regeneration.RT-qPCR and Western Blotting assays were conducted to detect the mRNA and protein expression pattern of Tmub1 and securin.2.The lentivirus vectors of Tmub1 overexpression and shRNA knockdown were conducted.Rat normal hepatocyte BRL-3A cells were infected respectively and screened for stably infected cell lines.CCK-8,Edu,flow cytometry assays were performed to study the effect of Tmub1 expression on BRL-3A cell proliferation and cell cycle.3.Cell cycle synchronization and immunofluorescence assays were conducted to investigate the effect of Tmub1 expression on the mitosis of BRL-3A cells.Western Blotting assay was used to detect the expression levels of cyclins during mitosis.4.Immunoprecipitation,Western Blotting and RT-qPCR assays were utilized to identify the influence of Tmub1 expression on securin expression and its ubiquitination level.5.The plasmid vectors of the mutant and the truncated mutant of Tmub1 UBL domain were constructed to identify the effect of UBL domain on securin ubiquitination.6.All statistical analyses were performed with SPSS 19.0 software and statistical graphs were generated with GraphPad Prism 6.0 software.All data were presented as mean ± SD,comparisons within groups were made by repeated measures ANOVA with LSD(or student t test when only two groups were compared).P< 0.05 was identified to be statistically significant.Results:1.After 70% hepatectomy,the mRNA and protein levels of Tmub1 increased,peaked at 24 h to 48 h,and then decreased.The mRNA expression of securin are not consistent with the protein expression.The mRNA level gradually increased 12 h after the hepatectomy,peaked at 48 h and then decreased,however,the protein level decreased after the surgery,bottomed at 48 h and then recovered at 7d.The expression pattern of the mRNA levels of Tmub1 and securin are temporally positively correlated,and the expression pattern of the protein levels were temporally reversely correlated.2.Tmub1 overexpression reduced the proliferation rate of BRL-3A cells,while Tmub1 knockdown increased the proliferation rate.Tmub1 overexpression significantly increased the G1 phase cell population and decreased the S and G2/M phase cell population,while Tmub1 knockdown significantly increased the S phase cell population and decreased the G1 phase cell population.3.Tmub1 overexpression interfered the mitosis of BRL-3A cells and influenced the expression of cyclins in M phase.Specifically,in the negative control BRL-3A cells,the protein levels of cyclin A and cyclin B1 significantly decreased at 0.5h after released from M phase synchronization,but in the Tmub1 overexpressed BRL-3A cells,the protein levels of cyclin A and cyclin B1 remained high till 1.5h after released from M phase synchronization.4.The securin mRNA levels of Tmub1 overexpression or knockdown groups were not significantly different from the control groups.However,Tmub1 overexpression significantly increased the protein level of securin,while Tmub1 knockdown significantly decreased the protein level of securin.5.Tmub1 overexpression decreased the ubiquitination level of securin,while Tmub1 knockdown significantly increased the ubiquitination level of securin.6.After transfected with the UBL domain mutant plasmid vector,the ubiquitination level of securin was not significantly different compared to the negative control group.After transfected with the UBL domain truncanted mutant plasmid vector,the ubiquitination level of securin was also not significantly different from the negative control group.Taken together,neither the UBL domain mutant nor the UBL domain truncant mutant led to the level change of securin ubiquitination.Conclusions:The expression pattern of Tmub1 shows obvious regularity during liver regeneration.Tmub1 represses hepatocyte proliferation,influences the cell cycle and inhibits hepatocyte mitosis via inhibiting securin ubiquitination by its UBL domain.In conclusion,Tmub1 may be an important negative regulator of liver regeneration. |
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