The Role Of Tmub1in Liver Cell Proliferation Induced By IL-6and Its Regulation Of Expression

Background:Hepatic failure is the end-stage status of the various liver diseases, common butdifficult to be effectively treated. We know that excessive liver resection will lead to thepostoperative hepatic failure, which greatly limits the practice of liver surgery. Because ofthe complexity of liver function, artificial liver technology does not completely replaceliver function, and its long-term efficacy is not satisfactory; due to the number of donors,immune rejection, treatment costs and other reasons, liver transplant cannot be used widely.Therefore, effectively stimulating regeneration of their own liver is an ideal way to solvethese problems. Normal adult liver cells are stable cells, mitosis occurs rarely, but activeproliferation occurs rapidly in the residual liver cells when they are damaged and stimulatedby other factors and it shows obvious regularity. If we can reasonably stimulate the liverregeneration of the hepatosis patients, then we can make up for the lack of bioartificial liverand liver transplantation, and effectively treat hepatic failure. A lot of researches on liverregeneration have been actively carried out. However, the progress is not satisfactory andmechanisms of liver regeneration is not entirely clear.Tmub1(transmemebrane and ubiquitin－like domain containing1), playing animportant role in the process of liver cell proliferation is a new molecule first reported byFazia in2005. The expression of Tmub1significantly increases in liver regeneration, andoverexpressed Tmub1has a disincentive effect on cell proliferation. We aso found theexpression of Tmub1significantly increases after partial hepatectomy. Tmub1has theability of inhibiting expression and activation of STAT3induced by IL-6,which suggestingTmub1may play the role of negative feedback in proliferation of liver cells, but its specificmechanism of action remains to be researched in depth. CAML (Calcium modulatingcyclophilin ligand), expressing in all organizations, plays an important role in proliferation,apoptosis and function. It is found in recent research that CAML is the target protein ofTmub1but there is no description on specific mechanism of their interaction, its impact on liver regeneration is still unknown. During the process of liver regeneration, if Tmub1andCAML interact with each other, the proliferation of liver cells will be impacted, which maybe one of the mechanisms by which Tmub1controls the proliferation of liver cells. IL-6isone of important signals which excites liver regeneration and plays a direct or indirect rolein regulating the expression of a variety of proliferation-specific genes. Our grouppreviously found that IL-6can up-regulate the expression of Tmub1, suggesting that theexpression and regulation of Tmub1is linked to IL-6signal passway. Therefore, definingthe relationship of Tmub1expression with IL-6and its downstream molecular is veryimportant. Identifing the molecular characteristics of Tmub1and regulation mechanism ofits expression, which will provide new evidence to explore the molecular regulationmechanism of liver regeneration and the proliferation of liver cells from a new perspective,has an important clinical significance.Objective:1. To understand affection of Tmub1on proliferation of liver cells, especially inducedby IL-6.2. To identify whether Tmub1and CAML interact with each other in the growthingliver cells and to know the probable mechanisms of the action.3. To know the probable mechanisms of IL-6to regulate expression of Tmub1andidentify transcription factors probably participating Tmub1transcription.4. To define the relationship between C/EBPβ, the downstream transcription factor ofIL-6, and Tmub1expression.Materials and Methods:1. The lentiviral vectors of Tmub1ShRNA were used to inhibit the expression ofTmub1in rat's hepatocytes; IL-6was used to induce the proliferation of liver cells;[3H]Thymidine incorporation assay was performed to assess changes in cell proliferationrates; the techniques of RT–PCR and Western-blot were used to exam the geneexpression of Tmub1.2. Laser scanning confocal microscopy and immunoprecipitation, followed by Westernblotting were used to assess the interaction between Tmub1and calcium modulatingcyclophilin ligand (CAML) protein. The effect of Tmub1on calcium ion influx intoBRL-3A cells was measured by inversion fluorescence microscopy. 3. The promoter on-line analysis software was used to analyze the5' upstreamsequence of Tmub1gene to find the potential binding sites of of transcription factors inrelation to liver regeneration.4. According to the results of bioinformatics analysis, a series of the luciferase reportergene vectors including the5' upstream sequence of Tmub1gene were constructed. In orderto detect the promoter activity of these deleted sequences, the reporter gene vectors weretransfected into hepatic cells and the activity of luciferase was detected. Influence of IL-6on Tmub1promoter activity were also examed with same method.5. The siRNA of rat C/EBPβ was synthetize and transfected into BRL-3A cells.under the condition of the inhibition of C/EBPβ expression, the affection of C/EBPβ onTmub1expression was observed.6. The overexpressed vectors of C/EBPβ were constructed. The overexpressed vectorsof C/EBPβ and Tmub1luciferase reporter gene vectors were transfected into BRL-3A cellsto observe the affection of C/EBPβ on the transcriptional activity of the5' upstreamsequence of Tmub1gene.Results:1. Cell lines transfected with Tmub1shRNA lentiviral vector were obtained.After infecting BRL-3A cells with Tmub1shRNA vectors, stably infected cells weresorted with a flow cytometer, expanded, and named BRL-3A/256cells. Negative controlcells were infected with the control vector, and named RL-3A/Neg cells.Tmub1expressionin BRL-3A/256cells was inhibited effectively.2. The optimum working conditions of IL-6in inducing hepatocyte proliferationwas confirmed.We found that the most effective concentration and treatment time of IL-6onproliferation of rat hepatocytes were15ng/ml for12h, respectively. At thecondition,incorporation efficiency of [3H]-TdR reached the top. The working conditionsof IL-6will be used in following experiments.3. Tmub1expression was up-regulated significantly by IL-6in rat liver cells.During the process of the proliferation of liver cells induced by IL-6, IL-6increasedmRNA and protein level of Tmub1obviously (p<0.05). In addition, IL-6induction reversedinhibitory effect of shRNA lentiviral vector on Tmub1expression partly (p<0.05). 4. Inhibiting Tmub1expression promoted proliferation of liver cells.We assessed the effect of Tmun1knockdown on hepatocyte proliferation in thepresence or absence of IL-6treatment. In the absence of IL-6treatment,[3H]thymidineincorporation into BRL-3A/256cells was significantly higher than in control cells, butdifferences were more significant after IL-6treatment.5. Interaction of Tmub1with CAML protein exists in rat liver cells.The confocal microscopy images showed colocalization of Tmub1with CAML proteinin the cytoplasm of rat liver cells. Results from our immunoprecipitation assays showedbinding between Tmub1and CAML proteins. Moreover,we also found that expression ofCAML protein was upregulated after Tmub1knockdown, compared to the parental andnegative control cells. Furthermore, Tmub1knockdown had the ability to regulate Ca2+influx in rat liver cells.6. Several potential binding sites of transcription factor were found in5'upstream sequence of Tmub1gene.Sequence between-2007～+50bp of Tmub1gene5' upstream sequence containedseveral potential binding sites of transcription factors related to liver regeneration, whichinclude5C/EBP binding sites,2AP-1binding sites, a STAT binding site and2CREBbinding sites.7. Luciferase report gene vector of rat Tmub1gene5' upstream deletion fragmentwas constructed.Iuciferase report gene vector of12deletion fragments which contained-2008~+50,-1734~+50,-1637~+50,-1559~+50,-1392~+50,-1267~+50,-876~+50,-679~+50,-467~+50,-273~+50,-152~+50,-57~+50sequence, was constructed and named T1-T12respectively. Furthermore, all vector were identified to be properly constructed by MluI andXhoI double-enzyme cleavage, PCR amplification and sequencing determination.8.-2008~+50sequence of Tmub1gene Tmub1showed obvious transcriptionalactivity.Luciferase activity of all report gene vectors were significantly higher than the controlgroup(P<0.05) indicating that-2008~+50sequence has obvious transcriptional activity.Except T4and T6vector, other deleting sequence transcriptional activities show descendingtendency successively. 9. Transcriptional activities of Tmub1gene promoter region was enhanced byIL-6obviously.When treated with IL-6, except cells transfected with T5and T6vector, luciferaseactivity of other groups were increased obviously(P<0.05)indicating that IL-6had theability of enhancing Tmub1gene transcription. According to the difference of luciferaseactivity in all groups, transcriptional activitiy of Tmub1promoter sequence was decreasedobviously after deleting sequence containing C/EBP β, STAT3, AP-1binding sites.10. Positive correlation of C/EBPβ and Tmub1expression was found in rat livercells.When C/EBP β was inhibited with siRNA, expression of Tmub1was also decreasedobviously (p<0.05), indicating that C/EBP β and Tmub1expression was related positively.11. Over expression vector of C/EBP β was constructed.Whole expression sequence of C/EBP β was inserted into pCMVTag2B vector, thenthe vector were identified by BamHIU and XhoI double-enzyme cleavage, PCRamplification and sequencing determination. The vector was proved corrected constructedand named pCMVTag2B-C/EBP.12. Transcriptional activities of Tmub1gene promoter region was enhanced byC/EBP βobviously.When pCMVTag2B-C/EBP vector and Tmub1gene luciferase report gene vectorswere co-transfected into BRL-3A cells, except cells transfected with T6vector, luciferaseactivity of most groups were increased obviously. The result indicated that C/EBP β hadability of increasing Tmub1gene transcription. However, compared with T1, T10and T11group respectively, the luciferase activity of T2, T11and T12group declined moreobviously, which indicated that enhancement of C/EBPβ on Tmub1transcription wererelated to-2008~-1735,-273~-153and-152~-58sequence in Tmub1gene.Conclusion:1. Tmub1has ability of inhibiting the proliferation of rat liver cells, and play anegative control role in the process of liver regeneration.2. Interaction of Tmub1and CAML exist in growthing liver cells, and Tmub1regulates hepatocyte proliferation through participating in degradation of CAML proteinand affecting Ca2+influx. 3. IL-6enhances Tmub1expression through activating its downstream transcriptionfactors such as C/EBP β, STAT3and AP-1.4. C/EBP β correlated with Tmub1expression positively, and may be one of the keytranscription factors which involved in regulation of Tmub1gene expression.