| BackgroundHepatic fibrosi（sHF）is a pathological syndrome of chronic liver disease causedby different etiologies. It is a common pathological process that different damagingfactors induce liver damage. Currently, it is considered that the activation of hepaticstellate cells (HSCs) play a key role in development of liver fibrosis, Activated HSCscould overexpress α-SMA, which further stimulates the numerous synthesis ofECM in the liver and exceeds the capacity of hepatic clearance. In the initial phase,HSCs activation is started mainly by neighboring cells (such as liver cells, Kupffercells, etc.) through paracrine effects. In addition, HSCs secrete some cytokines, thatpromote the activation of Kupffer cells and further promote themselves activation byautocrine and paracrine effects.Small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein. Differentfrom the degradation pathway mediated by ubiquitin-proteasome, the results ofSUMO is not to degradate the protein, but to enhance their stability, adjust theirsubcellular localization, or regulate their transcriptional activity by modifying them,that plays a more extensive features. Our previous study found that following thedevelopment of hepatic fibrosis, SUMO-1expression increased gradually in the liver.SUMO-1may contribute to the activation and proliferation of HSC and promote theirsecreting large amounts of collagen by enhancing the expression of TGF-β1andPDGF, then promoted hepatic fibrosis. The process of SUMO modification requiresthe participation of a variety of enzymes, but different from ubiquitin modificationwhich has several numbers of E2enzymes. SUMO modification has only one SUMOligase, which is ubc9protein. Therefore, we infer ubc9may regulate the developmentof liver fibrosis and even liver cancer by adjusting the SUMO modification process.To further study the role and mechanism of ubc9in the process of liver fibrosis and toprovide a new theoretical basis for liver fibrosis therapy, RT-PCR and Western blotanalysis were used to detect the expression of ubc9among normal liver cell line L02,human-derived hepatic stellate cells LX-2and HepG2, SMMC-7721hepatoma cells in this study. After transfection of pcDNA6.2-mir ubc9into LX-2, the expressionlevels of ubc9、α-SMA、IKBα、p65and Bcl-2were examined, the changes ofTNF-α and IL-6concentration were detected by ELISA, flow cytometry analysis wascarried out as well.ObjectiveTo explore the effects and mechanism of ubc9in the activation and proliferationof LX-2.MethodsNormal liver cell lines L02, hepatic stellate cell line LX-2and hepatoma cellsHepG2, SMMC-7721were cultured in vitro respectively. RNA and protein ofdifferent cell lines were extracted, and RT-PCR and western blotting were employedto detect the ubc9expression level in four cell lines. After transfecting pcDNA6.2-mirubc9into LX-2, RT-PCR and western blotting were employed to detect theexpression of ubc9in LX-2. Western blotting was further to detect the expressionlevel of α-SMA、IKBα、p65and Bcl-2in LX-2after transfecting pcDNA6.2-mirubc9, the changes of TNF-α and IL-6concentration were detected by ELISA. Andflow cytometry was to probe examine the cell cycle and apoptosis of LX-2.ResultsThe results of RT-PCR and Western blot showed that Ubc9were expressed innormal human liver cells, hepatic stellate cells and liver cancer cells. ThepcDNA6.2-mir ubc9could inhibit the expressions of ubc9. The expression differenceof α-SMA, Bcl-2, and p65in LX-2once the down-regulating of ubc9was statisticallysignificant（P <0.05）. After being transfected pcDNA6.2-mir ubc9, the cell cycle ofLX-2was blocked in G2/M phase, and cell apoptosis increased, the difference wasstatistically significant（P <0.05）. These results suggested that down-regulting ubc9expression could inhibit the proliferation of LX-2.Conclusions1. Down-regulting ubc9may inhibit the proliferation of LX-2by the action ofBcl-2;2. Ubc9may regulate NF-κB signaling pathway to participate in the process ofliver fibrosis by the action of IKBα and p65. |
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