The Effect Of FAT10 On HCC Cell Proliferation By Regulating Expression Of WISP1 And Its Mechanisms

Objective:Investigate the expression and biological function of WISP1 in hepatocellular carcinoma(HCC)and the effect of FAT10 on the biological characteristics of HCC cells by regulating WISP1 protein expression;Clarify the mechanism of the expression of WISP1 regulated by FAT10;Clarify FAT10 promote the expression of WISP1 mRNA through stablization ?-catenin;Next,clarify the mechanism that FAT10 leads to the discordance of WISP1 protein and mRNA expression;Finally,we will clarify the molecular mechanism of FAT10 regulating WISP1 expression and its effect on the biological characteristics of HCC cells.Methods: Fistly.Protein mass spectrometry and other experiments were used to detect the expression level of WISP1 protein in HCC tissues;The relationship between WISP1 expression and clinical pathological characteristics of patients was analyzed by single factor and multiple factor regression.The survival analysis and analysis of WISP1 Relative high expression and relatively low expression of the total survival rate;Secondly,WISP1 was overexpressed in HCC cell lines,cell proliferation and expression of proliferation associated antigen wrer detected;Nude mouse tumor test to detect the formation of WISP1 on the tumor cell line tumor ability of liver cancer;Alter the expression of FAT10 and alter the expression of WISP1,HCC cell proliferation ability was detected by EdU;In the knockout FAT10 cell line,the expression of WISP1 was detected by restoring the expression of FAT10 or inducing the expression of FAT10;Thirdly,Co-IP was used to detect the combination of endogenous and exogenous FAT10 and WISP1;confocal laser confocal detection of the co localization of FAT10 and WISP1;after silence UBA6 and USE1 expression,the Co-IP was used to detect the alteration of FAT10-WISP1 complex,and a Flag-FAT10?GG mutant overexpressed plasmid was constructed by the deletion of the FAT10-C terminal diglycine,and the Co-IP was carried out and the WISP1 expression was detected.The changes of the binding of FAT10 and WISP1 and the expression of WISP1 protein;Overexpression of wild type FAT10 and mutant FAT10?GG,WISP1 protein and mRNA were detected respectively;Overexpression wild type FAT10 in HCC cells,WISP1 protein and mRNA expression were detected.Fourthly.Alteration of the expression of ?-catenin,WISP1 protein,WISP1 mRNA,WISP1 promoter luciferase level were detected;Overexpression or silence of FAT10 expression,WB and qRT-PCR were used to detect ?-catenin,WISP1 protein and mRNA expression.At the same time,the changes of ?-catenin,WISP1 protein and mRNA were detected WB and qRT-PCR.;Silence FAT10 expression and overexpressed ?-catenin expression at the same time,WB and qRT-PCR were used to detect the expression of ?-catenin,WISP1 protein and ?-catenin.Fifthly,WB and qRT-PCR was used to detect expression of FAT10,?-catenin,WISP1 protein and mRNA;Gradually overexpress FAT10 or FAT10?GG,WB and qRT-PCR were used to detect the expression of FAT10,?-catenin,proteins and mRNA.Sixthly,the expression of FAT10,?-catenin,WISP1 protein and mRNA were detected by hybridization;The expression of FAT10,?-catenin,WISP1 protein and mRNA in fresh HCC tissues were detected,WB and qRT-PCR,the correlation between the four groups was analyzed;And the expression of FAT10,?-catenin,protein and WISP1 protein were detected by IHC and hybridization.Results: Firstly,the results showed that the expression of WISP1 protein was low in the tumor tissue of HCC;Further analysis showed that the expression of WISP1 protein was significantly correlated with tumor size and clinical stage,and the patients with low WISP1 protein expression had significantly shorter total survival time;WISP1 was an independent risk factor for poor prognosis of liver cancer patients.Secondly,WB results showed that the expression of WISP1 protein in HCC cells was significantly lower than that of normal hepatocytes;cell function experiments and WB results showed that overexpression of WISP1 reduced the proliferation ability of hepatoma cells and the expression of cyclin D1 and PCNA;The tumorigenesis experiment of nude mice showed that overexpression of WISP1 inhibited the tumor formation ability of HCC cells,IHC showed that the expression of Cyclin D1 and PCNA decreased significantly.Thirdly,WB results showed that the expression of FAT10 protein in hepatoma cells was higher,while the expression of WISP1 protein was lower;The expression of WISP1 protein was increased by the down regulation of FAT10 expression in the hepatoma cells,and the expression of WISP1 was obviously reduced by overexpression of FAT10.In vitro and in vitro results showed that the overexpression of WISP1 protein restore the expression of WISP1 protein in the HCC cells with FAT10 low expression and the proliferation ability of HCC cells;On the contrary,the downregulation expression of WISP1 protein inhibited the elevated WISP1 protein expression caused by FAT10 downregulation and significantly reduced he proliferation of HCC cells leaded by FAT10 overexpression in vivo and vitro;Restoration of the expression of FAT10 in the FAT10 knockout cell line,the expression of WISP1 protein decreased significantly,and there was no obvious change in the expression of WISP1 protein when inducing the expression of FAT10.Fourthly.The results of Co-IP and confocal microscopy showed that the direct interaction between FAT10 and WISP1 in HCC cells;Silence of the expression of E1 and E2,the WISP1-FAT10 complex decreased significantly,and WISP1 protein expression increased.;While wild-type FAT10 can bind to WISP1 and decrease WISP1 protein expression.FAT10?GG did not bind to WISP1,but WISP1 protein expression increased,;FAT10?GG increased WISP1 protein and mRNA expression;The expression of WISP1 mRNA was enhanced by overexpression of FAT10 and the expression of WISP1 protein was reduced.In addition,overexpression of FAT10 decreased WISP1 protein and increased WISP1 mRNA expression in varied HCC cells.Fifthly,the results show that the downregulation of beta-catenin protein expression leads to the reduction of WISP1 mRNA,protein expression and thepromoter luciferase activity;Overexpression of ?-catenin protein leads to the opposite effect;?-catenin does not affect the activity of the mutant WISP1 promoter luciferase activity.In the HCC cells,the ?-catenin protein was directly linked to the WISP1 promoter and regulated the expression of the WISP1 expression;The overexpression of FAT10 increased the expression of ?-catenin protein and WISP1 mRNA expression,did not significantly change the expression of ?-catenin mRNA;On the contrary,Downregulation FAT10 expression reduced ?-catenin protein and WISP1 mRNA expression,with no obvious change in the expression of ?-catenin mRNA;In cells with ?-catenin expression inhibition and overexpression FAT10 expression at the same time,WIS1 mRNA increased or did not change significantly.On the contrary,the overexpression ?-catenin in low FAT10 expression cells and the WISP1 mRNA expression were restored.Sixthly,without the inhibitor of ?-catenin protein,the overexpression of FAT10 resulted in the inconsistency of the decrease of WISP1 protein and the increase of mRNA.With the inhibitor of ?-catenin protein,the overexpression of FAT10 did not result in the WISP1 protein and mRNA inconsistency.FAT10 ?GG expression gradually increased,both WISP1 mRNA and protein expression incresased;WISP1 protein and mRNA expression inconsistency did not occur;In vivo experiment,this phenomenon was further confirmed;the correlation between FAT10 and WISP1 protein and mRNA expression in clinical specimens was detected;The negative correlation between FAT10 and WISP1 protein was found;FAT10 and WISP1 mRNA was positively correlated;WISP1 protein was negatively correlated with WISP1 mRNA expression.Conclusion:Firstly,WISP1 protein can inhibit the proliferation of hepatoma cells in the liver cancer tissue,and overexpression of WISP1 inhibits the proliferation of hepatoma cells;Secondly,FAT10 promotes the proliferation of hepatoma cells through FAT10 degradation of WISP1;Thirdly,FAT10 promotes the expression of WISP1mRNA by stabilizing the beta-catenin;Fourthly,FAT10 simultaneously exerts its degradation and stability function,which causes WISP1 protein and mRNA expression discordance.