| ObjectiveMutation in CTNNB1 gene encoding β-Catenin is one of the most common somatic genetic events in hepatocellular carcinoma(HCC).Once β-Catenin is activated,it will promote the expression of target genes associated with members of the T cell factor(TCF)family,of which T-box transcription factor 3(TBX3)is a known target gene for the Wnt/β-Catenin cascade.Multiple studies have shown that HCC tumors with CTNNB1 mutations are well differentiated with low serum AFP(Alpha-fetoprotein)level,less microvascular infiltration and better prognosis,but the mechanism remains unclear.The purpose of this study is to explore the regulatory effect of TBX3 on CTNNB1 mutant HCC and related pathways,with a view to provide a new therapeutic target basis for the pathogenesis and clinical medication of HCC.Methods1.Database analysisTBX3 mRNA expression and its correlation with CTNNB1 and other common gene mutations were analyzed by TCGA-LIHC human HCC database.HCC was divided into CTNNB 1 mutation group and non-CTNNB1 mutation group,and TBX3 expression pattern in human HCC was analyzed.Genes positively and negatively correlated with TBX3 mRNA expression level in human HCC were analyzed,with KEGG pathway enrichment analysis.2.Vitro experimentTBX3 expression in 9 human HCC cell lines and 1 hepatoblastoma(HB)cell line was analyzed by Western blot.TBX3 was transfected into the cell lines with low TBX3 expression,and the effect of TBX3 overexpression was detected by cell clone formation assay.3.Vivo experiment3.1 TBX3flox/flox mice were co-transfected with c-Met,continuously activatedβ-Catenin(ΔN90-β-Catenin),Cre recombinase and SB transposable enzyme plasmid into mice by hydrodynamic tail vein injection technique,so as to induce hepatocellular carcinoma in situ,and TBX3 gene was knocked out.Euthanasia was performed on mice when they developed huge abdominal lumps and were dying.Liver tumors in mice were collected and treated with H&E staining to observe the histopathological features.The expression of Ki67,SOX9,E-Cadherin,HNF4a and Vimentin was detected by immunohistochemical technique.The expression of ERK,AKT and STAT3 pathway proteins,p-YAPser127,p-YAPSer397,Lats2,YAP/TAZ,nuclear YAP/TAZ and other members of Hippo pathway including MST1/2,SAV1,MOB1 and p-MOB1 was detected by Western blot.The mRNA expression of AFP,Epcam and metabolic pathway related genes,as well as the mRNA expression of Ctgf,Cyr61 and Notch pathway target genes was detected by RT-qPCR.3.2 TBX3flox/flox mice were co-transfected with c-Met,continuously activatedβ-Catenin(ΔN90-β-Catenin),Cre recombinase,SB transposable enzyme plasmid and Lats2 plasmid into mice by hydrodynamic tail vein injection technique,so as to induce hepatocellular carcinoma in situ,and TBX3 gene was knocked out as well as YAP/TAZ negative regulatory gene Lats2 was overexpressed simultaneously.When the mice developed a large abdominal mass and were close to death,they were euthanized.Liver tumors of mice were collected and treated with H&E staining to observe the histopathological features.The expression of Ki67,SOX9,E-Cadherin,HNF4a and Vimentin was detected by immunohistochemical technique.The expression of p-YAPSer127,p-YAPSer397,Lats2,YAP/TAZ,nuclear YAP/TAZ,Notch2 and Sox9 was detected by Western blot.The mRNA expression of Lats2,Ctgf and Cyr61 was detected by RT-qPCR.Results1.Expression pattern of TBX3 in human HCCs1.1 The overall expression level of TBX3 in HCC was significantly lower than that in surrounding normal liver tissues,and CTNNB1 mutation was the main manifestation of higher TBX3 expression in liver cancer.1.2 TBX3 is highly expressed in CTNNB1 mutated HCC,while down-regulated in non-CTNNB1 mutated tumors.1.3 Genes positively correlated with TBX3 expression are highly abundant in metabolic pathways,while genes negatively related to TBX3 expression are highly abundant in cancer pathways.2.Role of TBX3 in CTNNB1 mutant HCCs2.1 TBX3 protein was expressed high level in HepG2,a HB cell line with CTNNB1 mutation,and low in other 9 HCC cell lines.2.2 Cell clone formation assay showed that TBX3 overexpression significantly inhibited the growth of HCC cells in vitro.2.3 TBX3flox/flox mice of c-Met/β-Catenin/Cre(experimental group,knocked out the TBX3 gene in tumors)had faster tumor growth,shorter survival time,higher liver weight and liver/body weight ratio than c-Met/β-Catenin/pCMV TBX3flox/flox mice(control group).2.4 In liver tissues of c-Met/β-Catenin/Cre TBX3flox/flox mice(experimental group),Ki67 and SOX9 expressions were enhanced;The mRNA expressions of AFP and Epcam were significantly up-regulated,while the mRNA expressions of metabolic pathway related genes Cyp1a1,Cyp1a2 and Cyp2e1 were markedly down-regulated;Nuclear protein YAP/TAZ levels were up-regulated,the mRNA expressions of YAP/TAZ downstream target genes Ctgf,Cyr61 as well as Notch pathway target genes were up-regulated prominently.3.Regulation of YAP/TAZ by TBX3 in CTNNB1 mutated HCCs3.1 TBX3flox/flox mice of c-Met/β-Catenin/Cre/Lats2(experimental group,knocked out the TBX3 gene in tumor and over-expressed YAP/TAZ negative regulatory gene Lats2)had slower tumor growth and longer survival than c-Met/β-Catenin/Cre/pT3 TBX3flox/flox mice(control group).3.2 The survival of c-Met/β-Catenin/Cre/Lats2 TBX3flox/flox mice(experimental group)was similar to that of c-Met/β-Catenin/pCMV TBX3flox/flox mice(control group in the second part);H&E staining showed that livers in mice of the two groups were histologically similar.3.3 In livers of c-Met/β-Catenin/Cre/Lats2 TBX3flox/flox mice(experimental group),Ki67 and SOX9 expression were down-regulated;total YAP/TAZ and nuclear YAP/TAZ protein expression were down-regulated;Ctgf and Cyr61 mRNA expression was down-regulated.Conclusions1.In human HCC,TBX3 has a bimodal expression pattern,with high expression in CTNNB1 mutant HCC,but low in non-CTNNB1 mutant HCC.2.TBX3 may acts as a tumor suppressor during the development of HCC in vitro and in vivo.3.In c-Met/β-catenin-induced mice HCC,TBX3 may exerts tumor suppressor function by regulating YAP/TAZ activity(figure 1). |
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