| ObjectivePrimary hepatocellular carcinoma is the sixth common cancer and the fourth mortality in cancer-related death worldwide.The prognosis of HCC patients is not optimistic due to the high incidence of intrahepatic and distant metastases.Therefore,early detection and treatment are the key factors to improve the prognosis.It is necessary to further explore the molecular mechanisms of HCC progression for seeking new early biomarkers or therapeutic targets,which is crucial to improve the prognosis of patients.Bit1 is an anoikis effector that participates in the development of a variety of tumors and may become a diagnostic marker or therapeutic target of the tumor.Previous studies have shown that the expression of Bit1 was remarkable specificity in different tumors,which played a vital role in promoting or inhibiting tumor development by regulating the cell proliferation,apoptosis,migration and invasion.To date,it has never been studied about Bit1 in HCC.The purpose of this study is to investigate the expression and clinical significance of Bit1 in HCC,and to elucidate the biological function and mechanism of Bit1 in HCC.Importantly,it is to reveal the molecular mechanism of cetuximab interfering with Bit1 to hinder the HCC progression,which provide new ideas and basis for exploring the research on HCC and provide personalized combined regimen mainly based on cetuximab for HCC patients with highly Bit1expression and expand the indications of cetuximab in the field of cancer treatment.Methods1.RT-qPCR,Western blot,immunohistochemistry of tissue chip and Bioinformatics analysis were used to investigate the Bit1 expression and clinical significance,Bit1-related signaling pathways and interacting molecules in HCC.2.The expression level of Bit1 in HCC cell lines was detected by Western blot.Bit1 shRNA interference plasmid and lentiviral vector were constructed and the endogenous Bit1 expression of HCC cells was interfered by the lentiviral transfection experiment.Wound healing assay and Transwell assay were performed to detect the effect of Bit1 on the migration and invasion of HCC cells.CCK-8 assay,flow cytometry,and TUNEL experiments were used to detect the effects of Bit1 on the proliferation and apoptosis of HCC cells.Transmission electron microscopy was used to detect the effects of Bit1 on the mitochondrial morphology of HCC cells.3.Detection of ZO-1,N-cadherin,Vimentin,PCNA,Ki67,Bax and Bcl-2expressions by Western blot and immunofluorescence revealed the relationship between Bit1 and EMT or molecules related to proliferation and apoptosis in HCC.4.Based on the bioinformatics analysis that there was a correlation between Bit1 and EGFR in HCC.Cetuximab,an EGFR inhibitor was selected as the intervention drug,and the drug concentration was determined by the IC50 value of the drug cytotoxicity test.The optimal intervention time of cetuximab was selected based on the results of the preliminary experiment.After treated LM3cells containing highly expressions of Bit1 and EGFR with cetuximab,the Bit1expression was detected by Western blot to determine the relationship between Bit1 and EGFR in HCC.The expressions of integrinβ1,MMP-2 and MMP-9were detected to define the relationship between Bit1 and key molecules related to extracellular matrix receptor pathway in HCC.The expressions of ZO-1,E-cadherin,Vimentin,PCNA,Bax and Bcl-2 were detected to analyze and compare the effect of Bit1 expression induced by cetuximab on EMT process or molecules related to proliferation and apoptosis in HCC.Results1.Expression and clinical significance of Bit1 in HCCThe Bit1 m RNA and protein level in HCC tumor tissues were significantly higher than those in matched pericarcinomatous tissues,and the protein level was positively correlated with the pathological grade of the tumor tissues.Immunohistochemistry of tissue chip showed that the Bit1 expression in HCC tumor tissues was closely related to pathological grade and recurrence and was negatively related to the postoperative survival of HCC patients.Bioinformatics analysis showed that PTRH2 was highly expressed in HCC and was positively correlated with the tumor stage.PTRH2 expression was negatively correlated with the overall survival of HCC patients,and was closely related to the postoperative metastatic recurrence.String database analysis found that PTRH2may be correlated with these genes encoding EMT-related proteins such as CDH1,CDH2,VIM and TJP1,and it may also be correlated with EGFR.Analysis of the KEGG pathway database found that Bit1 was negatively correlated with extracellular matrix receptor interaction pathway and positively correlated with ribosomes pathway.2.Biological function of Bit1 in HCC cellsMHCC-97H and LM3 cell lines with highly expression of Bit1 was selected for Bit1 shRNA lentiviral experiments to knock down endogenous Bit1expression.Wound healing assay showed that the wound healing rate of the HCC cells treated with Bit1-specific shRNA was significantly lower than the cells in WT and Vector groups（24 h:WT=39.4%,Vector=40.2%,Bit1 shRNA1=19.4%,Bit1 shRNA2=16.5%;48 h:WT=60.9%,Vector=60.9%,Bit1shRNA1=27.5%,Bit1 shRNA2=18.5%）.Transwell assay showed that the HCC cells transfected with Bit1-specific shRNA exhibited obviously reduced cells invading the underlying surface of the insert,compared with the cells in WT and Vector groups.CCK-8 assay revealed that Bit1 downregulation markedly lowered cell proliferation ability in HCC.Conversely,after transfected with Bit1-specific shRNA,total apoptosis rate was observably increased in HCC cells in flow cytometry analysis（WT=4.21%,Vector=7.45%,Bit1 shRNA1=11.75%,Bit1 shRNA1=19.31%）.TUNEL assay demonstrated that the number of late apoptotic cells was significantly increased in the HCC cells treated with Bit1-specific shRNA,compared with WT and Vector groups.Transmission electron microscopy results showed that transfected with Bit1-specific shRNA,the HCC cells displayed swollen and irregular mitochondria with gently dilated and disrupted crista.3.The mechanism of Bit1 in HCCWestern blot and immunofluorescence assay revealed that the expressions of ZO-1 and Bax were upregulated,while the expressions of N-cadherin,Vimentin,PCNA,and Bcl-2 were downregulated in HCC cells treated with Bit1-specific shRNA.It was concluded that Bit1 downregulation can inhibit EMT process and regulate the molecules related to proliferation and apoptosis to suppress HCC progression.4.Cetuximab suppresses the EMT via regulating the EGFR/integrinβ1/Bit1 axis to inhibit the HCC with high Bit1 expressionAccording to the bioinformatics analysis that Bit1 was closely related to EGFR,and the Bit1 expression was negatively correlated with ECM receptor interaction,we further explored the relationship between Bit1 and EGFR or key molecules related to extracellular matrix receptor pathway in HCC.Cetuximab was selected as the intervention drug.we decided to set 4 groups of concentrations:0,200,600,1800μg/m L by the IC50 value of the drug cytotoxicity test.According to the preliminary results,the optimal intervention time of cetuximab was 72 h.After treated LM3 cells containing highly expressions of Bit1 and EGFR with cetuximab,the expressions of EGFR,Bit1,MMP-2,MMP-9,Vimentin,PCNA and Bcl-2 were significantly reduced and the expressions of integrinβ1,E-cadherin,ZO-1 and Bax were obviously enhanced.The effects of cetuximab on EMT markers and the molecules related to proliferation and apoptosis were consistent with the results of Bit1downregulation.It was concluded that cetuximab suppresses the EMT via regulating the EGFR/integrinβ1/Bit1 axis to inhibit the HCC with high Bit1expression.Conclusions1.Bit1 is upregulated and predicts poor prognosis in HCC tissues.2.Bit1 downregulation obviously suppresses proliferation,migration,and invasion,promotes apoptosis and destroys the mitochondrial morphology in HCC cells.3.Bit1 downregulation inhibits the HCC progression by suppressing EMT process and regulating the molecules related to proliferation and apoptosis.4.Cetuximab suppresses the EMT via regulating the EGFR/integrinβ1/Bit1axis to inhibit the HCC with high Bit1 expression. |
PDF Full Text Request