| Background:Primary liver cancer(PLC)is a common malignant tumor of digestive system.According to the histological type,PLC can be divided into three types:Hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC)and HCC-ICC.HCC is the most common of PLC,accounting for more than 80%.The prevention and treatment of HCC has been a thorny problem at present because of its low early diagnosis and detection rate,easy invasion and metastasis,and complex pathological mechanism.Therefore,it is an urgent problem to explore the molecular factors related to the pathological process of liver cancer and to improve the diagnosis and treatment rate of patients.In recent years,more and more attention has been paid to the relationship between the imbalance of intercellular adhesion function and the occurrence and development of tumors.Desmocollin(DSC)is the main protein that mediates cell adhesion and is an important component of desmosome and semi-desmosome tissue.DSC2 is the most important subtype of DSC,which is widely distributed in desmosome and plays an important role in mediating cell-to-cell connection,adhesion and signal transduction.Previous studies have shown that there are structural or functional disorders of DSC2 in some tumors.However,the role of DSC2 in hepatocellular carcinoma and its molecular mechanism are not clear.Therefore,we detected the expression of DSC2 in liver cancer tissues,and further analyzed the relationship between DSC2 and clinical prognosis of patients with liver cancer in this study.At the same time,the study further explored its effect on the growth,proliferation,migration and invasion of liver cancer cells and its molecular mechanism,so as to provide theoretical basis and data support for the diagnosis and treatment of liver cancer in the future.Purpose:The purpose of this study was to detect the expression of DSC2 in HCC and to further analyze its relationship with clinical pathological parameters and overall survival of patients with HCC.At the same time,the effects of DSC2 expression on proliferation,clonogenesis,apoptosis,migration and invasion,EMT and other biological functions of HCC cells and its mechanism were investigated by in vitro cell experiments,and the effect of DSC2 expression on tumor growth in vivo was further verified by in vivo experiments.The study aims to provide new theoretical basis and data support for the treatment of liver cancer in the future.Methods:The cancer tissues and adjacent normal tissues of 82 patients with HCC were collected,and the expression of DSC2 in tumor tissues was detected by Immunohistochemica(IHC),tissue immunofluorescence and Western blot.The correlations between DSC2 and clinical pathological data,DSC2 and clinical prognosis were further analyzed.After detecting the expression of DSC2 protein in the HCC cell lines by Western blot,the suitable cell lines were selected for up-regulation or down-regulation of DSC2 gene expression,and the stable cell lines were further verified and screened by q RT-PCR and Western blot methods.The proliferation ability of transfected cells was detected by colony formation,CCK-8 and Ed U assays.The apoptosis of transfected cells was analyzed by AO/EB assay.The migration and invasion ability of transfected cells were determined by wound healing and Transwell assays.The subcutaneous tumor model of nude mice was established to further observe the effect of DSC2 on tumor growth.Western blotting was used to detect the expression of DSC2,apoptosis-related proteins,cell cycle-related proteins,EMT-related proteins,MMPs-related proteins and ERK1/2 pathway-related proteins in transfected HCC cells,so as to further analyze the role of DSC2 in the occurrence and malignant progression of HCC.Results:1.IHC,tissue immunofluorescence and Western blot were used to detect the expression of DSC2 in HCC cancer tissues and adjacent normal tissues,and it was found that the expression of DSC2 in HCC cancer tissues was significantly lower than that in adjacent tissues(p<0.001).2.The expression of DSC2 were not correlated with age,sex,AFP,HBs Ag,HBV DNA,smoking,drinking,liver cirrhosis,and BCLC stages(p>0.05 for all),but significantly correlated with tumor diameter(p<0.001),tumor differentiation(p=0.004)and portal vein tumor thrombus(p=0.002).3.Log-rank logarithmic rank test showed that the overall survival time(OS)of DSC2 negative HCC patients was significantly shorter than that of DSC2 positive patients(p<0.001).Cox proportional hazard regression model showed that the positive expression of DSC2(HR=0.447,95%CI: 0.222-0.902;p=0.025)was an independent protective factor of OS in patients with HCC.However,moderate-poor differentiated tumor(HR=2.071,95%CI: 1.160-3.698;p=0.014),portal vein tumor thrombus(HR=2.091,95%CI: 1.080-4.048;p=0.029)and BCLC stage B/C(HR=2.150,95%CI: 1.197-3.861;p=0.010)were independent risk factors for OS in patients with HCC.4.The expression of DSC2 protein in HCC cell lines(7721,Huh7,LM3 and97H)were significantly lower than that in normal hepatocyte cell line(L02 cell line)by Western blot(p<0.05 for all).5.Colony formation,CCK8 and Ed U assays showed that colony formation and cell proliferation were inhibited in Over Exp-DSC2 LM3 cells(p<0.01 for all),while promoting in Sh RNA-DSC2 7721 cells(p<0.05 for all).In addition,the expression of Cyclin D1,Cyclin B,CDK1 and CDK2 were down-regulated in Over Exp-DSC2LM3 cells(p<0.001),while up-regulating in Sh RNA-DSC2 7721 cells(p<0.05).6.Overexpression of DSC2 in LM3 cells significantly stimulated apoptosis(p<0.001),but interference with DSC2 significantly inhibited apoptosis in 7721 cells(p<0.01).The expression of Bax,c-Caspase-3 and Caspase-8 increased Over Exp-DSC2 LM3 cells(p<0.01 for all),while decreasing in the expression of Bcl-2 and Survivin(p<0.01 for both).The expression of Bcl-2 and Survivin increased in Sh RNA-DSC2 7721 cells(p<0.05 for both),but the expression of Bax and c-Caspase-3 decreased in that of cells(p<0.001 for all).7.Wound healing assay showed that overexpression of DSC2 in LM3 cells inhibited cell viability(24h p<0.05,48 h p<0.01),while increasing in Sh RNA-DSC27721 cells(24h p<0.05,48 h p<0.01).Transwell assays showed that the migration and invasion abilities decreased in Over Exp-DSC2 LM3 cells(p<0.01),and that of Sh RNA-DSC2 7721 cells increased(p<0.05).8.Animal experiments in vivo showed that the tumor volume and weight in Over Exp-DSC2 group were significantly lower than those in Vector group(p<0.001),while the tumor volume and weight in Sh RNA-DSC2 group were significantly higher than those in Sh Control group(p<0.001).9.The expression of E-cadherin protein was up-regulated in Over Exp-DSC2LM3 cells,while down-regulating in the expression of β-catenin,N-cadherin and Vimentin(p<0.01 for all).The expression of E-cadherin protein was decreased in Sh RNA-DSC2 7721 cells,while increasing in the expression of β-catenin,N-cadherin and Vimentin(p<0.01 for all).In addition,the expression of MMP2 and MMP9 in Over Exp-DSC2 LM3 cells were down-regulated(p<0.05 for both),while up-regulating in Sh RNA-DSC2 7721 cells(p<0.05 for both).10.Overexpression of DSC2 inhibited the expression of p-ERK1/2 and downstream proteins(c-MYC and Fos)in LM3 cells(p<0.01 for all),while inhibition of DSC2 increased the expression of p-ERK1/2,c-MYC and Fos in 7721 cells(p<0.01 for all).Conclusion:1.DSC2 affects the prognosis of liver cancer patients,and its low expression has a worse prognosis,and its low expression promotes the proliferation,EMT,migration and invasion of liver cancer.2.DSC2 may affect the proliferation and metastasis of HCC by regulating ERK signal pathway. |
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