| Background:Primary liver cancer is one of the most common malignancies worldwide.According to histological type,primary liver cancer can be divided into hepatocyte type,biliary cell type and mixed type,among which hepatocyte type is the most common,that is,hepatocellular carcinoma(HCC),referred to as liver cancer,its incidence rate is about accounts for 90%of the incidence of primary liver cancer.In 2015,there were about 370,000 new cases of hepatocellular carcinoma in China,with an incidence of 26.92/100,000,ranking fourth in all malignant tumors,326,000 deaths,and the mortality rate ranking second in malignant tumors.Although the survival rate of liver cancer patients has been greatly increased compared with the past,the treatment effect on patients with advanced disease is still not satisfactory.At the same time,due to the high recurrence rate of liver cancer,it also has a great impact on the survival of patients.The incidence of liver cancer in China has gradually increased in the past two decades,and the patient population has gradually developed to a younger age.Therefore,it is urgent to find new effective methods for diagnosis and treatment.The current treatment for liver cancer mainly includes surgical resection,liver transplantation and radiotherapy,etc.,among which radical resection still occupies the dominant position.There are many reasons for the risk of high recurrence after liver cancer patients undergo radical surgery,which is mainly related to the biological characteristics of liver cancer.The abnormal proliferation of liver cancer cells and the massive production of cell adhesion factors lead to the entry of liver cancer cells into the blood and surrounding tissues and organs.More and more liver cancer cells stay and proliferate in other tissues and organs,providing a favorable microenvironment for their own growth.Finally,it causes recurrence of liver cancer.At present,most researchers believe that the occurrence of tumor and its malignant process are closely related to the uncontrolled cell proliferation,invasion to surrounding normal tissues,distant metastasis,and abnormal cell differentiation.In addition,the tumor cell cycle and apoptosis mechanism are disordered.related.Therefore,in-depth study of malignant tumor cell proliferation,invasion,cycle and apoptosis mechanism,hope to lay the foundation for the development of new liver cancer treatment.Desmogleins(DSG)and Desmocollins(Dsc)are a group of transmembrane proteins that are members of the cadherin family and mediate intercellular adhesions between mucosal epithelial cells.The adhesion process plays an important role in the formation of the major glycoproteins of intercellular desmosomes.DSG includes four subtypes,of which DSG2 is the most widely distributed subtype.DSG2 is an adhesion protein located on the cell surface and belongs to the cadherin family.Its expression can be detected in all monolayer epithelial cells and tissues containing desmosome in the human body.In the DSG family,only high levels of endogenous expression of DSG2 occur.The study found that DSG2 is highly expressed in a variety of human malignancies,including skin cancer,colorectal cancer,and lung cancer.Other studies have shown that the expression of DSG2 in gastric cancer,prostate cancer,melanoma,pancreatic cancer and other malignant tumors is significantly down-regulated.The above results indicate that the expression level of DSG2 is up-regulated or down-regulated in malignant tumors of different tissue origins,and plays an role of oncogenes or tumor suppressor genes.Meanwhile,it has been showed that DSG2 plays an important role in the proliferation,differentiation and malignant transformation of human cells,and is also related to the invasive growth and metastasis of tumors.However,there is no research on the expression of DSG2 in liver cancer and its related mechanisms.Part 1 The expression of DSG2 in HCC and its clinical significanceObjective:The purpose of the study is to detect the expression of DSG2 in hepatocellular carcinoma and cell lines,and the degree of the expression related to clinicopathological parameters and survival time of cases with liver cancer.Methods:In this experiment,the expression level of DSG2 in HCC cancer tissues and adjacent noncancerous tissues were detected by immunohistochemistry stain,quantitative real time PCR(RT-PCR)and Western blot.The exression of DSG2 mRNA and rotein in human normal liver cell line HL-7702 and liver cancer cell lines(QGY-7404 and Bel-7402)were detected by real-time PCR and western blot The relationship between the expression of DSG2 and clinicopathological data.was analyzed.The relationship between DSG2 protein expression and the overall survival of liver cancer patients was also analyzed.Results:1.It was found that the expression of DSG2 in liver cancer tissues was considerably higher,compared to that in adjacent non cancerous tissues by immunohistochemical staining,qRT-PCR and Western blot,the difference was statistically significant(P<0.01)2.The results showed that the expression levels of DSG2 mRNA and protein in QGY-7404 cells(P<0.05)and Bel-7402 cells(P<0.01)were significantly up-regulated compared with HL-7702 cells3.104 patients were divided into high DSG2 expression grou and low DSG2 expression group.DSG2 expression was not associated with age,gender,HBsAg and smoking status of liver cancer patients(P>0.05),but with tumor size(P＝0.039),AFP((P=0.037)and tumor stage(P=0.020)related4.Log-rank test showed that OS of patients with high expression of DSG2 was significantly shorter than those with low expression of DSG2(P=0.036).5.Univariate analysis showed that DSG2 expression(P=0.027),tumor size(P=0.037),AFP and tumor stage(P=0.025)had significant effects on OS in patients with liver cancer;age,gender,HBsAg,smoking status,and liver cancer was not associated(P>0.05)6.In addition,a multivariate analysis of significant variables in Cox proportional hazard analysis univariate analysis showed that DSG2 expression was an independent prognostic factor for OS(P=0.021),along with tumor size(P=0.032)and tumor stage(P20.019).Conclusion:1.The expression of DSG2 in hepatocarcinoma tissue was significantly higher than that in adjacent non cancerous tissues.The expressions of DSG2 in HCC cell lines were significantly higher than human normal liver cell line HL-7702.2.The expression of DSG2 was related to the tumor stage,AFP and tumor size of patients with liver cancer.3.The overall survival of patients with high expression of DSG2 is significantly shorter than that of patients with low expression of DSG2.Independent factor.Part 2 Study on the the biological function and mechanism of DSG2 in hepatocellular carcinomaObjective:With attempts to investigate the role of DSG2 expression on the proliferation and clone formation and apoptosis of hepatoma cells by in vitro cell experiments,the study aims to clarify the role of DSG2 in the development and progression of hepatocarcinoma,and to lay a new molecular target for effective diagnosis and treatment of liver cancer.Theoretical and experimental basis.Methods:The proliferation activity of hepatic cell line HL-7702 and liver cancer cell lines was detected by CCK-8 method.The liver cancer cells QGY-7404 and Bel-7402 were routinely cultured,and each cell was randomly divided into two groups:NC siRNA group and si-DSG2 group.Detect the proliferation abilities of QGY-7404 and Bel-7402 with down-regulated DSG2 expression level by CCK-8 and cloning assays.Detect the cell apoptotic rate of QGY-7404 and Bel-7402 with down—regulated DSG2 expression level by flow cytometry.The expression and distribution of β-catenin and c-myc before and after DSG2 silencing was verified by western blot.Results:1.The results of CCK-8 assay showed that the proliferation activity of QGY-7404 and Bel-7402 cells increased on days 1,2 and 3 compared with HL-7702 cells,and on day 3(The proliferative activity of P<0.01)was significantly different from that of HL-7702 cells.2.The expression of DSG2 mRNA and proteinin in the liver cancer cells of each group after transfection showed that the expression level of DSG2 mRNA and proteinin in the cells of si-DSG2 group was significantly lower than that of NC siRNA group(P<0.01).3.The proliferation activity of each group after transfection showed that the proliferation activity of the cells in the si-DSG2 group decreased on the 1st,2nd and 3rd day,compared with the NC siRNA group.The proliferation activity at 3 days was significantly different from that of the NC siRNA group(P<0.01).4.The clone formation ability of DSG2 gene knockout group was notably lower,in contrast with the control group.(P<0.01)5.The results showed that compared with the control group,cell anoikis abilities of QGY-7404 and Bel-7402 had no significant difference(P>0.05).That is to say,down regulation of DSG2 in HCC cells has no significant effect on the anoikis of HCC cells in vitro.6.In QGY-7404 and Bel-7402 liver cancer cell lines,the expression of β-catenin and c-myc in silencing DSG2 group was significantly lower than that in control group.Conclusion:Silencing DSG2 gene can inhibit the proliferation and clone formation of HCC,but has no significant effect on apoptosis;DSG2 may regulate the proliferation of HCC by regulating Wnt/β-catenin signaling pathway. |
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