| Liver sarcomatoid carcinoma(sarcomatiod hepatocellular carcinoma, SHC)refers to the carcinoma morphologically similar to sarcoma, of which a part or most part was replaced by the cells of sarcomatoid spindle or spindle form. It is a type of primary hepatic epithelial tumors with high degree of malignancy and poor prognosis. The incidence of hepatocellular carcinoma transforming to sarcomatiod type after treatment could reach 20.9%. Currently sarcomatoid hepatocellular carcinoma lack the effective treatment systems so it is significant to clarify the pathogenesis of hepatocellular carcinoma sarcomatoid transformation and find the efficient, highly selective therapeutic targets.CtBP1(C-terminal binding protein 1,Ct BP1) as a transcriptional co-inhibitor, can inhibit the expression of epithelial markers, tumor suppressive genes and pro-apoptotic genes, therefore promoting epithelial-mesenchymal transition(epithelial-mesenchymal transition EMT), cell cycle progression and inhibiting apoptosis.Objective: Based on the background above and our previous study, we purpose to explore the role of Ct BP1 in sarcomatoid transformation process ofhepatocellular carcinoma, and further to reveal new molecular targets for diagnosis and treatment of the sarcomatoid carcinoma.Methods:I. The clinical significance of Ct BP1 expression in hepatocellular sarcomatoid carcinoma.1. Collect the cancer tissues, para-cancer tissues and clinicopathological parameters of HCC patients.Cancer tissues and para-cancer tissues of 13 cases diagnosed as sarcomatoid hepatocellular carcinoma and 23 cases diagnosed as hepatocellular carcinoma were collect the from surgical specimens of Guilin Medical university affiliated Hospital in 2008—2015.Corresponding para-cancer tissues were collected 5cm from edge of the tumor. All these samples are fixed with 10% formalin and paraffin-embedded. Every patient has received radiotherapy and chemotherapy before surgery.2. Detect the expression of Ct BP1 in tissue Detect the expression of Ct BP1 in tissue samples with HE and immunohistochemical staining.II. The influence of Ct BP1 Overexpression in hepatocellular carcinoma cell line1. Cell line culture conditions hepatocellular carcinoma cell lines HUH-7,7402,7404,7721 and Hep G2 were cultured with DMEM of 10% fetal bovine serum in cell incubator with37 ℃, 5% CO2, 95% relative humidity.2. Detection of Ct BP1 in hepatocellular carcinoma cell linesProtein are extracted from hepatocellular carcinoma cell lines and expression levels of Ct BP1 are measured through Western blot method.3. Extraction and purification of Plasmid.Overexpression vector were purchased from abcam company of United States. Transform, amplify the bacteria, extract plasmid, purify plasmid through Na AC-ethanol precipitation.4. establish stable transfected cell lines.Shuttle vector Ps PAX2 and packaging expression vector PMD2 G plasmids were co-transfected into 293 T cells to produce lentivirus. Lipofectamine2000 method are adopted to infect hepatocellular carcinoma cell lines 7402 and 7404 with lentivirus carrying Ct BP1.5. The impact of Ct BP1 overexpression on EMT, invasion, migration and proliferation of 7402,7404 cell lines.Ct BP1 are transfected into 7402,7404 cell lines and measured by Western blot assay. EMT related genes, migration, invasion and proliferation are measured by q RT-PCR, Western blot, Transwell, Boyden chamber, CCK-8and colony formation assayResults:1. The results of immunohistochemistry showed that in para-cancer tissues,hepatocellular carcinoma and sarcomatoid hepatocellular carcinoma tissues,Ct BP1 was negatively correlated E-cadherin expression and ki67 expression.2. Western blot detection showed that all liver cancer cell lines express a certain of Ct BP1,of which HUH7,7721 cells lines express considerably low and7402,7404 and Hep G2 cells lines considerably high.3. overexpressing the exogenous Ct BP1 in 7402 and 7404 cell lines, the expression of epithelial marker E-cadherin was downregulated and mesenchymal markers N-cadherin, Vimentin were upregulated. Therefore,Ct BP1 caused changes in the Ct BP1 overexpression 7402 and 7404 cells lines and induced the mesenchymal-like phenotype and alteration of molecular markers in epithelial-mesenchymal like cells.4. The results of Transwell chamber assay and boyden chamber assay further demonstrated that overexpression of Ct BP1 in7402 and 7404 promotes their migration and invasion ability.5. Results of cck8 and colony formation assay showed that overexpression of Ct BP1 improve the proliferation cell lines.Conclusions: On the current results, we think that Ct BP1 promotes the sarcomatoid transformation of hepatocellular carcinoma by mediated the EMT of hepatocellular carcinoma.This study will provide a theoretical basis for certain molecular targeted therapy in patients of late liver cancer. |
PDF Full Text Request