| Gastric cancer is one of the most common cancers in the world.The five-year survival rate of advanced gastric cancer is 30%.The main reason leading to the poor prognosis of advanced gastric cancer is that tumor cells have a high invasion and metastasis ability,tumor cells easily to the surrounding tissue and lymph nodes invasion,and ultimately transferred to other organs,which is a complex multi-step physiological process.Epithelial-mesenchymal transition(EMT)plays an important role in the invasion and metastasis of gastric cancer and is an early event in gastric cancer.At present,we found that many signal pathways were involved in the regulation of EMT by synergism and antagonism,such as Ras-MAPK,Wnt,Rho,Src,PI3K/AKT and Hedgehog pathway.Galectin-1(Gal-1)is a kind of animal lectin,which is distributed in a variety of mammals in a variety of organs and tissues.Gal-1 is present in a variety of ways,including monomeric or non-covalently bound homodimers.Gal-1 is closely related to tumor proliferation,invasion,metastasis and recurrence.The high expression of Gal-1 in gastric adenocarcinoma can promote tumor angiogenesis and high expression of Gal-1 in gastric adenocarcinoma tissues,suggesting a poor prognosis.However,the specific mechanism of Gal-1 protein in gastric adenocarcinoma is unknown.Hedgehog(Hh)signaling pathway plays an important role in the proliferation,cycle,differentiation,invasion and metastasis of tumor cells.The Hh pathway is composed mainly of Hh ligand,membrane receptor Ptch,transmembrane signal transduction protein Smo and Gli-1(Glioma-associated oncogenetranscription factors-1).Gli-1 is the end of cascade conduction and is recognized as a key factor in the activation of Hh signaling pathway,which plays an invaluable role in gene signal transduction.Studies have found that:the expression of Gal-1 in pancreatic cancer by directly affecting the Hh/Gli-1 pathway to promote the development of pancreatic cancer.However,there is little research on the mechanism of Gal-1 in gastric adenocarcinoma caused by invasion and metastasis of gastric adenocarcinoma by Hh pathway.DHA(dihydroartemisinin)is a kind of sesquiterpene lactone derivative extracted from Chinese herbal medicine artemisinin,and it is also the main active ingredient of artemisinin.DHA is one of the most effective drugs for the treatment of drug-resistant malaria,falciparum malaria and cerebral malaria.In recent years,many studies have shown that DHA can inhibit the growth of a variety of malignant tumor cells,and its mechanism is mainly through the induction of tumor cell apoptosis,cell cycle arrest,anti-angiogenesis and inhibition of EMT process to achieve.Although this effect has been confirmed in a variety of tumors of DHA for gastric adenocarcinoma,but the effect and mechanism are still not clear,between the Gal-1 and the antitumor effect of DHA the existence of relationship is rarely reported.This study was based on the preliminary study and aims to explore the relationship between Gal-1 and EMT markers in gastric adenocarcinoma tissues and to construct Gal-1 overexpression/interference with lentiviral vector-infected gastric adenocarcinoma cell lines.To investigate the molecular mechanism of EMT,to observe the effect of DHA on the proliferation,invasion and expression of Gal-1 protein and the activity of gastric adenocarcinoma,and to explore the mechanism of DHA in the treatment of gastric adenocarcinoma.This paper is divided into three parts.Part 1 The expression,correlation and clinical significance of Galectin-1 and EMT markers in gastric carcinoma and expression of Galectin-1 in gastric cancer cell linesObjective:This study aimed to investigate the part of Gal-1 and EMT markers of cell adhesion molecule E-cadherin(E-cadherin)and vimentin(Vimentin)correlation in gastric adenocarcinoma tissue and between,and between them and the tumor clinical features and prognosis of the relationship.The gastric cancer cells with higher and lower expression of Gal-1 were screened as the object of subsequent molecular and molecular biology experiments.Methods:Gastric adenocarcinoma tissues and normal gastric mucosal tissues were collected from Jiangsu Province People's Hospital of Jiangsu Province.The immunohistochemical expression of Gal-1 protein,E-cadherin protein and Vimentin protein was detected in 162 cases of patients with gastric adenocarcinoma tissues and normal tissues.Univariate analysis of Gal-1 and E-cadherin,Gal-1 and Vimentin protein expression differences.And the relationship between Gal-1 and E-cadherin,Gal-1 and Vimentin protein was analyzed.According to the patient's age,gender,tumor size,lymph node metastasis,histological type and clinical stage were classified,using single factor analysis of variance and the relationship between the expression of the clinical and pathological features of tumor.The relationship between two proteins by nonlinear correlation analysis.The expression of Gal-1 protein and mRNA in Human gastric adenocarcinoma cell line MKN-45 cells,human gastric adenocarcinoma SGC-7901 cells,human gastric adenocarcinoma MKN-74 cells and human gastric adenocarcinoma MGC-803 cells were detected by Western blot and real-time fluorescence quantitative-PCR.Results:Gal-1 and vimentin was highly expressed in gastric adenocarcinoma tissues,and E-cadherin was low expressed in gastric adenocarcinoma tissues.Gal-1 was low in normal gastric mucosa,and E-cadherin was highly expressed in normal gastric mucosa,and vimentin was low in normal gastric mucosa.The expression level of E-cadherin is related to the degree of differentiation,depth of invasion,TNM staging and lymph node metastasis of gastric adenocarcinoma.The expression of Vimentin is stronger than that of gastric adenocarcinoma.The expression of E-cadherin is related to the differentiation degree,depth of invasion,TNM staging and lymph node metastasis.Weak and the depth of invasion of gastric adenocarcinoma.TNM staging and lymph node metastasis.There was a negative correlation between the expression of Gal-1 and E-cadherin in gastric carcinoma.Gal-1 was positively correlated with vimentin expression.The expression of MGC-803 cells in human gastric adenocarcinoma AGS cells,human gastric adenocarcinoma MKN-45 cells,human gastric adenocarcinoma SGC-7901 cells,human gastric adenocarcinoma MKN-74 cells and human gastric adenocarcinoma MGC-803 cells Protein and mRNA levels were the highest,and the expression level was the lowest in MKN-74.Conclusion:Gal-1 and EMT marker protein are closely related to the invasion and metastasis of gastric adenocarcinoma.The expression of Gal-1 was the highest in aggressive gastric cancer cell line MGC-803,and the expression of Gal-1 was the lowest in invasive gastric cancer cell MKN-74.Part 2 Effect of Galectin-1 on Metastatic Transformation of Gastric Cancer Cells by Hedgehog PathwayObjective:To observe the effect of Gal-1 expression on EMT in gastric cancer cells and to explore the possible mechanism of Gal-1-induced EMT in gastric cancer cells.Method:Gal-1 shRNA and Gal-1 overexpressing lentiviral vector were constructed and infected with gastric cancer cells.The expression of Gal-1 was detected by Western blot.The morphology of Gal-1 was observed.Matrigel invasion assay was used to measure the invasive ability of cells through the number of gastric cancer cells in the pores of the polycarbonate membrane.Western blot and real-time quantitative PCR were used to detect the expression of E?cadherin and interstitial marker vimentin protein and mRNA in the cells.Western blot and real-time quantitative PCR were used to detect the expression of Gli-1 expression level;the use of ?-lactose(Gal-1 chemical competitive inhibitor)confirmed that endogenous Gal-1 by induction of Gli-1 expression increased EMT caused by the occurrence.Results:Successful construction of packaged lentiviral vectors(LV-Gall-shRNA1,LV-Gal1-shRNA2,LV-Gall-shRNA3,LV-Gal-1)and corresponding control vectors(LV-shRNA-Control,LV-Control)was achieved by observing green fluorescent expression in combination with antibiotic screens to obtain stable infection in cell lines.The expression of Gal-1 in MGC-803 cells infected with LV-shRNA-control was significantly decreased by Western blot(P<0.05).The expression of Gal-1 in MKN-74 cells was significantly increased by infection with LV-Gal-1(P<0.05).Transwell invasion assay showed that LV-Gall-shRNA3 infection significantly reduced the number of invasive cells of MGC-803 compared with MGC-803 cells infected with LV-shRNA-Control(P<0.05).LV-Gal-1 infection could significantly increase the number of invasive cells in MKN-74 cells(P<0.05).Western blot and real time RT PCR showed that the expression of E-cadherin in LVN-Gal-1-infected MKN-74 cells was significantly decreased and the expression of Vimentin was significantly increased(P<0.05).The expression of E-cadherin in MGC-803 cells infected with shRNA3 was significantly enhanced and Vimentin(P<0.05).Western blot and real time RT PCR showed that down-regulation of Gal-1 expression could decrease the expression of Gli-1 and up-regulate the expression of Gal-1 and the difference was statistically significant(P<0.05).To clarify the effect of endogenous Gal-1 on EMT in gastric cancer cells we used(3-lactose to inhibit the effect of exogenous Gal-1.The results showed that the expression of Gli-1 and Vimentin protein was significantly increased and the expression of E-cadherin protein was decreased in the ?-lactose treated group(P<0.05).Conclusion:Gal-1 regulates the EMT via activation of the Hh/Gli-1 pathway in gastric cancer cells.Part 3 Study on the Effect of dihydroartemisinic on the epithelial mesenchymal transition of human gastric cancer cells induced by Galectin-1Objective:To investigate the effect of dihydroartemisinin(DHA)on the inhibition of gastric cancer cell lines and the regulation of apoptosis and the expression of Gal-1,and to explore the possible mechanism of DHA anti-tumor effect.Methods:The gastric cancer MGC-803 cells cultured in vitro.The DHA was diluted with DMSO to 7 concentration gradients:1,2,4,8,16,32,64?M cells were treated for 12h,24h and 48h respectively.The final concentration of cis-diaminedichloroplatinum(DDP)was 10?gg/mL as the positive control group.MTT method was used to detect different concentrations of DHA on gastric cancer cell proliferation inhibition,then separately calculated half lethal dose(IC50 value)of DHA.Inhibition of DHA on the invasion and metastasis of human gastric cancer MGC-803 cells and shGall#3 cells were detected by cell scratch test.Effect of DHA on the expression of E-cadherin,Vimentin and Gal-1 in human gastric carcinoma MGC-803 cells and shGall#3 cells were detected by qPCR and Western blotting.Results:The MTT assay indicated that the viability of human gastric carcinoma MGC-803 cells was significantly decreased in a concentration-dependent manner following resveratrol treatment.The IC50 values of DHA for MGC-803 cells was 26.03?M,11.13?M and 6.784?M at 12h,24h and 48h,respectively.The activity of DDP(10?g/mL)was 55.29%,26.55%and 19.78%at 12h,24h and 48h,respectively.The effect of DDP(10?g/mL)on cell viability was equivalent to 26?M DHA.The cell scratch test showed slower cells invasion and metastasis in the DHA group.The qPCR and Western blotting showed a decrease in Gal-1 and Vimentin expression,and an increase in E-cadherin expression in the DHA.Conclusion:DHA can significantly inhibit the invasion and metastasis of human gastric cancer MGC-803 cells,and its mechanism may be related to the inhibition of Gal-1 expression by DHA in gastric cancer cells. |
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