| According to the latest report of GLOBOCAN,gastric cancer has been the 5th most frequently diagnosed cancer and 4th leading cause of cancer death worldwide,with more than 1 million new cases of gastric cancer each year.In recent years,with the development of endoscopic screening,the detection rate of early gastric cancer has gradually increased,and the cure rate has also improved dramatically.However,some patients are at the progressive stage once diagnosed,losing the chance of surgery,and some patients die of recurrent metastasis after radical gastric cancer surgery.At present,the mechanism of gastric cancer invasion and metastasis is still not well studied;therefore,finding specific molecular markers and targets for gastric cancer progression is of great significance for the diagnosis and treatment of gastric cancer and improving the survival prognosis of patients.Galectin-1 is an important member of the galectins family and is encoded by the LGALS-1 gene.Galectin-1 protein can be synthesized and secreted by a variety of cells.The literature reports that Galectin-1 can perform a variety of biological functions,including regulation of cell growth,apoptosis,pre-messenger RNA splicing,cell and cell-matrix adhesion,cell polarity,signal transduction,and innate/adaptive immunity.Studies have shown that Galectin-1 is involved in a variety of tumor invasive metastasis and tumor immune escape processes,and its aberrant overexpression is associated with low survival rates.Our team previously found high expression of Galectin-1 in gastric cancer tissues in a small sample size study,but the mechanism of Galectin-1 promoting gastric cancer progression has not been investigated in depth.Therefore,an in-depth study of Galectin-1 may provide potential therapeutic targets for gastric cancer treatment.The endoplasmic reticulum(ER)is a multifunctional organelle involved in regulating biological processes such as folding and modification of nascent proteins,calcium storage,and detoxification.Unfavorable external and internal factors,such as hypoxia,nutritional deficiencies,drug toxicity,acidic extracellular pH,and genetic mutations,lead to the accumulation of unfolded or misfolded proteins in the ER lumen and trigger endoplasmic reticulum stress(ERS)to re-establish intracellular homeostasis to promote cell survival.in the case of ERS,an unfolded protein response is activated,which aims to restore protein homeostasis in vivo.To maintain a highly proliferative state,tumor cells consume large amounts of material,and these cells often undergo aerobic glycolysis to support malignant expansion and form a unique cancer microenvironment.However,tumor cells can adapt to this hostile microenvironment by initiating ERS.GRP78 is the central molecule of ERS and studies have shown that GRP78 is overexpressed in a variety of human cancers and promotes tumor growth through multiple mechanisms.Therefore,GRP78 is considered as an attractive target for therapeutic tumors.However,Galectin-1 plays different functions in different tumors,and since the role played by Galectin-1 in cancer is controversial,this study is the first study comprehensively using public databases combined with multiple experimental approaches to explore the expression of Galectin-1 in gastric cancer and its relationship with patient prognosis.The effect of Galectin-1 on the proliferation and migration of gastric cancer was investigated through in vivo and in vitro experiments.bioinformatics and Co-Immunoprecipitation(CO-IP)were used to investigate the downstream mechanism of Galectin-1 in promoting gastric cancer progression.And whether the effects of Galectin-1 on gastric cancer are mediated through GRP78 signaling was explored,which provides a clinical and basic experimental basis for the development of future targeted therapeutic agents for gastric cancer.Part 1:Expression of Galectin-1 in gastric cancer and its correlation with clinicopathological factors and prognosisObjective:To explore the expression of Galectin-1 in public databases and in gastric cancer tissue samples from our center,and to analyze the correlation between Galectin-1 expression and clinicopathological factors and prognosis of patients.Methods:The mRNA expression differences of Galectin-1 in gastric cancer and adjacent tissues were analyzed using the GEPIA 2 database(http://gepia2.cancer-pku.cn/)and the Oncomine(www.Oncomine.org)database.Sixteen fresh frozen gastric cancer and adjacent tissue samples from our center were selected,and the expression of Galectin-1 protein was detected by Western blot.Furthermore,80 gastric cancer specimens were selected from patients who underwent radical gastrectomy in the Department of Gastrointestinal Surgery at clinical medical college Yangzhou University from June 2015 to May 2017.Immunohistochemistry was used to detect the expression of Galectin-1 protein in gastric cancer and adjacent tissues,and the correlation between Galectin-1 expression and clinicopathological factors of gastric cancer patients was analyzed.The relationship between Galectin-1 expression and the 5-year survival rate of patients was analyzed using the Kaplan-Meier method and Log-rank test,and COX univariate and multivariate analyses were performed to identify factors affecting the prognosis of gastric cancer patients.Results:Galectin-1 mRNA was highly expressed in the GEPIA and Oncomine databases.Western blot experiments showed that Galectin-1 protein was highly expressed in the 16 fresh gastric cancer compared with adjacent tissue samples from our center.Immunohistochemical experiments showed that Galecitn-1 was highly expressed in gastric cancer tissue and was significantly correlated with tumor size,lymph node metastasis,distant metastasis,TNM stage(P<0.05).Kaplan-Meier analysis revealed that patients with high Galectin-1 protein expression had a lower 5-year survival rate(P<0.05).COX univariate and multivariate analyses showed that tumor size,distant metastasis,TNM stage(Ⅰ,Ⅱ vs.Ⅲ,Ⅳ),Galectin-1 expression(low vs.high),and GRP78 expression(low vs.high)were independent factors affecting patient prognosis(P<0.05).qRT-PCR tests showed that Galectin-1 was highly expressed in gastric cancer cells compared to GES-1 cells.Conclusion:Galectin-1 is highly expressed in gastric cancer tissue,and patients with high Gal-1 expression have a poorer prognosis.Galectin-1 is also highly expressed in gastric cancer cells.Part 2:The effect of Galectin-1 expression on gastric cancer cell function.Objective:To explore the biological function of Galectin-1 in gastric cancer.Methods:Stable knockdown and overexpression cell lines of Galectin-1 were constructed in HGC-27 and AGS cells.CCK8 assay and clone formation assay were used to detect the effect of Galectin-1 on gastric cancer proliferation.Scratch assay and Transwell assay were used to detect the effect of Galectin-1 on gastric cancer migration and invasion.In vivo experiments were conducted by subcutaneously implanting tumor cells in nude mice to detect the effect of Galectin-1 on gastric cancer proliferation.Results:The CCK8 proliferation assay showed that the activity of Galectin-1-silenced HGC-27 cells was significantly lower than that of the control group,while the activity of Galectin-1-overexpressing AGS cells was significantly higher than that of the control group.The clone formation assay showed that knockdown of Galectin-1 decreased the number of clones formed by HGC-27 cells,while overexpression of Galectin-1 increased the number of clones formed by AGS cells.The scratch assay showed that knockdown of Galectin-1 inhibited gastric cancer cell migration,while overexpression of Galectin-1 enhanced gastric cancer cell migration.The Transwell assay showed that knockdown of Galectin-1 significantly inhibited gastric cancer cell migration and invasion,while overexpression of Galectin-1 significantly increased the migration and invasion ability of AGS cells compared to the control group.The nude mouse subcutaneous tumor model showed that the tumor growth rate and tumor volume and weight of the Galectin-1-overexpressing group were significantly higher than those of the control group.The in vivo experiment confirmed that Galectin-1 overexpression promotes tumor proliferation.Conclusion:Galectin-1 overexpression significantly enhances the proliferation,migration,and invasion ability of gastric cancer cells.Part 3:Galectin-1 promotes the progression of gastric cancer through interaction with GRP78Objective:To investigate the mechanism by which Galectin-1 promotes the progression of gastric cancer and whether Galectin-1 promotes gastric cancer cell proliferation,migration,and invasion through the GRP78 signaling pathway.Methods:The BioGRID database(https://thebiogrid.org/)was used to explore the protein interaction between Galectin-1 and GRP78.The interaction and subcellular localization were validated using CO-IP and fluorescence co-localization assays.Furthermore,the correlation between Galectin-1 mRNA and GRP78 mRNA was analyzed using the GEPIA online database.Lenti-viruses were constructed to knockdown GRP78,and a GRP7 8 overexpression plasmid was transfected to examine the efficiency of knockdown or overexpression of GRP78 in AGS and HGC-27 cells using Western blotting.The GRP78 overexpression plasmid and GRP78 knockdown lentivirus were transfected into gastric cancer cells with stable down-regulation or up-regulation of Galectin-1.Finally,the changes in cell proliferation,invasion and migration were analyzed using clone formation and Transwell assays.The expression of GRP78 protein in gastric cancer tissue was also detected to explore its effect on the progression of gastric cancer.Results:The BioGRID database confirmed the interaction between Galectin-1 and GRP78,while the GEPIA database showed no significant correlation between Galectin-1 mRNA and GRP78 mRNA.Cloning experiments demonstrated that overexpression of GRP78 restored the inhibitory effect of knocking down Galectin-1 on HGC-27 cell proliferation.Transwell migration and invasion experiments also confirmed that overexpression of GRP78 inhibited the decrease in migration and invasion of HGC-27 cells caused by knocking down Galectin-1.In addition,knocking down GRP78 protein in Galectin-1 overexpressing AGS cells was found to inhibit the promoting effect of Galectin-1 on AGS cell proliferation,migration,and invasion.The expression of GRP78 and Galectin-1 was positively correlated in gastric cancer tissue,with high expression of GRP78 in gastric cancer tissue being associated with tumor differentiation degree(P=0.004),lymph node metastasis(P=0.025),TNM stage(P=0.048),and infiltration depth(P=0.049).According to Kaplan-Meier analysis,patients with high expression of GRP78 had a poor prognosis.Univariate and multivariate analysis of factors affecting patient prognosis revealed that GRP78 expression level was an independent prognostic factor for 5-OS in gastric cancer patients.Conclusion:Galectin-1 promotes gastric cancer proliferation,migration and infiltration by regulating GRP78,which is overexpressed in gastric cancer tissue and indicates a poor prognosis. |
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