Mechanism Of Galectin-4 Regulating Invasion And Metastasis Of Lung Adenocarcinoma Cells

Background and objectivesLung cancer is one of the most common malignant tumors in the world and is a serious disease that threatens human health and life.Not only morbidity and mortality but also malignant metastasis are high in lung cancer and the prognosis is poor.Therefore,the search for markers of lung cancer invasion and metastasis and targeted therapy is a new strategy for the treatment of lung cancer.Galectin-4 is a tandem repeat galectin that has two similar carbohydrate cognition domains(CRDs).Galectin-4 is expressed both intracellularly,on cell surface and in circulation,and regulates cell proliferation,differentiation,apoptosis and adhesion.Studies have shown that galectin-4 is involved in the regulation of tumor invasion and metastasis in colorectal cancer and pancreatic cancer.In lung adenocarcinoma,high level expression of galectin-4 is an independent predictor of metastasis,which is associated with poor clinical prognosis.However,the regulation mechanism of galectin-4 in the invasion and metastasis of lung adenocarcinoma has not been reported in the literature.This project intends to reveal the relationship between galectin-4 and invasion and metastasis in lung adenocarcinoma A549 cells and H1299 cells,and to explore the underlying molecular mechanisms.Methods and ResultsMethods:1.The role of intracellular galectin-4 involved in the invasion,metastasis and adhesion to vascular endothelial cells of lung adenocarcinoma cells was determined.In this dissertation,lung adenocarcinoma A549 cells and H1299 cells with high expression of galectin-4 were selected.The siRNA technology was used to interfere with the expression of galectin-4 in A549 cells and H1299 cells.The expression of galectin-4 in these two cells was detected by Western blot method.The effect of galectin-4 on cell invasion and metastasis was measured by wound scratch assay,adhesion assay and Transwell assay after interfering with the expression of galectin-4 by siRNA.2.The effects of galectin-4 on the expression of adhesion molecules and invasion and metastasis related proteins were detected by Western blot method.To investigate the mechanism of galectin-4 modulating the adhesion,invasion and metastasis of lung adenocarcinoma cells,the expression of intercellular adhesion molecules,E-cadherin,N-cadherin,ICAM-1 and CD44,and the invasion and metastasis related proteins,EGFR,MMP-2 and p-FAK in A549 cells and H1299 cells was detected by Western blot method.To further reveal the molecular mechanism of galectin-4 regulating the expression of these proteins,the effects of galectin-4 on the expression of transcription factors P-catenin,NF-?B and p-STAT3 were examined.Meanwhile,p-STAT3 inhibitor HO-3867 and NF-?B inhibitor PDTC were used to uncover the upstream and downstream relationship between NF-?B and p-STAT3 in the signaling pathway.3.To detect the regulation mechanism of galectin-4 on the adhesion,invasion and metastasis of lung adenocarcinoma cells.NF-?B and ?-catenin are two important transcriptional regulatory factors.To explore the regulatory effects of both transcription factors on E-cadherin,N-cadherin,CD44 and MMP-2,NF-?B inhibitor PDTC and ?-catenin inhibitor ICG-001 were used.The role of NF-?B and ?-catenin in invasion and metastasis was further verified by scratch,adhesion and Transwell assays using the NF-?B inhibitor PDTC,the?-catenin inhibitor ICG-001 and galectin-4 siRNA.Molecular mechanism of galectin-4 regulating the invasion and metastasis of lung adenocarcinoma was further verified by Western blot method.Results:1.Intracellular galectin-4 regulated the invasion and metastasis of lung adenocarcinoma cells and adhesion to vascular endothelial cellsThe expression of galectin-4 in A549 cells and H1299 cells was suppressed by interfering with siRNA of galectin-4.The adhesion to human umbilical vein endothelial cells(HUVECs),migration and invasion were enhanced in A549 cells after transfected with galectin-4 siRNA,while that was inhibited in H1299 cells.2.Galectin-4 regulated the expression of adhesion molecules and invasion andmetastasis related proteinsThe results showed that the expression of E-cadherin was decreased,and the expression of N-cadherin and CD44 was increased in A549 cells after the interference of galectin-4.It indicated that galectin-4 could inhibit the adhesion of A549 cells to HUVECs by downregulating the expression of adhesion molecules,E-cadherin,N-cadherin and CD44.The expression of adhesion molecules N-cadherin and CD44 was decreased in H1299 cells after the interference of galectin-4.It indicated that galectin-4 could promote the adhesion of H1 299 cells by upregulating the expression of N-cadherin and CD44.Furthermore,galectin-4 could downregulate the expression of the invasion and metastasis related protein MMP-9 in A549 cells,upregulate the expression of MMP-2 and EGFR in H1299 cells.It showed that both EGFR and MMP-2 were involved in the invasion and metastasis regulated by galectin-4.Galectin-4 did not regulate the expression of ICAM-1 and p-FAK in both cells.In addition,we found that galectin-4 regulated the expression of transcription factors,?-catenin,NF-?B and p-STAT3.Adhesion molecules and invasion/metastasis related proteins might be regulated by these transcription factors.P-STAT3 inhibitor HO-3867 significantly inhibited the expression of NF-?B,whereas the NF-?B inhibitor PDTC had no effect on the expression of p-STAT3.This indicated that NF-?B was regulated by p-STAT3,that is,NF-?B is located downstream of p-STAT3.3.Galectin-4 involved in adhesion,invasion and metastasis of lung adenocarcinoma cells by regulating the expressions of adhesion moleculars and MMP-2 through regulating p-STAT3/NF-?B and ?-catenin expression.The expressions of E-cadherin,N-cadherin,CD44 and MMP-2 were changed after using NF-?B and ?-catenin inhibitors,indicating that NF-?B and P-catenin regulated the expressions of E-cadherin,N-cadherin,CD44 and MMP-2.Therefore,we speculated that galectin-4 affected the invasion and metastasis of cells by regulating the expression of E-cadherin,N-cadherin,CD44 and MMP-2 through P-STAT3/NF-?B and ?-catenin.The results showed that cell migration,adhesion,and invasion were inhibited in the presence of PDTC and ICG-001.That is,galectin-4 affected cell invasion and metastasis by regulating NF-?B and ?-catenin.In summary,it was proved that galectin-4 involved in the adhesion,invasion and metastasis of lung adenocarcinoma cells by regulating the expression of E-cadherin,N-cadherin,CD44 and MMP-2 through regulating p-STAT3/NF-?B and ?-catenin expression.Conclusion1.Galectin-4 inhibited the migration,adhesion and invasion of A549 cells and promoted the migration,adhesion and invasion of H1299 cells.2.Galectin-4 downregulated P-catenin to reduce the expression of N-cadherin,CD44 and MMP-2 and increase the expression of E-cadherin and downregulated P-STAT3/NF-?B to reduce the expression of N-cadherin and CD44 to inhibit invasion and metastasis in A549 cells.Galectin-4 upregulated the expression of N-cadherin,CD44 and MMP-2 through upregualting ?-catenin and p-STAT3/NF-?B to promote invasion and metastasis in H1 299 cells.3.Galectin-4 upregulated the expression of EGFR,but did not promote EGFR entry into the cytoplasm in H1299 cells.However,galectin-4 could not regulate the expression of EGFR in A549 cells.EGFR may be an important regulator of the opposite action of galectin-4 in A549 cells and H1299 cells.