The Function And Regulation Mechanisms Of LRG1 In The Metastasis Of Colorectal Cancer

PART ? THE EXPRESSION OF LRG1 IN COLORECTAL CANCER TISSUEAims:To investigate the expression of LRG1 in colorectal cancer(CRC)tissue and normal tissue,and to explore the relationship between LRG1 expression and clinicopathological parameters.Methods:Public microarray databases were used to analyse the expression of LRG1 in colorectal cancer and normal tissue.Real-time PCR was performed to assess LRG1 mRNA expression in 30 cases of fresh CRC and matched normal tissue.Immunohistochemistry was performed to examine LRG1 protein in 68 CRC and 32 normal paraffin-embedded tissues.LRG1 expression and the clinicopathologic features were analysed.Results:1.Microarray databases of GSE20916 and GSE20842 revealed significant overexpression of LRG1 in CRC tissues than normal tissues.2.Real-time PCR showed that LRG1 was significantly overexpressed in CRC tissues,compared with corresponding normal tissues(P<0.0001).3.Results of immunohistochemistry showed that positive expression rate of LRG1 was 66.18%(45/68)in CRC tissues,significantly higher than that of normal colorectal mucosa(31.25%,12/32)(P=0.007).LRG1 expression was significantly correlated with deeper invasion depth(P=0.032)and lymph node metastasis(P=0.013).Conclusions:LRG1 expression was remarkably increased in CRC tissues compared with normal tissues.Expression of LRG1 was positively associated with deeper invasion depth and lymphatic metastasis.These results imply that LRG1 may be involved in CRC initiation and progression,especially in the metastasis of CRC.PART II THE FUNCTION OF LRG1 ON THE MALIGNANT BEHAVIOR OF CRC CELLSAims:To investigate the influence of LRG1 on the migration,invasion,and EMT phenotype of CRC cells.To explore the effect of LRG1 on the expression of VEGF-A in CRC cells,and the function of CRC cells-derived conditioned medium on endothelial cells.Methods:LRG1 siRNA and negative control siRNA were transfected into CRC cells.Real-time PCR and Western blot were used to verify the efficacy of LRG1 siRNA.Transwell invasion assays and wound healing assays were performed to evaluate the invasion and migration of CRC cells.Epithelial-to-mesenchymal transition(EMT)markers of E-cadherin,VDR,N-cadherin,?-SMA,Vimentin and Twistl were detected by Real-time PCR and Western blot.CRC cells were stimulated with indicated time and concentration of LRG1,and ELISA was used to measure the secretion level of VEGF-A.Conditioned medium(CM)from CRC cells was collected for endothelial cell migration and tube formation assay.Results:1.Both mRNA and protein levels of LRG1 were significantly downregulated in HCT116 and SW480 cells by LRG1 siRNA.In wound healing assays,wound closure ratio was markedly reduced in LRG1 siRNA transfected cells.Knockdown of LRG1 significantly reduced the number of cells invaded through the Matrigel.2.Real-time PCR and Western blot analysis showed that epithelial biomarkers of E-cadherin and VDR were increased in LRG1-depleted CRC cells,whereas mesenchymal biomarkers of N-cadherin,Vimentin,?-SMA and Twist1 were decreased.3.The mRNA and protein of VEGF-A were upregulated with the stimulation of LRG1 in time-and concentration-dependent manner.CM from rLRG1-stimulated HCT116 cells promoted the migration and tube formation capacity of endothelial cell.Conclusions:Transfection of LRG1 siRNA decreased the migration and invasion capacity of CRC cells,and reversed the EMT phenotype of CRC cells.LRG1 promoted the mRNA and protein of VEGF-A in CRC cells,and thus contributing to CRC angiogenesis.PART ? THE MECHANISM OF LRG1 ON CRC INVASION AND METASTASISAims:The mechanism of LRG1 on CRC invasion and angiogenesis were unclear.This study was to explore the downstream gene and signal pathway associated with LRG1.To verify the role of HIF-1? in LRG1 induced EMT and VEGF-A.Methods:Microarray was used to screen for differentially expressed genes in SW480 cells with transfection of LRG1 or negative control siRNA,and HIF-1? was choosed as the downstream gene of LRG1.To investigate the relationship between LRG1 and HIF-1?,CRC cells were stimulated with different concentration of LRG1,Real-time PCR and Western blot were performed to evaluate the mRNA and protein expression of HIF-1?.SW480 cells were pretreated with HIF-1? siRNA and then stimulated with LRG1.ELISA was performed to measure the secretion of VEGF-A.Western blot was used to detect the expression of HIF-1?,VEGF-A,N-cadherin,E-cadherin and Twistl.Invasion ability of SW480 cells was evaluated by Transwell assay.Results:Microarray data showed that LRG1 was associated with several pathways related to cancer.HIF-la mRNA and protein were upregulated with stimulation of LRG1.Knockdown of HIF-la significantly reversed the increased YEGF-A expression,EMT phenotype,and the invasion capability of SW480 cells.Conclusions:LRG1 can modulate the mRNA and protein expression of HIF-1?.HIF-la was responsible for LRG1-induced VEGF-A expression and EMT.