| Background:The prominent pathological feature and leading cause death of colorectal cancer patients is metastasis.The disruption of cell junctions is the primary factor of invasions and metastasis in epithelial-derived malignant tumors such as colorectal cancer.The hallmark of metastasis is the downregulation of epithelial-cadherin(E-cadherin).Preventing the down-regulation of E-cadherin is an important way to inhibit the invasions and metastasis of colorectal cancer.Therefore,the molecular mechanism of E-cadherin down-regulation is expected to deeply understand incolorectal cancer,and then provide a new choice for the treatment of invasions and metastasis in colorectal cancer.The level of E-cadherin in tumor cells is regulated by a variety of factors.Previously,it was mainly existed some pre-transcription/transcription regulation mechanisms that dominated the decline in E-cadherin levels,such as mutations in the coding gene CDH1,epigenetic regulation,and transcriptional repression..However,more and more results indicate that the pre-transcription/transcription regulation of CDH1 is not consistent with changes in E-cadherin levels,suggesting that there may be mechanisms other than pre-transcription/transcription levels involved in the down-regulation of E-cadherin levels.Therefore,starting from a mechanism other than the pre-transcription/transcription level,it is helpful to find new strategies to prevent E-cadherin down-regulation.Lysosomes are mainly responsible for degradation of various macromolecules or organelles including proteins by internalization,phagocytosis and autophagy pathways,and are at the core of protein abundance regulation.Studies have shown that lysosomesdegradation is another important way to regulate E-cadherin level in liver cancer.However,the regulation factors and evolution mechanism of lysosomes in colorectal cancer,as well as the regulation effects of lysosomes on E-cadherin level,remain to be explored.To clarify the regulation effect of lysosomes on E-cadherin level in colorectal cancer and its influence on the prognosis of patients is one of the important premises for the intervention of colorectal cancer metastasis and prolonging the prognosis of patients with colorectal cancer.The microenvironment of solid tumors is acidic.Previous studies had found that under the stimulation of the acidic microenvironment,the number of lysosomes in tumor cells increased and was mostly distributed around the cell membrane.In addition,the results suggested that the acidic microenvironment mediated a decrease in E-cadherin levels.However,the relationship between the numbers/spatial distributions of lysosomes and the level of E-cadherin and its metastasis in colorectal cancer is still unclear.From the perspectives of lysosomal-E-cadheirn regulation,looking for the regulatory factors affecting the degradation of E-cadherin,it is expected to find a new strategy to target the invasion and metastasis of colorectal cancer.Methods:1.Using the database(Colon Adenocarcinoma TCGA-632,GSE41258,GSE40967)to analyze the level of E-cadherin mRNA and methylation in metastatic and non-metastatic colorectal cancer.Immunohistochemical analysis of E-cadherin was performed by tissue microarray Hlin-Ade075 ME t-01(including 14 primary/paracance/metastasis,14 primary/metastatic,and 4 metastases),and E-cadherin protein expression was detected by Imageproplus?.2.Staining of E-cadherin and Lamp2 in serial sections of paraffin tissues of colorectal cancer(n=4,>5 high power fields/slices),immunofluorescence double staining of Lamp2/E-cadherin in frozen tissues of colorectal cancer(n=8,5 fields/section)was performed to estimate the numbers of lysosomes by Lamp2 staining area,and the correlation between the number of lysosomes and E-cadherin was calculated.3.Lamp2 staining was performed in colorectal cancer tissue microarrays(HCol-Ade180Sur-08,HCol-A180Su11-T-067 each containing 90 cases of cancer/cancer tissue)to analyze the effect of lysosomal number and localization on clinical pathological parameters and prognosis of colorectal cancer,and the relationship between the number and distribution of lysosomes and the invasion and metastasis potential of colorectal cancer were analyzed by morphology.Combined lysosome number,distribution and clinical pathological parameters,Cox regression was performed by SPSS to test the role of lysosomal number and localization in the prognosis of colorectal cancer.4.Searching for lysosomal related genes by KEGG,a protein-protein interaction network of lysosomes-associated proteins is constructed by String to analyze the intrinsic interactions.Proteins with degree,NC and NDE greater than the median were screened according to the degree of interaction,neighborhood Connectivity(NC),and the number of directed edges(NDE),which is considered to be key proteins in the lysosome.Meanwhile,the mRNA expression of lysosomal-related genes was extracted from the TCGA databas(Colon Adenocarcinoma TCGA-632),then the receiver operating characteristic curve(ROC)was drawn according to the mRNA expression and survival status.The area under the ROC curve(AUR)of each gene was calculated,and screen the gene with AUR greater than 0.5,which is considered to have diagnostic significance for the prognosis of colorectal cancer.The gene name corresponding to the lysosomal key protein and the gene name having diagnostic significance in colorectal cancer were intersected by Wayne map.Differential expression of LAMP2 in metastatic and in situ colorectal cancer was analyzed by GEO database;Survival analysis of LAMP2 was performed in TCGA and GEO datasets,and COX regression was performed to test the predictive value of LAMP2 in the prognosis of colorectal cancer.5.Add chloroquine to human colorectal cancer HT29 cell line and HCT116 cell line medium for 0,4,8 and 12 hours,and using siRNA to interfere LAMP2 in HT29 cell line and HCT116 cell line.The expression of E-cadherin protein was detected by western blot,and the expression of CDH1 mRNA level was detected by real time qPCR.6.The human colorectal cancer HT29 cell line and HCT116 cell line were cultured in acidic medium(pH=6.5)for 2 weeks.These cells were considered to be chronic acid-adaptive cells(Acidic Adaptation,AA),and siRNA was used to interfere the expression of LAMP2 in HT29(AA cells and NA cells)and HCT116(AA cells and NA cells)to detect changes in E-cadherin protein levels and mRNA levels.Results:1.Transcription/pre-transcriptional regulation of CDH1 in metastatic and non-metastatic colorectal cancer could not fully explain the changes in E-cadherin protein levels2.The numbers and locations of lysosomes were negatively correlated with E-cadherin protein levels,and were associated with clinical pathological parameters and prognosis of colorectal cancer.3.High expression of lysosomal membrane protein,Lamp2,was associated with metastasis and poor prognosis in patients with colorectal cancer.4.Down-regulation of Lamp2 could up-regulates E-cadherin protein levels in colorectal cancer cells.Conclusions:1.The numbers and distributions of lysosomes in colorectal cancer tissues were closely related to E-cadherin protein levels,clinical pathological parameters and prognosis.2.The lysosomal membrane associated protein,Lamp2,could be an independent predictor of colorectal cancer patients.3.Down-regulation of Lamp2 could up-regulates E-cadherin protein level. |
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