The Function And Molecular Mechanisms Of EPB41L5 In The Development,Progression And Metastasis Of Colorectal Cance

BACKGROUND ADN OBJECTIVEColorectal cancer(CRC)is one of the most common malignancies worldwide,ranking the third among all kinds of cancer.With the changes of population aging,lifestyle and diet,the incidence rate of CRC in china is increasing by 3.9%per year,significantly higher than the world average rate,2%per year.Metastasis is the primary cause of death of CRC.It is estimated that over 50%of patients diagnosed with CRC would died due to complications related to metastasis.Liver is a major and the first target organ of the metastatic CRC.Metastasis tumor of liver could be found in 25%patients in the first diagnosis.Unfortunately,about 25%-50%patients without metastasis in the first diagnosis would gradually develop into metastatic tumor althouth reveicing treatment.Thus,the study of molecular mechanism in the development and metastasis of colorectal cancer has great scientific significance and social value.The molecular mechanism of tumorigenesis and metastasis of CRC is a very complicated process which remains largely unknown.Carcinogenesis of CRC is a process of multisteps,multistages,multiple gene mutations of oncogene activation,inactivation of tumorsuppressor genes,apoptosis-regulating genes and DNA repair gene changes,as well as the disorder of microRNA adjustment which has been recognized in recent years.Metastasis is a complex multistep malignant process involved with the detachment of tumor cells from the primary site,interaction of tumor cells with the surrounding extracellular matrix,entering into the circulation or lymph system,adhesion to the vascular wall,migration of tumor cells into secondary sites,angiogenesis,and formation of new metastases.The molecular mechanism of metastasis is extremely complicated involving decline of adhesion among tumor cells,degradation of extracellular matrix,changes in the cytoskeleton,EMT,variation in the tumor microenvironment,enhancement of cell motility and migration.However,the mechanisms of CRC in the development,progression and metastasis are still unclear.Therefore,figuring out the molecular mechanisms of development,progression and metastasis of colorectal cancer could provide a new therapeutic target for the treatment and prognosis of CRC.EPB41L5(erythrocyte membrane protein band 4.1 like 5),also as known as BE37 or YMO1,maps to a region of the long arm of human chromosome 2q14.2.EPB41L5 was first discovered related to cell skeleton and movemen,widely expressed in the mesoderm and ectoderm of mammalian embryos.EPB41L5 has a FREM domian in the N-ternimus,which is conserved in the band 4.1 protein surper family.EPB41L5 was reported to be involved in variuos processes about cell motility,including regulation of cytoskeletal structure system,modification of the cell adhesion molecule,formation of basement membrane and cell polarity.Researches showed that there was crosslinking interaction between the FERM domain of EPB41L5 and the MyTh4(myosin tail homology 4)domain of myosins,which was involved in the regulation of cellular pseudopod formation and cell movement,and EPB41L5 could also combine with the cytoskeleton protein including Talinl andPTK to regulate the actin cytoskeleton.Hirano's study showed that the overexpression of EPB41L5 could decrease the expression of E-cadherin in the membrane surface through the function of FERM domain,thus promoting the EMT process.Gosens has found that the CRB-MPP5-EPB41L5 complex could regulate many kinds of cell adhesion factors,which played an important role in the formation of the tight junction and cell polarity,and the complex has closely relationship with the formation of epithelial cell polarity and membrane basement.Recent studies showed that Arf6-EPB41L5-AMAP1 complex could promote the promote the mesenchymal malignancy of renal cancer.Our previous analysis of gene chip of CRC tissue showed that EPB41L5 was a different expression gene in the.We also found that the expression of EPB41L5 was upregulated in CRC and was positively correlated with invasion and metastasis of CRC,which indicated that the up-regulation of EPB41L5 may be involved in the development,invasion and metastasis of CRC.However,little is known about EPB41L5 in CRC.However,the influence and molecular mechanisms of EPB41L5 deregulation during the development,progression and metastasis of CRC are still unclear.METHODS:1.GEO and CCLE databases were used to analyse the expression of EPB41L5 in CRC and other malignant cancers.2.Immunohistochemistry(IHC),immunoblotting(westem blot)and Real-time quantitative PCR(RT-PCR)were used to detect EPB41L5 expression in CRC tissues and matched normal tissues.The relationship between EPB41L5 expression and clinicopathological characteristics,including tumor differentiation,stage,metastasis and clinical prognosis was analysed.3.Stable EPB41L5-overexpressing cell lines(SW480/FLAG-EPB41L5,and HCT15/FLAG-EPB41L5),and control cell lines(SW480/PSIN and4.HCT15/PSIN)were established using retroviral expression system.Similarly,stable EPB41L5-silencing cell lines(SW620/shRNA2,SW620/shRNA3 and LOVO/shRNA2,LOVO/shRNA3),and control cell lines(SW620/GV248and LOVO/GV248)were established using retroviral expression system.5.MTT assay,colony formation assay,Transwell invasion assay and wound healing assay were used to evaluate the proliferation,invasive and migratory abilities of CRC cells.6.Western blot assay was used to detect the relative makers of EMT expression in CRC cells with EPB41L5 overexpression or silencing.7.GEO and Pre-PPI databases were used to seek out potential proteins and signaling pathways which were related to relatively high expression of EPB41L5.8.Western blot and confocal laserscanning microscopy assay were used to detect the impact of EPB41L5 expression on Racl-GTP,the downstream of Racl and the changes of cell cytoskeleton in CRC cells.9.Western blot,RT-PCR,confocal and TOP/FOP assays were used to investigate the impact of EPB41L5-Racl regulation axis on Wnt signaling pathway in CRC cells10.All statistical analyses were performed using SPSS 20.0 software.Paired-samples T test was used to analyse the data of CRC tissues and matched normal tissues detected by RT-PCR and IHC assays.The Independent-samples T test was used to analyse the data of Transwell invasion assays,wound healing assays,MTT assay and colony formation assay.Chi-square test,Fisher exact probability test or Kruskal Wallis H test were used to analyse categorical data.Spearman(two-way)was used to analyse correlation between EPB41L5 expression and clinicopathological characteristics.With P<0.05 as threshold of statistical significance.RESULTS:1.The expression of EPB41L5 in CRC1)The expression of EPB41L5 in CRC and other malignant in the CCLE databaseCCLE(Cancer Cell Line Encyclopedia)database and cBioPortal for Cancer Genomics searching tool were used to analyze the expression and gene mutations of EPB41L5 in CRC and other malignant cancers.Results suggested that the expression of EPB41L5 was higher in T-cell ALL cell,prostate cancer cell,lung small cell cancer cell and colorectal cancer cell than other cancer cell.Besides,3 colorectal cancer datasets showed that there were gene mutations of EPB41L5 in CRC.2)The expression of EPB41L5 in CRC in the public databaseThe GSEA analyses from GEO database showed that EPB41L5 was increasely expressed in normal colorectal tissues,carcinoma in situ,and metastatic colon cancer,and there were statistic signficance of EPB41L5 expression between different stages of CRC(p<0.05).3)The relationship between expression of EPB41L5 and metastasis of CRC in the public databaseThe result from the GSEA analysis of GEO database of metastasis colorectal cancer suggested that the metastasis colorectal cancer related gene set 'RICK MAN METASTASIS DN' was enriched and upregulated when the expression of EPB41L5 was relatively low(p<0.05),while another gene set 'RICK MAN METASTASIS UP'was enriched(p<0.05)and upregulated when the expression of EPB41L5 was relatively high.4)EPB41L5 expression in colorectal cancer tissueRT-PCR assay IHC and western blot were respectively used to detect the mRNA and protein expression of EPB41L5 in fresh CRC tissues and matched normal tissues.The results showed that the mRNA and protein expression of EPB41L5 were averagely higher in CRC tissues compared with matched normal tissues(p<0.05).The results of IHC showed that EPB41L5 was mainly expressed in membranes and cytoplasm,marked by yellow-brown.The expression of EPB41L5 in CRC tissues was higher than that in paired normal colon tissues from CRC patients,and the high expression of EPB41L5 in CRC was also related to the poor differentination,high Ducks' stage and metastasis of CRC(p<0.05).5)The relationship between EPB41L5 expression and the clinicopathological characteristicsThere were no significant differences of EPB41L5 expression between different age groups,different genders and different T classifications(p>0.05),but high expression of EPB41L5 was positively associated with Dukes stages,NM classification and tumor type(p<0.05).The high expression of EPB41L5 in CRC was also related to the poor differentination,high Ducks stage,lymph metastasis and distance metastasis of CRC(p<0.05).Kaplan-Meier Survival analysis revealed that 5-year overall survival rate of CRC patients especially the Ducks' C-D stages and T3-T4 stages patients with high EPB41L5 expression was significantly shorter than that with low EPB41L5 expression(p<0.05).2.The role of EPB41L5 in proliferation,EMT and invasion of CRC1)The construction of the EPB41L5 overexpression and knockdown Lentiviral vectors,and the establishment of stable expression cell linesFisrstly,western blot and RT-PCR were used to detect the expression of EPB41L5 in 7 CRC cell lines.Western blot and RT-PCR assays suggested that EPB41L5 expression was higher in SW620 and LOVO cell lines but lower in SW480 and HCT15 CRC cells.Then,Lentiviral vectors were used to establish stable expression cell lines,and the results of western blot showed that stable expression cell lines were successfully established.2)The effects of EPB41L5 overexpression and knockdown on proliferation in CRC cellsThe results from MTT assay revealed that,the growth of CRC cells with EPB41L5 overexpression increased markedly compared with the control group(P<0.05),while the growth of CRC cells with EPB41L5 knockdown decreased dramatically(P<0.05).The results of colony formation assay and softagar colony formation assay indicated that the colony numbers of CRC cells with EPB41L5 overexpression increased(P<0.05)while the colony numbers of CRC cells with EPB41L5 knockdown decreased(P<0.05).3)The effects of EPB41L5 overexpression and knockdown on invasion and migration abilities of CRC cellsTranswell invasion assays showed that the numbers of invasive CRC cells with EPB41L5 overexpression increased markedly compared with the control group(p<0.05),whereas the number of invasive CRC cells of EPB41L5 silencing decreased significantly compared with the control group(p<0.05).Wound healing assays revealed that the speed of wound healing by migratory CRC cells with EPB41L5 overexpression was faster than that in the control group,while the speed of wound healing by migratory CRC cells of EPB41L5 silencing was slower than that in the control group(p<0.05).4)The effects of EPB41L5 overexpression and knockdown on EMT-related proteins in CRC cellsWestern blot was used to detect the effects of EPB41L5 overexpression and knockdown on EMT-related proteins in CRC cells.The results of western blot suggested that the expression of the epithelial marker of EMT,E-cadherin increased,while the expression of the mesenchymal marker,Vimentin decreased in SW480 with EPB41L5 overexpression;however,the expression of E-cadherin and Vimentin showed opposite results in SW620 with EPB41L5 knockdown.3.The molecular mechanism of EPB41L5 promoting development and metastasis of CRC1)The analysis of upregulated signaling pathways related to the high expression of EPB41L5 in CRCPre-PPI and GEO database were used to analyze up-regulated signaling pathways related to the high expression of EPB41L5 in CRC.Pre-PPI database analysis showed that proteins related to regulation of actin cytoskeleton,colorectal cancer,adherens junction and wnt signaling pathway had interactions with EPB41L5.GSEA analysis of dataset GSE17536 showed that the CYTOSKELETON "MICROTUBLE C YTOSKELETON","CORTICAL CYTOSKELETON" and "RAC1 REG PATHWAY" gene sets were up-regulated and enriched in EPB41L5 relatively high expression group(ES=0.5,ES=-0.61,ES=0.48,P<0.05),and analysis of dataset GSE131294 showed that "WNT BATA CATENIN PATHWAY",gene set was also up-regulated and enriched in EPB41L5 relatively high expression group(ES=-0.51,P<0.05).2)The regulation of Racl activity by EPB41L5Western blot was used to detect the expression of Racl-GTP and its downstream proteins in CRC cells with EPB41L5 overexpression or knockdown.Western blot results showed that the expression of Racl-GTP and the downstream signaling target molecules of Racl pathway such as PAK,P-PAK,LIMK,P-LIMK,cortactin,P-cortactin,WAVE2 and WAVE3 were increased significantly in colorectal cancer cell line SW480 with EPB41L5 overexpression compared with control group.Correspondingly,the expression of Racl-GTP and the downstream signaling target molecules of Racl pathway were decreased in CRC cells with EPB41L5 knockdown compared with control group.3)EPB41L5-Racl signaling pathway regulated cell morphology and cytoskeletal remodelingThe results of confocal laserscanning microscopy assay indicated that HCT15 and SW480 with EPB41L5 overexpression showed elongated morphology and more protrusion compared with control cells that showed round morphology and fewer protrusion.By contrast,LOVO and SW620 with EPB41L5 knockdown showed round morphology and fewer protrusion compared with control cells that showed elongated morphology and more protrusion.4)EPB41L5-Racl signaling pathway regulated Wnt pathwayGSEA analysis,TOP/FOP assays,western blot assays,RT-PCR assays and confocal laserscanning microscopy assays were used to detect effects of the EPB41L5-Racl signaling pathway on the regulation of Wnt pathway.The results of GSEA analysis suggest that genes related to Wnt/?-catenin pathway were enriched in the group of EPB41L5 overexpression.The result of TOP/FOP assays dual luciferase reporter gene assay and western blot assay showed that the activity of Wnt signaling pathway and the nuclear expression of ?-catenin increase when EPB41L5 was overexpressed compared with the control group.By contrast,the activity of Wnt signaling pathway and the nuclear expression of ?-catenin were decreased when EPB41L5 was knockdown compared with the control group.Moreover,the results of western blot showed that the expression of c-Myc,CyclinD1 ?-catenin and the ?-catenin in the nucleus increased in CRC cells with EPB41L5 overexpression compared with the control group.However,the expression of c-Myc,CyclinD1 ?-catenin and the?-catenin in the nucleus showed opposite results in CRC cells with EPB41L5 knockdown.CONCLUSION:1.EPB41L5 expression in CRC tissue is significantly up-regulated and correlated with metastasis.High expression level of EPB41L5 is positively correlated with clinicopathological characteristics including tumor type,Dukes stage,NM stage,metastasis and prognosis.2.The overexpression of EPB41L5 promotes proliferation,invasion and migration in colorectal cancer cell,while the knockdown of EPB41L5 inhibits tumor cell proliferation,invasion and migration.3.EPB41L5 could influence the cytoskeleton and EMT progress by regulating the activation of Rac1 and Wnt pathway.