The Function And Molecular Mechanisms Of EPB49in The Development, Progression And Metastasis Of Colorectal Cancer

BACKGROUND AND OBJECTIVE:Colorectal cancer (CRC) is one of the most commonly digestive malignant tumors, and the morbidity and cancer-related mortality are among the upper third of all tumors. In recent years, with the change of the people’s lifestyle and diet, the incidence of CRC has increased in the world, and age at oneset is becoming younger and younger. Metastasis is the leading cause of death in the patients with cancer, metastatic tumors could be found in the40-50%patients at the process of initial diagnosis and treatment. Liver is a major target organ of the metastatic colorectal cancer, the prognosis of CRC patients with liver metastasis is very poor. CRC has serious harmful effects on people’s physical health, quality of life, but also brings the heavy economic burdens on our society.The molecular mechanisms of tumorigenesis and metastasis of CRC have been unknown completely. Carcinogenesis of CRC is a process with multistep, multistage, multiple gene mutations, accompanied by oncogene activation, inactivation of tumor suppressor genes, apoptosis-regulating genes and DNA repair gene changes, as well as increased telomerase activity. The molecular mechanism of metastasis is also complicated, involving interaction between cancer cells and host, decline in cell adhesion, enhancement of cell movement capacity, changes in the cytoskeleton, EMT, changes in the tumor microenvironment, and the dyregulation of the secretory vesicles of tumor cell and mesenchymal cell. For these reasons, it is necessary to further explore the mechanisms of tumorigenesis and metastasis of CRC for developing effective treatments and improving survival rates.EPB49gene maps to a region of the short arm of human chromosome8p21.1,15kb length, encoding the EPB49protein, also known as Dematin (DMTN). EPB49is an actin-binding/bundling protein that was originally isolated from human erythrocyte membrane, which can directly bind F-actin through actin binding sites to regulate cytoskeleton remodeling. Dyregulation in the structure and function of the cytoskeleton is an important factor in the development and progression of malignant tumors, and the abnormal expression of actin-related proteins are also close to malignant phenotype transformation of normal cells.EPB49is responsible for maintaining the shape and integrity of the erythrocyte. Biochemical characterization of EPB49has shown that it exhibits phosphorylation dependent actin bundling activity. EPB49is expressed predominantly in the hematopoietic(erythrocytes, platelets, and lymphocytes), cardiac, and vascular cells, brain, endothelial and epithelial cells, skeletal muscle, and kidney cells. The broad expression of EPB49suggests that it may play a significant role in the regulation of the actin cytoskeleton in nonerythroid cells.Dyregulation and deletion of EPB49are closely related to the carcinogenesis and metastasis of cancer. EPB49gene maps to chromosome8p21.1, which region is often accompanied by the loss of heterozygosity in prostate cancer patients. In prostate cancer PC-3cell, overexpression of EPB49can restore epithelioid cell morphological phenotypes. Recent studies have shown that EPB49regulates cell shape, motility, and wound healing by modulating RhoA activation. Our preliminary work suggested that EPB49expression was downregulated in CRC, which indicated that the dyregulation of EPB49may be involved in the development and metastasis of CRC. However, the relationship between EPB49and colorectal cancer, the upstream regulation mechanisms of EPB49still remain unclear.Therefore, in our study we will detect EPB49expression in CRC, analyse the relationship between EPB49expression and clinical pathologic parameter, illustrate the function and associated molecular mechanism of EPB49in the development and metastasis of CRC, and explore the upstream regulating mechanisms of EPB49.METHODS:1. GEO and Oncomine database was used to analyse the expression of EPB49in CRC and other malignant cancer.2. Immunohistochemistry (IHC), immunoblotting (westernblot) and Real-time quantitative PCR (RT-PCR) were used to detect EPB49expression in paraffin-embedded and fresh CRC samples; the relationship between EPB49expression and clinical pathology parameters, including tumor differentiation, stage, metastasis and clinical prognosis, was analysed.3. The colorectal caner cell lines with EPB49overexpression and knockdown were constructed; cell proliferation was measured by MTT assay, colony formation assay, softagar colony formation assay and Xenograft model in nude mice; the distribution of cell cycle was detected by flow cytornetry; cell invasion ability was tested through transwell invasion assay, scratch wound healing assay and3-D cell culture assay, surgical orthotopic transplantation (SOI), tail vein injection of nude mice.4. Confocal laser scanning technique was used to observe cell morphology, adhesion and cytoskeleton of CRC cells with EPB49overexpression and knockdown.5. GEO database was used to analyse the enrichment of Rac1, RhoA and CDC42signaling pathway in CRC patiens with low expression of EPB49.6. Westernblot was used to detect the expression of Racl-GTP in CRC cells with EPB49overexpression and knockdown; confocal laser scanning technique and Co-IP were used to check the interaction and specific sites between EPB49and Tiaml; resuming experiment was used to further test the effect of their interaction on activity of Racl.7. GEO database was used to analyse the enrichment of Wnt_beta-catenin and MAPK signaling pathway in CRC patiens with low expression of EPB49.8. The activation of Wnt_beta-catenin and MAPK signaling pathway were measured by dual luciferase reporter gene assay; the expression of EMT markers was detected by Immunofluorescence assay and westernblot assay; the target genes expression of Wnt_beta-catenin and MAPK signaling pathway was checked by westernblot assay.9. The UCSC database was used to predict CpG Island of EPB49; bisulfite genomic sequence (BSP) assay was used to test CpG Island methylation in CRC tissue samples; the expression of EPB49was measured by RT-PCR; the correlation between EPB49expression and the degree of CpG Island methylation was analysed.RESULTS:1. The expression of EPB49in CRC tissue1) The expression of EPB49in CRC in the public databaseThe results from Oncomine database analysis showed that, there were12databases met the inclusion criteria, including5EPB49-upregulation database and7EPB49-downregulation database. It should be noted that downregulation of EPB49was showed in a large sample database, TCGA database (881samples). The results from GSEA analysis suggested that CRC related gene set,"KEGG_COLORECTAL CANCER11, was upregulated in low EPB49CRC patients, and related genes were enriched significantly (ES=0.4, P<0.05)2) The expression of EPB49in CRC tissueThe results from IHC showed that EPB49positive expression mainly localized in membranes and cytoplasm, marked by yellow-brown. The expression of EPB49in CRC tissue was lower than that in paired normal colon tissue in79.5%(169/200) CRC patients. The results from westernblot and RT-PCR also suggested that the protein and mRNA level of EPB49in CRC tissue were lower than that in paired normal colon tissue.3) The relationship between EPB49expression and clinicopathological parametersThere were no significant differences of EPB49expression in different age groups, different genders(P>0.05); but there were significant differences of EPB49expression in different differentiation, Dukes stage, TNM stage, metastasis and prognosis(P<0.05), and there was a negative correlation between EPB49and these clinicopathological parameters. Kaplan-Meier Survival analysis revealed that5-year overall survival rate in high EPB49expression CRC patients was significantly higher than that in low EPB49expression CRC patients(Log-Rank, P<0.05), and5-year overall survival rate declined with the decreasing of EPB49expression.2. The role of EPB49in proliferation, EMT and invasion of CRC1) The construction of the EPB49overexpression and knockdown Lentiviral vectors, the establishment of stable expression cell linesMolecular cloning techniques were used to construct the EPB49overexpression and knockdown Lentiviral vectors, the results from sequencing show that insertion sequence was correct, there was no mutation, loss, or frameshift. Then, Lentiviral vectors were used to establish stable expression cell lines, and the results from westernblot showed that stable expression cell lines were successfully established.2) The effects of EPB49overexpression and knockdown on proliferation in CRC cellsThe results from MTT assay revealed that, the growth of CRC cells with EPB49overexpression decreased markedly compared with the control group (P<0.05); but the growth of CRC cells with EPB49knockdown increased dramatically (P<0.05). The results from colony formation assay and softagar colony formation assay indicated that, the colony numbers of CRC cells with EPB49overexpression decreased (P<0.05); but the colony numbers of CRC cells with EPB49knockdown increased (P<0.05). The results from Xenograft model in nude mice exhibited that, the growth of tumor from CRC cells with EPB49overexpression decreased (P<0.05), Ki67positive rate of the tumor (33.33%) was lower than that in the control group (86.67%)(P<0.05); but the growth of tumor from CRC cells with EPB49knockdown increased (P<0.05), Ki67positive rate of the tumor (89.67%%) was higher than that in the control group (63.67)(P<0.05).3) The effects of EPB49overexpression and knockdown on invasion and migration in CRC cellsThe results of westernblot showed that, the expression of EPB49was downregulated dramatically in CRC cell treated with TGF-β for72h. The results of westernblot and immunofluorescence also suggested that the expression of the epithelial marker of EMT, E-cadherin increased, and the expression of the mesenchymal marker, Vimentin decreased in HCT116and SW480with EPB49overexpression; however, the expression of E-cadherin and Vimentin showed opposite results in HT29and SW620with EPB49knockdown.The results from transwell migration assay showed that, the migration cell numbers of CRC cells with EPB49overexpression decreased markedly compared with the control group (P<0.05); but the migration cell numbers of CRC cells with EPB49knockdown increased dramatically (P<0.05). The results from scratch wound healing assay displayed that the speed of wound healing of CRC cells with EPB49overexpression slower than that in the control group; the speed of wound healing of CRC cells with EPB49knockdown faster than that in the control group. The results from3-D cell culture assay revealed that, the number of protrusion of CRC cells with EPB49overexpression decreased markedly compared with the control group; but the number of protrusion of CRC cells with EPB49knockdown increased dramatically.3. The molecular functions of EPB49in the development and metastasis of CRC1) The analysis of upregulation signaling pathways in CRC with low EPB49expressionThe results of GSEA assay suggested that, RHO_GTPASES and Racl signaling pathways was upregulated in CRC tissues with low EPB49expression in GSE13067and GSE35896, and the pathway-related genes were enriched significantly (P<0.05)2) The regulation of the activity of Racl by EPB49 The results from confocal laser scanning microscopy and westernblot showed that, the expression of EPB49in HCT116and SW480with a round morphology is lower than that in HT29SW620with an elongated morphology; however, the expression of Racl-GTP in HCT116and SW480with a round morphology is higher than that in HT29and SW620with an elongated morphology. Moreover, the results of westernblot displayed that the expression of Racl-GTP decreased in HCT116and SW480with EPB49overexpression decreased compared with the control group; however, he expression of Racl-GTP increased in HT29and SW620with EPB49knockdown compared with the control group.3) EPB49inhibits Racl activity by interacting with TiamlThe result of westerblot exhibited that the expression of Racl-GTP increased in SW480transfected by Tiaml vector compared with control vector; however, the expression of Racl-GTP decreased in SW480cotransfected by Tiaml and EPB49vector.The results of immunofluorescence showed that EPB49co-localized with Tiaml in the cell membrane and cytoplasm in HCT116, SW480, HT29and SW620. The results of Co-IP indicated that EPB49interacted with Tiaml through core domain (226-336aa), and Tiaml bound to EPB49through the domain DH, PHc and PHn. Then Tiaml and EPB49truncated mutant, including M1, M2, M3, M4and M5, were cotransfected in SW480respectively, the results of westernblot displayed that the expression of Racl-GTP decreased in the M1, M3and M5groups, which suggested that EPB49(226-336aa) could interact with domain DH, PHc and PHn of Tiaml, and inhibit the activation of Rac1.4) EPB49-Tiaml-Racl signaling pathway regulated cell morphology and cytoskeletal remodelingThe results from confocal laser scanning microscopy assay indicated that, HCT116and SW480with EPB49overexpression showed round morphology and fewer protrusion compared with parent cells, which showed elongated morphology and more protrusion; by contrast HT29and SW620with EPB49knockdown showed elongated morphology and more protrusion compared with parent cells, which showed round morphology and fewer protrusion.5) EPB49-Tiaml-Racl signaling pathway regulated Wnt and MAPK pathwayThe results of GSEA suggested that Wnt_beta-catenin signaling pathway was upregulated in CRC with low EPB49expression, and pathway-related genes were enriched significantly (P<0.05). The results of dual luciferase reporter gene assay of TOP/FOP showed that, the activity of Wnt signaling pathway decreased in HCT116and SW480with EPB49overexpression compared with the control group; by contrast, the activity of Wnt signaling pathway increased in HT29and SW620with EPB49knockdown compared with the control group. Moreover, the results of westernblot showed that the expression of CyclinDland p-JNK decreased in HCT116and SW480with EPB49overexpression compared with the control group; but the expression of CyclinD1and p-JNK showed opposite results in HT29and SW620with EPB49knockdown. The expression of p27was upregulated in n HCT116and SW480with EPB49overexpression,but downregulated in HT29and SW620with EPB49knockdown.4. The upstream regulating mechanism of EPB49in CRC1) The analysis of deletion and mutant of EPB49in CRCThe results of bioinformatics analysis showed that the deletion rate of EPB49is6.3%(37/589) and2%(5/257) in TCGA (Provisional) and the TCGA (Nature2012) colorectal cancer databases respectively; the mutant rate of EPB49is1.4%(1/72) in Colorectal (Genentech) databases.2) The analysis of CpG Island of EPB49geneThe prediction results of UCSC database displayed that there are two CpG Island in promoter region and the first exon of EPB49gene. GC content is72%and71.2%, the size is250bp and793bp, the ratio of observed to expected CpG is0.89and0.62, the CpG number is20and89, respectively. The analysis results of GEO showed that the degree of CpG Island methylation in CRC is higher than that in the normal intestinal mucosa.3) The expression of EPB49in CRC cells treated with5-Aza-CdRThe results of westernblot and RT-PCR revealed that the expression of EPB49 increased in CRC cells treated with5-Aza-CdR in a concentration dependent manner.4) The detection of the degree of CpG Island methylation in CRC tissuesThe results of BSP suggested that the degree of CpG Island methylation was5%,15%,10%,25%,20%in the normal intestinal mucosa tissues respectively, and the degree of CpG Island methylation was15%,50%,45%,70%and65%in CRC tissue. The degree of CpG Island methylation in CRC tissues was higher than that in the normal intestinal mucosa tissues.5) The relationship between the expression of EPB49and the degree of CpG Island methylationCombining the results of RT-PCR and BSP, we found that there was a negative correlation between the expression of EPB49and the degree of CpG Island methylation, the expression of EPB49decreased with increasing the degree of CpG Island methylation.CONCLUSION:1. The expression of EPB49is downregulated in CRC; there is a negative correlation between EPB49and clinicopathological parameters, including differentiation, Dukes stage, TNM stage, metastasis and prognosis.2. The overexpression of EPB49inhibits proliferation, EMT, invasion and migration in colorectal cancer cell; while the knockdown of EPB49promotes tumor cell proliferation, EMT invasion and migration.3. EPB49inhibits the activity of Racl by interacting with Tiaml in CRC cells.4. EPB49regulates cytoskeletal remodeling, the activity of Wnt and MAPK signaling pathways by modulating the activation of Tiaml-Racl signaling pathway.5. The expression of EPB49can be regulated by methylation of CpG Island...