| B-Myb is a member of the Myb family of transcription factors,located at chromosome 20q13.B-Myb is widely expressed in the developing cells.Through determining the cultured cells in vitro,the B-Myb expresion level is the highest at S-phase.Moreover aberrant B-Myb expression can affect the cell cycle progress,apoptosis,carcinogenesis and senescence.The datas of gene expresion profile suggest that the B-Myb expresion level is more higher in the cancers of numerous types than in the normal tissues of the same histologic origin.B-Myb promotes the cell clone formation,cell cycle progress,migration and invasion in the breast cancer.Moreover B-Myb promotes the cell proliferation,transition of G1-S and G2-M phase in the hepatocellular carcinoma.These studies indicate that B-Myb has the role of promoting tumor development and progression.To date,the function of B-Myb and its molecular mechanism impact in colon cancer have remained elusive.In the present study,we investigate the expression of B-Myb,the function and its molecular mechanism in colon cancer.Part 1: The expression of B-Myb in colon cancer samplesObjectives: To detect the expression of B-Myb in colon cancer tissues,normal colon tissues and different colon cancer cell lines.To analysis the relationship between B-Myb expression level and clinicopathologic features.Methods: The expression of B-Myb mRNA and protein in colon tissues were respectively determined by qRT-PCR,Western blot and immunohistochemistry(IHC).The statistical analysis was used for relationship with the pathologic grade,clinical stage and size of the tumour,lymph node metastasis.Results: The B-Myb expression was determind in a panel of colon cDNA arrays including 43 patients with colon cancer and six healthy controls,six colon cancer cell lines and colon tissue microarray including 110 patients with colon cancer and ten healthy controls.The statistical analysis showed that the expression level of B-Myb mRNA was significantly up-regulated in colon cancer samples compared with normal colon tissues(p=0.002),and has relationship to clinical stage of the tumour(p=0.005).The expression level of B-Myb was significantly different in six colon cancer cell lines,being the highest in SW620 cell and the lowest in RKO cell.The datas of IHC showed that the main expressional location of B-Myb is in cytoplasm.The expression level of B-Myb protein was significantly up-regulated in colon cancer samples compared with normal colon tissues(p<0.05),and has significant relationship to pathologic grade(p<0.05)and size(p<0.001)of the tumour.Conclusion: The expression level of B-Myb was significantly up-regulated in colon cancer samples compared with normal colon tissues,correlating with pathologic grade,clinical stage and size of the tumour,and being significant different in six colon cancer cell lines,being the highest in SW620 cell and the lowest in RKO cell.Part 2: The function of B-Myb in the development and progression of colon cancerObjectives: To study the effects of overexpression or knockdown B-Myb on cell proliferation,cell cycle,cell apoptosis,migration,invasion,colony formation in colon cancer cell HCT116,HT29 and/or SW620,and tumorigenesis in vivo.Methods: The expression levels of B-Myb were detected by qRT-PCR and Western blot in the colon cancer cell HCT116,HT29 and/or SW620 of overexpression/knockdown B-Myb.After overexpression or knockdown of B-Myb,The cell proliferation,the cell cycle and apoptosis,migration and invasion,colony formation and tumorigenesis were respectively detected by CCK8 assays,flow cytometry and immunofluorescence,transwell,plate colony-forming assay and tumor-forming assay in vivo.Results: Lentivirus-mediated and siRNA-mediated establishment of colon cancer cell HCT116,HT29 and SW620 of B-Myb overexpression and knockdown was finished.The results by cck8 assay have showed that the proliferation was significantly increased in HCT116(P<0.001)and HT29 cells(P<0.001)of stable B-Myb overexpression compared with lentivirus-mediated control stable cells from 72 h to 96 h,increased in HCT116(P<0.01),HT29(P<0.01)and SW620 cells(P<0.05)of siRNA knockdown of B-Myb compared with siRNA control cells from 48 h to 96 h,significantly reduced in HCT116(P<0.01)and HT29 cells(P<0.01)of stable B-Myb knockdown compared with lentivirus-mediated control stable cells from 72 h to 96 h.The results by flow cytometry assay have showed that the cell numbers at G1(p<0.05),S(p<0.01)and G2/M(p<0.05)phase were respectively increased,reduced and increased in HCT116 and HT29 cells of stable B-Myb overexpression compared with lentivirus-mediated control stable cells;the cell numbers at S(p<0.05)and G2/M(p<0.001,p<0.01)phase were respectively reduced and increased in HCT116 and SW620 cells of siRNA knockdown of B-Myb compared with siRNA control cells;the cell numbers at G1(p<0.05,p<0.001)and G2/M(p<0.001,p<0.01)phase were respectively reduced and increased in HCT116 and HT29 cells of stable B-Myb knockdown compared with lentivirus-mediated control stable cells,the cell numbers at S phase(p<0.05)reduced in only HCT116 cells.The results by flow immunofluorescence assay have showed that the cell numbers at S(p<0.01,P<0.001)and M(p<0.01,P<0.001)phase were all increased in HCT116 and HT29 cells of stable B-Myb overexpression compared with lentivirus-mediated control stable cells;the cell numbers at M(p<0.01,p<0.05)phase were increased in HCT116 and HT29 cells of stable B-Myb knockdown compared with lentivirus-mediated control stable cells,the cell numbers at S phase reduced in HCT116 cells(p<0.001),and increased in HT29 cells(p<0.05).The results of apotosis determination have showed that the percentage of cell apotosis was significantly reduced in HCT116 and HT29 cells of stable B-Myb overexpression compared with lentivirus-mediated control stable cells,increased in HCT116 and HT29 cells of stable B-Myb knockdown compared with lentivirus-mediated control stable cells.The results of wound healing assay have showed that the lateral migration was significantly increased in HCT116 and HT29 cells of stable B-Myb overexpression compared with lentivirus-mediated control stable cells,reduced in B-Myb knockdown cells compared with control cells.The results of transwell assay have showed that the motility was significantly increased in HCT116(migration,P<0.001;invasion,P<0.001)and HT29(migration,P<0.001;invasion,P<0.001)cells of stable B-Myb overexpression compared with lentivirus-mediated control stable cells;the migration was significantly reduced in HCT116(P<0.001)and HT29(P <0.01)cells of siRNA knockdown of B-Myb compared with siRNA control cells;the motility was significantly reduced in HCT116(migration,P<0.001;invasion,P<0.001)and HT29(migration,P<0.05;invasion,P<0.001)cells of stable B-Myb knockdown compared with lentivirus-mediated control stable cells.The results of plate colony formation assay have showed that stable B-Myb overexpression in the HCT116(P<0.01)and HT29(P<0.05)cells remarkably enhanced colony forming ability compared with lentivirus-mediated control stable cells;stable B-Myb knockdown in the HCT116(P<0.05)and HT29(P<0.05)cells remarkably inhibited colony forming ability compared with lentivirus-mediated control stable cells.The results of tumorigenesis of HCT116 cells in vivo assay have showed that both tumor volume(P<0.05)and tumor weight(P<0.01)were significantly increased in the three nude mice with stable B-Myb overexpression;both tumor volume(P<0.05)and tumor weight(P<0.001)were significantly increased in the five nude mice with stable B-Myb knockdown.Conclusion: B-Myb enhances cell proliferation,G1/S phase transition,motility,colony forming ability and tumorigenesis ability of HCT116 cells in vivo,inhibiting cell apoptosis.Part 3: The molecular mechanism of the development and progression of colon cancer induced by B-MybObjectives: To verify the molecular mechanism of the development and progression of colon cancer induced by B-Myb.Methods: Downstream target genes and pathways were Screening by GO and pathway enrichment analysis.Downstream target genes and ERK signaling pathway were respectively verified by qRT-PCR and Western blot assay.B-Myb gene deletion and point mutation bodies were established to explore transcriptional activation domain of B-Myb.Results: RNA-seq analysis was conducted to compare the differential gene expressing profiles between LV-B-Myb overexpression and LV-vector HCT116 cells,shB-Myb knockdown and shNC HCT116 cells.Differential gene expression analysis revealed that totally 1558 genes were differentially up-regulated in response to B-Myb overexpression and down-regulated in response to B-Myb knockdown;totally 1962 genes were differentially down-regulated in response to B-Myb overexpression and up-regulated in response to B-Myb knockdown.GO enrichment analysis for these genes revealed that the functions of downstream target genes included signal transduction,regulation of biological process,cell proliferation,regulation of cell cycle and apoptosis,regulation of cellular process,cell differentiation and growth,regulation of cell motility.The qRT-PCR analysis further confirmed target genes of differential expression,including E2F1、E2F2、E2F3、E2F8、ZNF473、ESCO2、KIF11、NRP1、WISP2、DLC1、KBTBD6、IGFBP3.The qRT-PCR results corresponded well to the RNA-seq data.Pathway enrichment analysis revealed that signaling pathway included MAPK signaling pathway,cytokine-cytokine receptor interaction,pathways in cancer,Rap1 signaling pathway and Jak-STAT signaling pathway.Immunoblot analysis demonstrated that overexpression of B-Myb significantly increased the levels of phosphorylated ERK and AKT.Conversely,knockdown of B-Myb significantly reduced the levels of phosphorylated ERK and AKT.B-Myb gene deletion and point mutation bodies,including B-Myb(aa 1-359),B-Myb(aa 1-561),B-Myb(aa 360-700)and B-Myb(N174A),were established.The results of wound healing assay have showed that B-Myb(aa 1-359)didn’t have significant changes on the lateral migration of stable B-Myb knockdown HCT116 cells.However B-Myb(aa 1-561),B-Myb(aa 360-700)and B-Myb(N174A)all promoted the lateral migration of stable B-Myb knockdown HCT116 cells.Conclusion: B-Myb might promote colon cancer HCT116 cell development and progression at least partially through downstream target genes positively regulating ERK and AKT signaling pathway.Moreover,B-Myb genes playing functions in colon cancer HCT116 cell may mainly depend on the CR(aa 369-614)domain. |
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