Interaction And Regulatory Mechanism Between TUSC3 And E2F1 In Colorectal Carcinoma

BACKGROUND&OBJECTIVEIn our previous study,we found that TUSC3 was up-regulated and promoted tumor cell proliferation and invasion in colorectal cancer(CRC).However the detailed regulatory mechanism of TUSC3 was still unclear in CRC.Here we aimed to figure out the transcriptional regulatory of TUSC3 by E2F1 and the relationship between TUSC3 and E2F1.We further explored the molecular mechanism underlying how TUSC3 regulating E2F1 in CRC.METHODS1.Bioinformatic analysisWe first predicted the transcriptional factors of TUSC3 by informatic method and chose E2F1 to validate.The probable binding sites between E2F1 and TUSC3 promoter were also predicted through online database.Four of the most possible binding sequences were selected to construct plasmids for dual-luciferase reporter system and design primer for Chip-qPCR experiment.2.Analysis of the correlation between TUSC3 and E2F1 expression in colorectal cancer cells in vivo and in vitro2.1 120 cases of colorectal cancer tissues and paired normal colon mucosa were collected from Nanfang Hospital,Southern Medical University.Then Western blotting,qRT-PCR and immunohistochemical assays were used to detect the expression levels of TUSC3 and E2F1 in CRC cells and tissues,Then the expression correlation between E2F1 and TUSC3 was analyzed.The relationship between E2F1 expression and the clinicopathological parameters of CRC was also detected.2.2 Over-expression and si-RNA induced down-regulation of E2F1 cell lines were achieved by transient transfection.The efficiencies of silencing and over-expression of E2F1 were examined using western blotting.Then CCK8 assay,transwell migration assay,wound healing assay,and colony formation assay were performed to detect the effects of ectogenic over-expression or down-regulation of E2F1 on the biological behaviors of CRC cell in vitro.3 TUSC3 was transcriptionally regulated by transcription factor E2F13.1 Dual-luciferase report system was used to explore the effects of E2F1 on the transcriptional active of TUSC3 promoter.Furthermore the best binding sequence between E2F1 and TUSC3 promoter was also identified.3.2 qRT-PCR,western blot,nuclear plasma separation and immunofluorescence technique were used to analysis the expression level of TUSC3 in E2F1-interferent and over-expressed CRC cell lines.4.Explore the mechanism of E2F1 regulated by TUSC3Over-expression and si-RNA induced down-regulation of TUSC3 cell lines were achieved using lentiviral vector.The silencing and over-expression efficiencies were assessed by western blotting.The interaction between E2F2 protein and TUSC3 protein was tested by co-immunoprecipitation assay in CRC cell.Western blotting,qRT-PCR,nuclear plasma separation and immunofluorescence technique were used to analyze expression of E2F1 in TUSC3 knockdown and over-expression CRC cell lines.Then detect E2F1 expression by Western blotting in TUSC3-overexpressed CRC cell lines after blocking Wnt/?-catenin and PI3K/AKT pathway.In the end,the molecular weight change of E2F1 protein was detected by Westerm blotting after treated with pGNase F.4.Statistical analysisStatistical analysis was performed by SPSS20.0.A threshold value was set at 0.05(two tailed).RESULTS1.Consistent expression and biological function of TUSC3 and E2F1 show that TUSC3 may correlate with E2F1 in CRC patient.1.1 Over-expression of E2F1 and TUSC3 in CRC cell and tissue1.1.1 Western blotting revealed that both TUSC3 and E2F1 proteins highly expressed in CRC cell lines and tissues.The expression of TUSC3 protein positively correlates with that of E2F1 protein in CRC cells(P<0.05).1.1.2 Real-time PCR revealed that both TUSC3 and E2F1 mRNA was over-expressed in CRC cell lines and tissues.However there was no statistical correlation between TUSC3 and E2F1 mRNA expression in CRC cells and tissues(P>0.05).1.1.3 Immunohistochemical results show that positive rate of E2F1 was higher in CRC group(101/120,84.2%)than normal group(30/120,25%).High expression of E2F1 was observed in 57 cases(47.5%)of CRC while low expression was in the other 63 cases(52.5%).In the total 120 cases,positive rate of TUSC3(95/120,79.1%)was higher in CRC group than normal group(18/120,15%).High expression of TUSC3 was observed in 62 cases(51.7%)of CRC,while low expression was in the other 58 cases(48.3%).The positive rate of TUSC3 and E2F1 in CRC patient was significantly higher than normal tissues(p<0.05).Positive correlation was found between TUSC3 and E2F1 in all paired cases using Spearson statistic analysis(P<0.05).E2F1 expression was significantly associated with TNM stage in CRC patients(P<0.05).However,no relationship was found between E2F1 expression and age,gender,Duke stage and tumor differentiation.1.2.E2F1 promotes cellular proliferation,invasion and migration in CRC in vitroKnockdown of E2F1 could repress the proliferation,invasion and migration ability of SW480 and SW620 cells in vitro(P<0.05),Conversely,over-expression of E2F1 could promote the proliferation,invasion and migration ability of LS174T and HCT116 cells in vitro(P<0.05).2.The expression of TUSC3 was transcriptionally regulated by E2F1 in CRC cell2.1 Four of the most possible E2F1-binding sequences of TUSC3 promoter were predicted through www.Genecard.com and http://biodev.hgen.pitt.edu.database.2.2 Dual-luciferase report system and ChIP-qPCR assays results indicate that E2F1 is able to activate TUSC3 transcription in CRC cells through the specific E2F1-binding site of TUSC3 promoter.2.3 Western blot,qRT-PCR and immunofluorescence technique results illustrated that knockdown or over-expression of E271 colud up-regulate or down-regulate TUSC3 expression in CRC cell lines.The results of nuclear protein extraction assay showed that over-expression of E2F1 enhanced the expression level of TUSC3,especially in cytoplasm.3.TUSC3 up-regulates E2F1 expression through Wnt/?-catenin and PI3K/akt pathwayTUSC3 and E2F1 can mostly colocalize at cytoplasm while a little at nuclear as detected by confocal laser scanning microscopy.Protein co-immunoprecipitation and Western blotting results showed that TUSC3 and E2F1 could combine to each other.Western blot,qRT-PCR immunofluorescence assays results illustrated that silencing or over-expressing TUSC3 in CRC cell lines could up-regulate or down-regulate E2F1 expression.Nuclear protein extraction assay result showed that over-expression of TUSC3 could elevate the expression level of E2F1,especially in nuclear.When Wnt/?-catenin and PI3K/akt pathway was blocked respectively,the expression level of E2F1 decreased obviously in TUSC3 over-expressed cell lines,indicating that TUSC3 may regualte E2F1 expression via Wnt/?-catenin and PI3K/akt pathway.No molecular weight change of E2F1 protein was found after the protein was treated with pGNase F in CRC cell,indication no glycosylation change was observed in E2F1 protein..conclusionIn this study,we have found for the first time that E2F1 activates TUSC3 expression via binding to TUSC3 promoter region(+296bp-+319bp)directly.E2F1 promote the proliferation,invasion and migration ability of CRC cells in vitro.Interestingly,TUSC3 up-regulates E2F1 through Wnt/?-catenin and PI3K/Akt pathway,indicating a positive feedback regulation between TUSC3 and E2F1.