The Role And Molecular Mechanism Of SNEP1 On Colorectal Cancer Cells Growth By Regulating Of Hedgehog Signaling Pathway

Background:Colorectal cancer is one of the common gastrointestinal malignancies in the world,and its morbidity and mortality are the third and second in malignant tumors respectively.Its morbidity and mortality ranks fourth in malignant tumors in China.And its morbidity presents a trend of increasing gradually with the change of environment and lifestyle.Because of the pathogenesis of colorectal cancer is not clear,no major breakthrough in the treatment of colorectal cancer.Therefore,further study the molecular mechanism of colorectal cancer occurrence and development,and find new molecular markers and therapeutic targets for colorectal cancer is of great significance for the early diagnosis,clinical treatment and prognosis of colorectal cancer.The Hedgehog(Hh)signaling pathway is a widely distributed and evolutionarily-conserved pathway.This pathway plays an important role in the development of embryos,the formation of tissues and organs,and the maintenance of homeostasis.In addition,aberrant activation of Hh signaling pathway has been associated to the occurrence and development of several types of cancers,including basal cell carcinoma,medulloblastoma,gastric cancer,colorectal cancer,liver cancer and prostate cancer.Significant progress has been made in the understanding of the role and mechanism of Hh signaling pathway in tumor growth.However,due to the tumor heterogeneity and different stages of tumor development,the role and mechanism of Hh signaling pathway in tumorigenesis and development still need to be further clarified.Recently,studies have shown that abnormal activation of Hh signaling pathway is closely related to the occurrence and development of colorectal cancer.However,the role and mechanism of Hh signaling pathway in colorectal cancer development remains unclear,and the molecular mechanism of abnormal activation of Hh signaling pathway in colorectal cancer is also unclear.Therefore,in order to study the role of Hh signaling pathway in the growth of colorectal cancer.We detected the changes in the expression profile of Hh signaling inhibitor-treated colorectal cancer cells,screened the differentially expressed genes,and selected SNEP1(SuFu negating protein 1)for subsequent studies,to clarify the role and molecular mechanism of the abnormal activation of Hh signaling pathway in the occurrence and development of colorectal cancer.Part Ⅰ:SNEP1:a new downstream target gene for the Hedgehog signaling pathwayMethods:1.Colorectal cancer cell line HT-29 cells were treated with cyclopamine and Gant61,that are the upstream and downstream inhibitors of the Hh signaling pathway,respectively.Then used the gene expression profile chip to detect the differences of gene.2.SNEP1 was selected as candidate of a new downstream target gene for Hedgehog signaling pathway.HT-29 or Caco2 cells were treated with Shh ligand,cyclopeptidamine and Gant61,respectively.This target gene was verified by Real-time PCR and Western blot.3.Detection of the changes of SNEP1 expression level by overexpression or interference the transcription factor Gli2 to studied whether SNEP1 was regulated by Gli2.The transcription factor binding sequence was analyzed by Genomatics(http://www.genomatix.de/)to analyze the binding site of Gli2 in the promoter region of SNEP1 gene.It was verified by chromatin immunoprecipitation(ChIP).4.The DNA fragments containing Gli binding sites were cloned to construct the pGL3.0-SNEP1-Luci reporters.This plasmid was used as a deletion mutation,and each binding site was deleted in turn,whether these binding sites were regulated by Gli2 was verified by dual-luciferase reporter system.Results:1.We identified 41 genes as potential Hh responsive targets through cluster analysis,SNEP1 is one of the genes down-regulated by both of the Hh antagonistit.2.The expression of SNEP1 was up-regulated under Shh ligand stimulation,and down-regulated under both cyclopamine and Gant61 treatment.3.Overexpression Gli2 enhance the expression of SNEP1,and knock down of Gli2 by shRNA interference inhibit the expression of SNEP1.4.Three Gli2 binding sites(BS1,BS2,BS3)were identified in SNEP1 promoter region by bioinformatics software analysis.And Gli2 directly bind to these three sites were shown by chromatin immunoprecipitation.5.The dual luciferase reporter system results showed that Gli2 was able to activated the expression of a promoter region containing three binding sites.Luciferase reporter assays driven by different fragments of the SNEP1 promoter region revealed that the Gli2-binding site 1(BS1)was the most important for Gli2 to regulate SNEP1 expression.And BS2 can enhance the activity of BS1,while BS3 attenuates the activity of the BS1 and BS2.Part Ⅱ:SNEP1 promotes colorectal cancer cell proliferation by activating Hedgehog signaling pathwayMethods:1.SNEP1 was overexpressed in HEK293T cells,and the protein levels of important members and target genes in Hh signaling pathway(Gli2,SuFu,Bcl2)were detected by Western blot.It was verified whether SNEP1 could feedback regulate Hh signaling pathway.2.Lentivirus were used to construct HCT-116 and Caco2 stable cell lines that overexpress SNEP1,and HT-29 and SW620 used for SNEP1 knockdown.Western blot was used to verify whether stable cells were successfully constructed,and whether SNEP1 can feedback regulate Hh signaling pathway in colorectal cancer cells.3.Overexpression SNEP1 stable cell lines(HCT-116 and Caco2)were used to detect proliferation capacities of colorectal cancer cells by colony formation experiments and xenograft experiments.To detection the effect of overexpression SNEP1 on proliferation of colorectal cancer cells.4.SNEP1 knockdown stable cell lines(HT-29 and SW620)were used to detect proliferation capacities of colorectal cancer cells by colony formation experiments and xenograft experiments.To detection the effect of SNEP1 knockdown on proliferation of colorectal cancer cells.Results:1.Overexpression SNEP1 results in upregulation of Gli2 and Bcl2 protein levels,and downregulation of SuFu protein level.2.Western blot results showed that the stable cell lines of colorectal cancer that overexpressed and interfered with SNEP1 were successfully constructed.Meanwhile,SNEP1 can also positively feedback to regulate Hh signaling pathway in colorectal cancer cells.3.Colony formation assays and xenograft mice model showed that overexpression SNEP1 promote colorectal cancer cell proliferation.4.Colony formation assays and xenograft mice model showed that SNEP1 knockdown inhibit colorectal cancer cell proliferation.Part Ⅲ:Molecular mechanism of SNEP1 activating Hedgehog signaling pathway to promote colorectal cancer cell proliferationMethods:1.Co-Immunoprecipitation(Co-IP)assay and GST-pulldown assay were used to confirm the interaction between SNEP1 and SuFu protein,and identifying protein interaction sites by deletion mutation the SuFu protein.2.Overexpression SNEP1 cells were treated with MG132 to detect whether SNEP1-induced down-regulation of SuFu through the proteasome pathway.3.SNEP1 was used as a bait protein to screen SNEP1-interacting proteins through the yeast two-hybrid system.We found a candidate protein LNX1(E3 ubiquitin ligase).Co-Immunoprecipitation(Co-IP)assay and GST-pulldown assay were used to confirm the interaction between SNEP1 and LNX1,and identifying protein interaction sites by deletion mutation the LNX1 protein.4.Western blot assay was used to detect the changes of SuFu protein level by overexpression or interference LNX1.5.To investigate whether SNEP1 induce SuFu down-regulation through mediating LNX1.SNEP1 and LNX1 were overexpressed,and SNEP1 was overexpressed while interfering with LNX1,LNX1 was overexpressed while interfering with SNEP1,and the SuFu protein level was detected by Western blot.6.Overexpression LNX1,or overexpression of LNX1 while interfering with SNEP1 to detect the half-life of SuFu protein.7.To detection the effect of nuclear fractions of Gli2 by overexpression of LNX1 or SNEP1 through nuclear separation experiment.8.Co-Immunoprecipitation(Co-IP)assay and GST-pulldown assay were used to confirm the interaction between LNX1 and SuFu,and detected the effect of overexpression or interference of SNEP1 on the interaction between LNX1 and SuFu.9.The effects of SNEP1 and LNX1 on the SuFu ubiquitination level were examined by in vivo and in vitro ubiquitination assay.10.The lysine residue(K),which may be a ubiquitinati on site,is predicted by conservation analysis of the amino acid sequence of SuFu protein.These residues were then mutated to arginine(R),and detect if the mutant is still regulated by LNX1.11.To further investigate whether the SuFu ubiquitination site mutation affects ubiquitination level by ubiquitination assay,and the protein half-life experiment was used to detest whether the mutation of SuFu ubiquitination site affected the half-life of SuFu protein.12.To detection the effect of LNX1 induced down-regulation of SuFu on colorectal cancer cell proliferation in HT-29 cells by the EdU assay.Results:1.SNEP1 interacts with SuFu,and the 110-174 aa sequence of SuFu protein is the core sequence that binds to SNEP1.2.MG132 inhibit the SNEP1-induced down-regulation of SuFu,and SNEP1 induce down-regulate of SuFu through proteasome degradation pathway.3.The result shows that LNX1 interacts with SNEP1 and binds to SNEP1 via the PDZ1 domain.4.Overexpression LNX1 promotes down-regulation of SuFu protein levels,while interference with LNX1 leads to elevated levels of SuFu protein.5.SNEP1 and LNX1 can synergistically promote down-regulation of SuFu,knockdown of SNEP1 can inhibit LNX1-induced down-regulation of SuFu,and knockdown of LNX1 also can inhibit SNEP1-induced down-regulation of SuFu.6.Overexpression LNX1 can significantly shorten the half-life of SuFu,while knockdown of SNEP1 with overexpression of LNX1 can lengthen the half-life of SuFu.7.Overexpression of either SNEP1 or LNX1 resulted in the increase of nuclear fractions of Gli2 in a way similar to that of SuFu knockdown.8.LNX1 interacts with SuFu mainly through RING and PDZ1 domain.Overexpression SNEP1 can enhance the interaction between LNX1 and SuFu,while knockdown of SNEP1 can significantly attenuate the interaction between LNX1 and SuFu.9.LNX1 knockdown decreased the level of polyubiquitination of SuFu,overexpression SNEP1 promotes LNX1-mediated SuFu ubiquitination,and SNEP1 knockdown also reduced LNX1-mediated SuFu ubiquitination.Meanwhile,we confirmed that SNEP1 promotes LNX1-mediated SuFu ubiquitination in vitro ubiquitination assay.10.We found that SuFu-K59 and SuFu-K470 are SNEP1/LNX1-mediates SuFu ubiquitination sites.11.SuFu(K59/470R),a double mutant contains both mutations at K59 and K470,can significantly inhibit the ubiquitination of SuFu,and the half-life of SuFu(K59/470R)was markedly prolonged even in the presence of SNEP1 or LNX1 expression.12.LNX1 enhances the proliferation of colorectal cancer cells by promoting the downregulation of SuFu(WT),not SuFu(K59/470R).Part Ⅳ:Expression and relation with clinical pathological features and prognosis of SNEP1 in colorectal cancerMethods:1.To detect the expression of SNEP1 and SuFu in 395 colorectal cancer tissues and matched adjacent tissues by immunohistochemistry,and then scored.At the same time,Western blot was used to detect the protein expression of SNEP1 and SuFu in 4 colorectal cancer tissues and matched adjacent tissues.2.We analyzed the correlation between the expression of SNEP1/SuFu and clinical pathological features.3.The expression levels of SNEP1 and SuFu were divided into a low expression group and a high expression group.Kaplan-Meier analysis was used to analyze the survival curves of patients with low-expression and high-expression colorectal cancer,and the statistical difference between the two survival curves was tested by Log-Rank.Results:1.The results of immunohistochemistry showed that the expression of SNEP1 in colorectal cancer tissues was significantly higher than that in adjacent tissues(n=395,p<0.001).The expression of SuFu in colorectal cancer tissues was significantly lower than that in adjacent tissues(n=395,p<0.001).The results of Western blot were consistent with the IHC finding.2.The expression level of SNEP1 was significantly correlated with the saccharide antigen CA-125,the general type,the size of the primary lesion,the degree of differentiation,the Dukes grade,and whether there was chemotherapy or not.The expression level of SuFu was significantly correlated with age,differentiation degree and Dukes classification.Moreover,the expression of SNEP1 is negatively correlated with the degree of differentiation,while the expression of SuFu is positively correlated with the degree of differentiation.3.The expression of SNEP1 was negatively correlated with the overall survival and disease-free survival of colorectal cancer patients.The expression of SuFu was only positively correlated with overall survival.Conclusions:1.SNEP1 is a new downstream target gene of Hh signaling pathway,and Gli2 directly regulates SNEP1 transcription.2.SNEP1 promotes colorectal cancer cell proliferation by activating Hedgehog signaling pathway.3.The molecular mechanism of SNEPl promoting colon cancer cell proliferation is that SNEP1,as a scaffold protein,activates Hh signaling pathway through promotes LNX1-mediates SuFu degradation.4.SNEP1 promotes LNX1-mediates SuFu degradation through the ubiquitinproteasome pathway,and ubiquitination at K59 and K470.5.SNEP1 is highly expressed in colorectal cancer tissues,and SNEP1 can be considered an independent prognostic marker for the clinical outcomes of CRC.