Functional Study Of DKK4 In Colorectal Cancer And The Underlying Mechanisms

【Background】Colorectal cancer is one of the most common gastrointestinal malignant tumors.The2018 Global Cancer Statistics found that the incidence of colorectal cancer ranks third among all malignant tumors,while the mortality rate has jumped to the second place.Since the early symptoms of colorectal cancer are atypical,most patients have metastases at the time of diagnosis.Patients with advanced colorectal cancer did not respond well to treatment,the five-year survival rate is only 10%.However,the main cause of death in patients with advanced colorectal cancer is tumor invasion and metastasis.Therefore,the study of pivotal molecular changes in the occurrence and development process of colorectal cancer,the elucidation of molecular regulation mechanism and the search for new therapeutic targets for advanced colorectal cancer may bring greater hope for their survival.The Wnt signaling pathway plays an important role in the occurrence and development of colorectal cancer,and the Dickkopf family(DKKs)is a classical inhibitor of Wnt signaling pathway.The previous study in our experimental group found that miR-100 and miR-125 b regulated the Wnt signaling pathway by targeting DKK1/DKK3,so as to promote the resistance of cetuximab monoclonal antibody.We found that the expression of DKK4 was correlated with the prognosis of patients with colorectal cancer,and patients with high expression of DKK4 had a better prognosis.However,the molecular mechanism was still unclear.Therefore,this study aims to reveal the expression of DKK4 in colorectal cancer and its molecular mechanism.【Objectives】1.To detect the expression level of DKK4 in colorectal cancer tissues and cells,and to establish the up-regulated and down-regulated cell model of DKK4.2.To observe the effect of DKK4 on the malignant phenotype of colorectal cancer cells.3.To explore the molecular mechanism of DKK4 regulating the malignant phenotype of colorectal cancer.4.To explore the regulation mode of DKK4,and to reveal the regulatory effect of miRNA on DKK4.【Methods】1.Detecting the expression of DKK4 in colorectal cancer tissues and cells.1)Real-time quantitative fluorescence PCR was used to detect the RNA expression level of DKK4 in normal intestinal epithelial cells and a variety of colorectal cancer cell lines,from which cell lines suitable for building cell function models were screened out.2)Immunohistochemistry was used to detect the expression of DKK4 in the primary and adjacent tissues of colorectal cancer microarray,and the difference of expression intensity of DKK4 in adjacent tissues and colorectal cancer tissues.And analyzed the relationship of DKK4 with tumor stage,metastasis and prognosis.2.Establishment of colorectal cancer cell models with down-regulated and overexpressed DKK4.1)Transient transfection of colorectal cancer cells with DKK4-si RNA and verificated their interference efficiency.2)DKK4-sh RNA lentivirus infected SW480 and HCT116 cell lines,and to build DKK4 down-regulated cell model and verify their interference efficiency.3)DKK4 lentivirus infected Caco-2 and HCT8 cell lines,and to construct DKK4 overexpression cell model and verify their overexpression efficiency.3.To verify the effect of DKK4 on the proliferation and metastasis ability of colorectal cancer cells.1)Transwell migration and Transwell invasion assays were used to detect the migration and invasion ability of colorectal cancer cells in the DKK4 downregulated and up-regulated cell models in vitro.2)The metastasis ability of DKK4 down-regulated cell model was detected by tail vein injection of tumor cells in nude mice and small animal live imaging technique in vivo.3)CCK8(Cell Counting Kit-8)was used to detect the proliferation ability of tumor cells in the DKK4 down-regulated and up-regulated cell models.The subcutaneous tumor formation in nude mice is further verified.4.To explore the molecular mechanisms of DKK4 inhibiting the malignant phenotype of colorectal cancer.1)Real-time quantitative fluorescence PCR was used to detect the expression of CD133 and FZD6 in the up-regulated and down-regulated colorectal cancer cell models.2)Western blot was used to detect the expressions of β-catenin,active β-catenin,p-β-catenin(Ser552),AKT1,AKT2,Phospho-AKT2,MMP2 and MMP3 in cell models with DKK4 downregulated and up-regulated.3)Real-time quantitative fluorescence PCR was used to detect the expression of DKK4 in colon cancer cell lines stimulated by EGF,HGF and Wnt3 a.5.Regulation of DKK4 by miRNA in colorectal cancer cells.1)Establishment of overexpressed cell models of miR-106 a,miR-299-3p and miR-450b-5p.2)Dual luciferase reporter assay performed to verify whether miR-299-3p and miR-450b-5p directly act on DKK4.3)Transwell migration and Transwell invasion assays were used to detect the migration and invasion ability of CRC cells with miR-299-3p and miR-450b-5p.4)The relationship between miR-299-3p,miR-450b-5p and the proliferative capacity of CRC was detected by CCK-8 method in vitro.5)To verify the relationship between miR-299-3p,miR-450b-5p and the proliferation,migration and invasion ability of CRC cells in DKK4 overexpressed cell lines.【Results】1.DKK4 was highly expressed in a variety of colorectal cancer cell lines.The expression of DKK4 in most colorectal cancer tissues was higher than that in adjacent tissues,and the survival time of colorectal cancer patients with high expression of DKK4 was longer.DKK4 expression in 24 colorectal cancer cell lines was significantly higher than that in normal intestinal epithelial cell FHC.A total of 229 pairs of matched colorectal cancer tissues and adjacent tissues were detected on the three microarrays.DKK4 expression in colorectal cancer tissues was significantly higher than that in the adjacent tissues(P<0.0001),and the difference was statistically significant.The survival time of patients in the high expression group was higher than that in the middle-low expression group,and the results showed a statistical difference(P =0.0467).There were no statistically significant differences in gender,age,T stage,M stage,N stage,tumor site,tumor size,or clinical stages between high DKK4 expression group and the middle-low expression group which from 294 patients with colorectal cancer in the three microchips.2.By establishing the up-regulation and down-regulation cell models of DKK4,it was confirmed that DKK4 could inhibit the invasion and metastasis of colorectal cancer cells,but had no significant effect on the proliferation of colorectal cancer cells.Lentiviruses were packed with si RNA sequences whose interference efficiency to SW480 and HCT116 cell lines was greater than 60%.DKK4-sh RNA lentivirus transfected SW480 and HCT116 cell lines,and the expression level of DKK4 was down-regulated by 61% and 78%,respectively(P<0.05,P<0.01).Westen blot also confirmed that DKK4 was significantly down-regulated compared with the control group.And the DKK4 down-regulated cell line model was successfully established.Similarly,DKK4 lentivirus transfected HCT8 and Caco-2 cell lines,and the expression level of DKK4 were increased by more than 5,000 times and 100,000 times,respectively(P<0.001).Western blot results showed that DKK4 was significantly upregulated in protein levels compared with the control group.And the DKK4 upregulation cell line model was successfully established.In vitro experiments showed that down-regulation of DKK4 could enhance the migration and invasion ability of colon cancer cells.Conversely,upregulation of DKK4 inhibits migration and invasion of colon cancer cells.The tail vein metastasis experiment of nude mice confirmed that the ability to form metastatic tumor in lung of nude mice was stronger than that of the control group after down-regulation of DKK4.The results of HE staining showed that the number of HCT116 cells with down-regulation of DKK4 formed metastasis foci in nude mice lung was more than that in the control group.Both experiments showed that DKK4 could inhibit the metastasis of colorectal cancer cells.In colorectal cancer cell lines,up-regulation and down-regulation of DKK4 had no significant effect on cell proliferation ability.The subcutaneous xenograft experiment of nude mice also confirmed that compared with the control group,DKK4 was down-regulated stably,and there were no statistically significant differences in tumor volume,growth rate and tumor weight formed by HCT116-sh DKK4 cells.3.DKK4 can inhibit Wnt/β-catenin signaling pathway and AKT signaling pathway,and AKT phosphorylation of β-catenin at Ser552 site enhances the transcriptional activity of β-catenin,thereby inhibiting the expression of MMP2 and MMP3.The ligand Wnt3 a and the cytokine EGF of the Wnt pathway can promote the expression of DKK4.The results of Western blot showed that there was no significant difference between the up-regulated and down-regulated expression of β-catenin in DKK4 cells compared with the control group.The expression of active β-catenin and p-β-catenin(Ser552)in DKK4 overexpressed cell lines was lower than that in the control group.The expression of active β-catenin and p-β-catenin(Ser552)in DKK4 down-regulated cell lines was higher than that in the control group.The expression of AKT2 and Phospho-AKT2 in DKK4 overexpressed cell lines was lower than that of the control group.The expression of AKT2 and Phospho-AKT2 in DKK4 down-regulated cell lines was higher than that of the control group.The expression of AKT1 in DKK4 up-regulated and downregulated cell lines showed no significant difference compared with the control group.The expression of MMP2 and MMP3 in DKK4 overexpressed cell lines was lower than that in the control group.The expression of MMP2 and MMP3 in DKK4 downregulated cell lines was higher than that in the control group.EGF stimulated CC,CCCR,Caco-2,293 T cell lines,and the expression of DKK4 was significantly higher at48 h than at 0 h,with statistical significance(P < 0.001).In addition,after HGF stimulated colorectal cancer cell lines CC,CC-CR and Caco-2,the expression of DKK4 showed no significant change at 48 h compared with that at 0 h.However,the expression of 293 T at 48 h was significantly higher than that at 0 h,which was statistically significant(P<0.001).Wnt3 a stimulated CC,CC-CR,Caco-2 and 293 T cell lines,the expression of DKK4 was increased on 48 h compared with that of 0 h,with statistically significant difference(P<0.05,P<0.001).4.miR-299-3p and miR-450b-5p promote the migration and invasion of colorectal cells by negatively regulating DKK4 expression,but have no effect on the proliferation phenotype of colorectal cells.Through bioinformatics analysis,the binding sites of miR-299-3p and miR-450b-5p were found in the 3’UTR region of DKK4.After the overexpression of miR-299-3p and miR-450b-5p,the expression of DKK4 in cell lines was significantly lower than in the control group,with statistical difference(P<0.01).Dual luciferase reporter assay showed that miR-299-3p and miR-450b-5p could affect the changes of fluorescence activity by binding DKK4 3’UTR.Colorectal cancer cell lines SW480 and HCT116 were transfected with miR-299-3p and miR-450b-5p,and the migration and invasion ability of colorectal cancer cells were significantly enhanced compared with the control group,with statistical significance.There was no significant effect on the proliferation of colorectal cancer cells.The DKK4 overexpressed cell lines Caco-2-NC,Caco-2-DKK4,HCT8-NC and HCT8-DKK4 were selected to overexpress miR-299-3p and miR-450b-5p.Compared with the control group,both miR-299-3p and miR-450b-5p promoted the migration and invasion of cells in colorectal cancer overexpressed cell lines,with statistically significant differences(P<0.05,P<0.01,P<0.001).However,there was no significant effect on the proliferation of overexpressed cell lines.【Conclusions】1.DKK4 was highly expressed in most colorectal cancer cell lines and tissues,and the survival time of patients with high DKK4 expression was longer than others.DKK4 overexpression can inhibit the migration and invasion of colorectal cancer cells,but has no significant effect on the proliferation of colorectal cancer cells.DKK4 inhibits Wnt/β-catenin signaling and AKT signaling,and AKT phosphorylation of β-catenin at Ser552 enhances the transcriptional activity of β-catenin,and then inhibits the expression of MMP2 and MMP3.2.Wnt pathway ligands Wnt3 a and cytokines EGF can promote the expression of DKK4.Activation of Wnt/β-catenin pathway and promotion of DKK4 expression is the negative feedback regulation mechanism of this pathway.During the occurrence and progression of colorectal cancer,abnormal activation of Wnt/β-catenin pathway leads to increased expression of DKK4 for the inhibitory negative feedback regulation of this pathway.This mechanism may explain the phenomenon that "DKK4 is highly expressed in most colorectal cancer tissues,but patients in the group with high DKK4 expression have a longer survival time".3.The expression level of DKK4 in colorectal cancer cells is also regulated by miRNA.We found that miR-299-3p and miR-450b-5p promoted the migration and invasion of colorectal cells by directly regulating DKK4,but had no significant effect on the proliferation ability of colorectal cancer cells.