Studies Of The Biological Role Of DIXDC1 And Its Related MicroRNA In The Progression Of Human Colon Carcinogenesis

DIXDC1 is the human homolog of Ccdl (Coiled-coil-DIX1), a third type of DIX (Dishevelled-Axin) domain-possessing protein and a positive regulator in the Wnt signaling pathway during zebrafish neural patterning. DIX domains of Dvl and Axin, two major Wnt downstream mediators, are involved in homomeric or heteromeric protein interactions and are essential for the signal transduction. Frameshift mutations of Axin2 gene resulting in complete deletion of C-terminal DIX domain of the Axin2 protein were detected in more than 25% of colorectal cancers (CRC) with micro-satellite instability (MSI). Human DIXDC1 was recently isolated as an Axin2 C-terminus binding protein. DIXDC1 was postulated to be associated with CRC tumorigenesis.Full-length DIXDC1 was cloned and rabbit polyclonal antibody was generated. However, there has been very little research on human DIXDC1 gene since it was firstly identified as an actin-binding protein in 2006. The biological role of DIXDC1 remained largely unknown.As a positive regulator in the Wnt pathway, what's the biological role of DIXDC1 in the formation and progression of human colorectal cancer? Is DIXDC1 also regulated by Wnt signaling pathway? Is microRNA also involved in regulation of DIXDC1 expression? What's the relationship between its related miRNA and colorectal cancer tumorigensis? All of these remain unknown. In our study, we explored the biological roles of DIXDC1 and its related microRNA in the progression of human colorectal cancer tumorigensis. DIXDC1 might be used as a potential target for colon cancer prevention and therapy. Our study also shed some new light on the mechanism of Wnt pathway gene activation in human colon carcinogenesis. Purpose To explore the biological role of DIXDC1 in the progression of human colon carcinogenesis and its mechanisms.Methods Lipofectin-mediated gene transfection method was used to establish DIXDC1 overexpression DLD1 cells. Cells proliferation viability was tested by MTT assay in vitro and Tumorigenicity assay in nude mice in vivo. Immunohistochemical staining was used to detect the expression of DIXDC1 in human colorectal cancer tissues and its correlation with high proliferation index. Flow cytometry was used to assay the cell cycle and apoptosis. Western blot was used to detect the cell cycle-related protein and the activity of PI3K and MAPK signaling pathway. Luciferase assay was performed to observe the effect of DIXDC1 on the TCF binding site reporter gene activity. The interaction between DIXDC1 and Phospho-AKT was demonstrated by immunoprecipitation assay. PI3K pathway inhibitor LY294002 was used to detect whether blocking this pathwasy can affect the cell proliferation, cell cycle and cell cycle-related protein expression in DIXDC1 overexpression cells. DIXDC1 siRNA was transfected to knock down DIXDC1 preotein expression, and its effect on cell proliferation, cell cycle, cell cycle-related protein expression and the activity of PI3K signaling pathway was also detected.Results In the current study, ectopic over-expression of DIXDC1 resulted in increased cell proliferation in vitro and accelerated tumorigenesis on nude mice in vivo. Up-regulation of DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated well with high cell proliferation index. We also showed that DIXDC1 promoted G0/G1 to S phase transition concomitantly with up-regulation of cyclinDl and down-regulation of p21 protein. DIXDC1 over-expression cells showed activation of PI3K/AKT pathway.β-catenin was also found to be upregulated in DIXDC1 overexpression cells. The TCF binding site reporter gene activity was higher in DIXDC 1 transfection cells compared with the control group. DIXDC1 might up-regulateβ-catenin and the Wnt pathway target gene cyclin D1. Both siRNA knockdown of DIXDCl and blocking PI3K pathway using specific inhibitor caused G1-S phase arrest, as well as down-regulation of CyclinD1 and up-regulation of p21 in DIXDC1 over-expression colon cancer cells. Conclusion Our study demonstrates that over-expression of DIXDC1 might target p21 and CyclinDl to promote colon cancer cell proliferation and tumorigenesis at least partially through activation of PI3K/Akt pathway.Purpose To clarify whether DIXDC1 protein expression is regulated by Wnt signaling pathway and elucidate the mechanisms involved in this process.Methods Immunohistoshemical staining was used to examine whether DIXDC1 andβ-catenin protein co-localized in human colorectal cancer tissue. DIXDC1 mRNA expression in the human colon tumoral and matched normal tissues was evaluated by Realtmie RT-PCR. In vitro experiment was performed to detect if Wnt-3a affected DIXDC1 mRNA and protein expression using Realtmie RT-PCR and Western blot. The decay rate of DIXDC1 protein in the presence or absence of Wnt-3a was measured by CHX chase assay. Western blot was used to detect the effect of MG132 on DIXDC1 protein expression. To determine whether DIXDC1 protein was targeted for ubiquitination, co-immunoprecipitation was performed to detect the association of DIXDC1 and ubiquitin with and without Wnt-3a treatment. Anti-phosphoserine, anti-phosphothreonine and anti-phosphotyrosine antibodies were used to detect the phosphorylation of endogenous DIXDC 1 after immunoprecipitation by anti-DIXDC1 antibody.Results Positive DIXDC1 staining was detected in colon cancer cells and was co-localized well withβ-catenin staining. However, DIXDC1 mRNA expression was decreased in human colon cancer cells comparing to the matched normal colon epithelial cells. We also found that DIXDC1 protein was induced upon Wnt-3a stimulation, whereas DIXDC1 mRNA level was not significantly increased after Wnt-3a treatment. Our further investigation showed that DIXDC1 protein was degraded through the proteasome pathway, and activation of canonical Wnt signaling decreased the ubiquitin-dependent degradation of both ectopic and endogenous DIXDC 1 protein. In order to explore the possible mechanism of ubiquitination of DIXDC 1, we found that the phosphorylation of DIXDC1 was inhibited by Wnt-3a. Conclution Canonical Wnt/β-catenin pathway activation might up-regulate DIXDC1 through post-translational mechanism by inhibiting the ubiquitin-mediated degradation of DIXDC1 protein. It is possible that Wnt-3a affected the phosphorylation of DIXDC1, resulting in the accumulation of DIXDC1.Purpose To detect the differential expression of DIXDC1 interaction miRNAs in the progression of colon carcinogenesis and evaluate their effects on the expression of DIXDC1 and colon tumorigenesis.Methods Human miRNA microarrays from Agilent Technologies were used in our study. Replicate experiments were performed to determine the variability of array hybridization. The microarray was used to analyze the miRNA expression pattern of normal colon mucosa, inflammatory polyps, colorectal adenoma and carcinoma. Real-time RT-PCR was performed to validate the data from array using 17 miRNAs probes. miRNAs that were differentially expressed between groups were identified using significance analysis of microarray (SAM). miRNAs that predicted to target DIXDC1 and conserved across various species were selected according to TargetScan 4.2. Luciferase assay was perfomed to detect the interaction between miRNAs and the 3'UTR of DIXDC1. miRNAs were transfected into colon cancer cells, the effect of miRNAs on the DIXDC1 protein expression was evaluated by Western blot. Corrected t test:Bonferroni correction was used to estimate differential expression of miRNAs contained the miRNAs related to DIXDC1 during the disease state progression.Results Each miRNA chip from Agilent Technologies contains 8 identical arrays, the average intra-array variation across 112 arrays was 6.3%; Inter-array correlation was 0.999, while inter-chip correlation with reference RNA was also 0.999. The quantitative correlation of fold change between Agilent miRNA microarrays and Real-time RT-PCR was 0.90. The SAM application identified 63 miRNAs that were significantly differentially expressed (40 over-expressed,23 under-expressed) in adenoma-carcinoma compared with normal-polyps tissue, while there were 38 miRNAs significantly differentially expressed (11 over-expressed,27 under-expressed) in adenoma compared with carcinoma. miR-195 and miR-497 were chosen for further investigation, because they were the most two significantly under-expressed. We found that miR-195 and miR-497 can interact with the 3'UTR of DIXDC1 and inhibit DIXDC1 protein expression. Differential expression of miRNAs among different stages of colorectal cancer was deteted. Dynamically, these miRNAs gave us a vision of the transformation and progression of colorectal cancer. Some of them differentially expressed from normal to adenoma transition, some of them differentially expressed from adenoma to carcinoma transition, while others differentially expressed from normal to adenoma to carcinoma transition. miR-195 and miR-497 were progressively downregulated from normal to adenoma to carcinoma, which indicated that miR-195 and miR-497 related to DIXDC1 might have some impacts on the transformation and progression of colorectal cancer.Conclution Series of miRNAs were significantly differentially expressed in the progression of colon carcinogenesis. miR-195 and miR-497 were progressively downregulated from normal to adenoma to carcinoma, while also interacted with the 3'UTR of DIXDC1 and inhibit DIXDC1 protein expression. miR-195 and miR-497 may have some impacts on the progression of colon carcinogenesis through interaction with DIXDC1.