| BACKGROUNDColorectal cancer is one of the malignant tumors of the world. New cases has been increased to1million every year, the speed of incidence is increasing to4.2%. Colorectal cancer has no obvious symptoms at early stage, Metastasis is the main cause leading to death of paitents with CRC. Therefore, to work out the mechanism of develo pment and progression of CRC and to find out the preventive and therapeutic methods is the main task in the field of colorectal cacer.The occurrence and development of colorectal cancer is an extremely complex biological phenomena.It required polygenic participation and multiple stages.The discovery of oncogenes and tumor suppressor genes, cell signaling pathways, has greatly enriched our understanding of cancer. APC, MCC, K-ras, MMR and p53gene mutation or inactivation is the classical molecular genetics of colorectal.Wnt signaling pathway is highly conservative in biological evolution.It regulate many life activities. It is not only determines the cell growth, differentiation, but also regulating the differentiation of important organs such as cardiovascular, central nervous. WNT signal pathway directly controls the cell’s polarization, proliferation, differentiation and apoptosis. The disorders of Wnt signaling pathway have been linked to a variety of tumors. cervical cancer, breast cancer, gastric cancer, liver cancer, melanoma, glioma, etc. Disorder Wnt signaling cause intracellular protein β-catenin accumulation, free β-Catenin can enter the nucleus, regulating downstream gene expression, thus promotes tumorigenesis.HOXB7is a member of the Antp homeobox family, and encodes a protein with a homeobox DNA-binding domain. Homeobox gene (homeoboxgene) first discovered in Drosophila. It functions as a sequence-specific transcription factor that is involved in cell proliferation and differentiation. Increased expression of this gene is associated with some cases of melanoma, ovarian carcinoma, ovarian cancer, oral cancer and prostate cancer.. In recent years, many studied found that HOXB7play an important role in tumor cell proliferation, movement, and invasion. It has close relationship with clinical pathological stage.Affymetrix gene microassay analysis for detection of distinguishing patterns of gene expression was conducted our previous research, which showed that HOXB7was upregulated in the cells with high metastatic potency, which suggests a possible link between HOXB7and the genesis, development and metastasis of CRC. In our early research, we detected the expression and biological function of HOXB7in colorectal cancer cells and tissues, and found that HOXB7was upregulated in CRC cell lines and surgical CRC specimans. HOXB7was positively correlated with Dukes stage, T stage, distant metastasis and Ki-67. Patients with high HOXB7expression exhibited poor prognosis. Further analysis on the biological functon of HOXB7through in-vivo and in-vitro experiment showed that HOXB7promoted the proliferation and tumorigenesis of human CRC cells.In our early analysis of the connection between HOXB7expression and clinicopathological characteristics in224CRC cases, we also found that overexpression of HOXB7in CRC was significantly associated with distant metastasis. These results suggested that HOXB7not only plays an important role in the growth and proliferation of human CRC, but also regulates the process of invasion and metastasis of CRC.NimbleGen genome-wide expression microarray and mass spectrometry analysis suggest HOXB7has the cloes relationship with β-Catenin and wnt signaling pathway, we aim to clarify the possible role and mechanism of HOXB7gene in the proliferation, invasion and metastasis of CRC. It will be helpful to understand the molecular basis of CRC, and establish HOXB7as a new target for metastatic diagnostic markers and novel therapeutic strategies.METHODS1. Stable cell lines were established by Lentiviral expression system. Nimble Gen genome-wide expression microarray and mass spectrometry analyze the paired cell lines:HOXB7overexpress cell line (SW480/HOXB7) and control group (SW480/vector).2. The expression of HOXB7was examined in specimens of100CRC with follow-up visit information by immunohistochemical method. HOXB7overexpress cell line (293FT/HOXB7), control group (293FT/vector) and HOXB7silenced cell lines (SW620/shHOXB7and HCT115/shHOXB7) and control groups (SW620/Scramble and HCT115/Scramble) were established by Lentiviral expression system.. Immunofluorescence, Real-time PCR and western blot were used to analysing the Colocalization between HOXB7and β-Catenin.3.Construction of the HOXB7and P-Catenin mutant plasmids.Protein co-immunoprecipitation(CO-IP),chromosome immune co-precipitation (CHIP) and laser scanning confocal microscope were used to explore the mechanism of HOXB7carry β-Catenin enter the nuleus and located at TCF4/LEF1promoter region.4. TOP FLASH/FOP FLASH dual luciferase report system was used to detecte Wnt signaling pathway activity of SW480/HOXB7,293FT/HOXB7, HCT15/shHOXB7, SW620/shHOXB7and control cell line SW480/vector,293FT/vector,HCT15/Scramble and SW620/Scramble.Western blot, Real-time PCR and SP Flow technology were used to detected expression of OCT-4、SOX-2、 CD133and CD44genes of these cell lines.RESULTS1.Nimble Gen genome-wide expression microarray and Mass Spectrometry analyze preliminary mechanism of HOXB7promoting CRC cell invasion and migration Nimble Gen genome-wide expression microarray and Mass Spectrometry analyze the HOXB7overexpressed cell line SW480/HOXB7and control cell line SW480/vector. We found that stable overexpressed HOXB7can directly combine with β-catenin and activate Wnt signaling pathway.2.HOXB7can regulate β-CateninImmunohistochemical detect the expression of HOXB7and β-Catenn in100colorectal cancer specimens. Results showed that HOXB7is lowly expressed in8cases of samples, and33cases samples showed moderate expression. HOXB7is highly expressed in59cases of samples. The expression of β-Catenin is consistent with HOXB7.Confocal laser scanning microscopy analyze the stable overexpressed HOXB7cell line (SW480/HOXB7,293ft/HOXB7) and control cell line SW480/vector,293ftvector), and stable interference HOXB7cell lines (HCT15/shHOXB7and SW620/shHOXB7) and control cell line (HCT15/Scramble and SW620/Scramble).We found that HOXB7and β-Catenin can Colocalize at nuclear and cytoplasm.Western blot and Quantitative PCR technology result illustrate that overexpressing HOXB7could upregulate β-Catenin expression, and inversely, knowndown HOXB7repressed the expression of β-Catenin.3.Exploration the reaction mechanism between HOXB7and β-CateninHOXB7-FLAG and β-Catenin-HA plasmids were co-transfected into SW480cells. Protein co-immunoprecipitation and Western blot results showed that HOXB7and β-Catenin could combine together.Nuclear protein extraction and Protein co-immunoprecipitation assays analyzed the SW480/HOXB7,293ft/HOXB7and control group SW480/vector,293ft/vector cell lines,results illustrated that both HOXB7and β-Catenin could locate at nuclear. We determine the effect of HOXB7mutants on β-Catenin Nuclear accumulation. β-Catenin accumulated in the nuclei of Cells expressing full-length HOXB7or the mutant with both the Homeobox domains. Incontrast,β-Catenin failed to accumulate in the nuclei of cells expressing HOXB7Mutants lacking the HOMEOBOX domain.Thus, the nuclear localization of β-Catenin depends on the interaction of β-Catenin with HOXB7and on HOXB7nuclear translocation. Also,co-IP experiments in SW480cells revealed that Arm repeats11-12of β-Catenin interacted with HOXB7. Chromatin immunoprecipitation assay showed that both HOXB7and β-Catenin can enter the nuclear and located at TCF4/LEF1promoter region.4.4.HOXB7influence the activity of the Wnt signaling pathway and downstream target genesTOP FLASH/FOP FLASH dual luciferase report system was used testing stable expression HOXB7(SW480/HOXB7,293ft/HOXB7) cell lines and controls cell line SW480/vector,293ft/vector) the stable interfered cell line (HCT15/shHOXB7and SW620/shHOXB7) and the control cell lines (HCT15/Scramble and SW620/Scramble). We found that Wnt signaling pathway activity was consistent with HOXB7expression. Western blot and Quantitative PCR analyzed the expression of DKK1、 LEF1及CyclinD1in SW480/HOXB7,293ft/HOXB7cell lines and controls cell line SW480/vector,293ft/vector) HCT15/shHOXB7and SW620/shHOXB7and the control cell lines HCT15/Scramble and SW620/Scramble). The results showed that the expression of DKK1、LEF1及CyclinD1were consistent with HOXB7.TOP FLASH/FOP FLASH dual luciferase report, Western blot, Quantitative PCR and SP Flow technology detected SW480-vector,SW480-HOXB7, SW480-HOXB7-scramble, SW480-HOXB7-sh-β-Catenin cells. Results revealed HOXB7and P-Catenin together regulated wnt signal activities.More important, they not only affected the wnt target gene’s expression, but also influented the stem cells number.CONCLUSION1. HOXB7has a close relationship with the β-Catenin and wnt signaling pathway..HOXB7promote β-Catenin locate at nuclear and activate Wnt signaling pathway. 2. Homologous sequence and repeat arm sites are the mainly reaction region of HOXB7-β-Catenin complex.Deletion or mutation of these areas may block β-Catenin entry into the nucleus and inhibit the activities of Wnt signaling pathway. These findings provide a new theoretical basis for the treatment of colorectal cancer.3. HOXB7and β-Catenin can locate at TCF4/LEF1promoter region, together regulate the wnt targetgene, and finally affect colorectal cancer cell proliferation, invasion, migration and transfer ability. |
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