The Roles And Mechanisms Of DAXX In Epithelial Mesenchymal Transformation And Tumor Stem Cells In Colorectal Cancer

Part Ⅰ Expression of DAXX in colorectal cancer and its effect on prognosisObjective: To explore the expression changes of DAXX in colorectal tumor tissues,analyze the correlation between DAXX expression level and tumor metastasis and prognosis,and explore the correlation between DAXX expression level and signal pathway and biological function of colorectal tumor cells.Methods: Rt-qPCR,Western-blot and immunohistochemistry were used to detect the m RNA and protein expression levels of DAXX in tumor tissues,paracancer tissues and liver metastasis tissues.The correlation between DAXX expression level in tumor tissues of colorectal cancer patients and clinical data was analyzed.The expression profile of colorectal cancer samples was searched and downloaded from TCGA database,and the potential pathway or mechanism of DAXX’s involvement in the malignant progression of colorectal cancer was preliminarily explored by bioinformatics methods such as gene set enrichment analysis.Results: The expression of DAXX in tumor tissues was significantly lower than that in adjacent tissues,and the low expression of DAXX was significantly positively correlated with the depth of tumor invasion,that is,the depth of primary tumor invasion was often deeper in patients with low expression of DAXX.The expression of DAXX in liver metastasis tissues of CRC patients is lower than that of primary tumor tissues,and the low expression of DAXX is also significantly positively correlated with the occurrence of liver metastasis,that is,patients with low expression of DAXX are more likely to develop liver metastasis.However,the expression of DAXX was not related to lymph node metastasis,age,gender,etc.In addition,whether liver metastasis occurs or not,low expression or loss of DAXX is an important indicator of poor prognosis in colorectal cancer patients.Based on gene set enrichment analysis,it was found that cellular adhesion molecular pathway,CRC signaling pathway,ECM receptor interaction pathway and TGF-Bata signaling pathway were activated in patients with low DAXX expression.Conclusion: DAXX expression was decreased in CRC tumor tissues,and DAXX expression level was negatively correlated with prognosis of CRC patients.CRC patients with low DAXX expression had a higher incidence of liver metastasis.Part Ⅱ DAXX regulates EMT and CSCs characteristics of colorectal cancerObjective: To investigate the effects of DAXX intervention on biological functions,tumor-formation and metastasis of CRC cells,and to investigate the effects of DAXX intervention on EMT and CSCs characteristics of CRC cells.Methods: Western Blot was used to detect the expression level of DAXX in CRC cell lines.Liposome transfection was used to intervene the expression of DAXX in CRC cells.CCK8 proliferation assay,nicks assay,Transwell migration and invasion assay were used to detect the effects of low expression of DAXX on the proliferation,migration and invasion ability of CRC cells.Sirna-daxx and Overexpression-ecardherin(pcDNA3.1 eukaryotic expression plasmid)were used to intervene the expression levels of DAXX and e-cadherin in CRC cells.Scratch test and Transwell migration and invasion test were used to verify that DAXX affected the migration and invasion ability of CRC cells by EMT.The effect of low expression of DAXX on dryness acquisition of CRC cells was investigated by stem cell pellet-forming assay.To GDSC database(http//:www.cancerrxgene.org/)as the training set,TCGA database through the calculation of R packages "pp Rophetic" CRC samples of 6 kinds of common IC50 of chemotherapy drugs,and to explore DAXX expression and the relationship between the 6 kinds of chemotherapy drug resistance;Flow cytometry was used to observe the influence of low expression of DAXX on drug resistance of CRC cells according to screening results.The model of subcutaneous tumorigenesis and liver metastasis in nude mice was established.The size and number of tumors were detected.Immunohistochemistry was used to detect e-cadherin and CD133 to analyze the expression changes of EMT and the characteristics of tumor stem cells.Results: By detecting the expression of DAXX in different colorectal cancer cell lines,SW48 and HT29 cells with high expression of DAXX were selected and cell lines with stable and low expression of DAXX were established by siRNA.It was found that the cell morphology of SW48 and HT29 with endogenous knockdown DAXX changed from the original round shape to spindle shape,and the cell-to-cell contact was reduced,showing typical EMT.Western blot and RT-qPCR showed that the expression of epithelial marker E-cadherin decreased in SW48 and HT29 cells with low DAXX expression,while the expression of interstitial marker Vimentin increased significantly.By scratch test,Transwell migration test and invasion test,it was found that DAXX knockdown colorectal cancer cells had significantly improved migration ability and invasiveness.Low expression of DAXX can increase the expression of stem cell markers and transcription factors in SW48 and HT29 cells by co-transfection of sir NA-DAXX and PCDNA3.1-e-cadherin.Cell pellet-forming experiments showed that the number and diameter of pellet formation of SW48 cells with low DAXX expression were significantly larger than those of the control group.The sensitivity of SW48 to cisplatin was detected,and it was found that low expression of DAXX can reduce the sensitivity of colorectal cancer cells to cisplatin,and increase the resistance to cisplatin.The tumorigenesis experiment in nude mice showed that the volume and weight of subcutaneous tumorigenesis in SW48-Sidax X #2 cell group were significantly larger than that in SW48-SINC cell group.In addition,the expression of epithelial marker e-cadherin decreased in sw48-Sidaxx #2 cell group,while the expression of tumor stem cell marker protein CD133 increased.The expression levels of E-cadherin and CD133 in colorectal cancer specimens were consistent with those in mouse tumor specimens.The results of nude mouse liver metastasis model showed that the number of liver metastasis in SW48-Sidaxx #2 cell group was significantly higher than that in control group,and the expression of DAXX and e-cadherin in SW48-Sidax X #2 cell group was significantly lower than that in control group,while the expression of CD133 was higher than that in control group.The results were consistent with those of human colorectal cancer patients.Conclusion: Low expression of DAXX can reduce the expression of E-cadherin in SW48 and HT29 cells,promote EMT of CRC cells,and enhance cell proliferation,migration and invasion.Reverse intervention of E-cadherin can reverse the above changes.Low expression of DAXX can promote the stemification of CRC cells and enhance drug resistance of cell tumors.Low expression of DAXX can promote tumorformation of CRC cells in vivo and liver metastases,and is associated with cell EMT and stem cell transformation.Part Ⅲ Study on the mechanism of DAXX regulating EMT and CSCs characteristics of colorectal cancerObjective: Screen and explore the possible action sites of DAXX regulating EMT and tumor stem cell transformation,and explore the specific mechanism of DAXX regulating EMT and tumor stem cell characteristics through downstream molecules,by intervening in the change of EMT level of CRC cells,the change of tumor stem cell characteristics and drug resistance under DAXX and downstream molecular conditions.Methods: EMT and stem cell pathway related transcription factors were screened by literature study.Immunoprecipitation and confocal fluorescence were used to detect the interaction between DAXX and downstream signal molecules.Sirna-daxx and sir NA-downstream molecules were used to intervene DAXX and downstream signal molecules,respectively.Luciferase reporter gene detection and Western Blot were used to detect the transcription activity and expression level of E-cadherin.After the intervention,CRC cells were pelleted and cisplatin resistance were detected by flow cytometry.Results: Through literature study,ZEB1,ZEB2 and SLUG were selected as possible downstream molecules of DAXX.Through co-IP and fluorescence confocal detection,it was found that ZEB1 could directly bind to DAXX.The expression of DAXX in SW48 cells was knocked down by SIRNA-DAxx #2,and the activation of TGF-β pathway was detected by RT-qPCR.The results showed that low expression of DAXX could significantly promote the m RNA expression of TGF-β signaling pathway protein in SW48 cells.By luciferase reporter gene assay,inhibition of DAXX could induce the high expression of e-cadherin,and inhibition of DAXX and ZEB1 could reverse the above changes.Western Blot analysis verified the above results.Through low expression of DAXX,it was found that CRC cells had increased pellet-forming ability and drug resistance,while inhibition of ZEB1 could reverse these changes.Conclusion: DAXX can directly bind to ZEB1 and regulate the expression level of ZEB1 in CRC cells in this way.DAXX can regulate the transcription and expression of e-cadherin through ZEB1,and then affect the EMT level of CRC cells.DAXX can regulate the dry changes of CRC cells through ZEB1,and affect the drug resistance of CRC cells.