| Colorectal cancer is one of the common malignant tumors.In recent years,with the dietary structure and living habits of our people changed,the incidence of colorectal cancer has also increased year by year.According to the data from China Cancer Report 2017,the incidence and mortality rate of colorectal cancer in men ranks fifth in China,the incidence of colorectal cancer in women ranks third,and the death rate ranks fifth.The 2018 American Cancer Society statistics show that colorectal cancer ranked third in the incidence of malignant tumors.Tumor invasion,migration is the leading cause of high mortality in patients with colorectal cancer.Epithelial-Mesenchymal Transition(EMT)plays an important role in tumor invasion and migration.The occurrence of EMT can lead to the loss of polarity of epithelial-derived tumor cells,reduce the adhesion between cells,destroy the connexin between cells and obtain the relevant characteristics of interstitial cells,thereby increasing the invasion and migration ability of individual cells and further promoting the tumor Invasion and migration.The occurrence of EMT involves the regulation of multiple signal pathways,such as Notch,TGF-β and Wnt pathway.Among them,Wnt/β-catenin signaling pathway in a variety of tumors has been confirmed with the occurrence of EMT are closely related.Activation of Wnt/β-catenin signaling pathway can promote the binding of β-catenin nucleus to T cell-specific transcription factor/lymphatic upregulation factor(TCF/LEF)to regulate the expression of E-cadherin,a characteristic factor of EMT,happened.Therefore,the activation of Wnt/β-catenin signaling pathway is crucial for the development of EMT.The study found that Wnt signaling pathway activation plays an important role in the development of colorectal cancer,pancreatic cancer,gastric cancer and breast cancer.SETDB1,also known as histone H 3 lysine 9(H3K9)methyltransferase,has a 50 kbp gene and locates on human chromosome 1q21.Its main function is to regulate the activity of cell-related proliferation genes.A large number of studies both at home and abroad have confirmed that SETDB1 is highly expressed in various tumor cells such as hepatocellular carcinoma,non-small cell lung cancer,prostate cancer,kidney cancer,glioma,breast cancer,melanoma,and so,SETDB1 is considered as a candidate Oncogene.The preliminary results of this study suggest that SETDB1 expression is increased in colorectal cancer tissues,and in vitro experiments suggest that silencing SETDB1 gene can significantly inhibit the proliferation of colorectal cancer Lovo cell lines and promote its apoptosis.However,whether SETDB1 is also closely related to tumor invasion and migration in colorectal cancer? There are still few reports in the literature.Therefore,in this study,the expression of SETDB1 gene was silenced by using SiRNA technique.The effect of silencing SETDB1 expression on the epithelial-mesenchymal transition and invasion and metastasis of colorectal cancer Lovo cells was observed.To investigate the effect of Wnt/β-catenin pathway on SETDB1-induced colorectal cancer Role of Lovo cells in epithelial-mesenchymal transition.Methods1.Effect of Silencing SETDB1 on Migration,Invasion Ability and EMT of Colon Cancer Lovo Cells in Vitro(1)SiRNA was used to transfect Lovo cells,and transwell invasion assay was used to observe the in vitro invasion ability of transfection group and control group;(2)Scratch test was used to observe the changes of migration in vitro in transfected and control cells;(3)Immunofluorescence was used to detect the change of cytoskeleton of F-actin in transfected and control groups;(4)The mRNA and protein expressions of EMT-related markers E-cadherin and Vimentin in transfected and control cells were detected by RT-PCR and Western-Blot respectively,and the effect of SETDB1 silencing on the EMT of colon cancer Lovo cells was analyzed.2.SETDB1 can induce EMT and promote its migration and invasion in colorectal cancer Lovo cells by regulating Wnt/β-catenin signaling pathway(1)Inhibition of Wnt/β-catenin signaling pathway on colon cancer Lovo cells in vitro migration,invasion and EMT:(1)Xav-939,a specific inhibitor of β-catenin,was used in colorectal cancer Lovo cells,transwell invasive assay was used to observe the changes of invasiv eness of cells in XAV-939 and DMSO control groups,and the migration ability was observed by scratch test;(2)Immunofluorescence was used to detect the morphological changes of cytoskeleton F-actin in the experimental group and the control group;(3)The mRNA and protein expressions of EMT-related markers E-cadherin and Vimentin in XAV-939 and DMSO control groups were detected by RT-PCR and Western-Blot.The effects of inhibiting Wnt/β-catenin signaling pathway on colon Effect of EMT in cancer Lovo cells;(2)The effect of SETDB1 silencing on the localization of β-catenin in colon cancer Lovo cells was observed by immunofluorescence technique;(3)After the expression of SETDB1 was silenced by SiRNA,the expression of β-catenin in the nucleus was detected by Western-Blot.Results1.Silencing SETDB1 on colon cancer Lovo cells in vitro migration,invasion ability(1)Transwell invasion assay and scratch assay showed that the migration and invasion ability of SETDB1-SiRNA-1 and SETDB1-SiRNA-2 cells were significantly lower than those of SETDB1-SiRNA-NC negative control group P <0.05);(2)Immunofluorescence results showed that F-actin was dense,regular and longitudinally arranged in the negative control group.F-actin protein was significantly decreased in the SETDB1-SiRNA-1 and SETDB1-SiRNA-Beam-like arrangement disappears,the edge is damaged,irregular arrangement;(3)The mRNA and protein expression of E-cadherin in SETDB1-SiRNA-1,SETDB1-SiRNA-2 transfected cells were significantly higher than those in SETDB1-SiRNA-NC negative control group,while the expression of Vimentin was lower than that in negative control group Significantly lower(P <0.05).2.SETDB1 regulates EMT induced by colon cancer cell line Lovo via Wnt/β-catenin pathway and promotes its migration and invasion in vitro.(1)Inhibition of Wnt/β-catenin signaling pathway on colon cancer Lovo cells in vitro migration,invasion and EMT:(1)Transwell invasion test and stratch assay showed that the cell migration and invasion ability of XAV-939-Lovo group were significantly lower than that of DMSO-Lovo group(P <0.05);(2)The immunofluorescence results showed that the F-actin fibers in the DMSO-Lovo group were arranged in a neat manner.However,the F-actin fibers in the cells of the XAV-939-Lovo group were sparse and the fluorescence was weaker and arranged irregularly;(3)The results of RT-PCR and Western-Blot showed that the expression of E-cadherin and Vimentin were down-regulated in XAV-939-Lovo group,and downregulated in hippocampus,and Vimentin was up-regulated in DMSO group Significance(P <0.05).(2)The results of immunofluorescence showed that the expression of β-catenin of SETDB1-Si RNA-1 and SETDB1-SiRNA-2 transfected groups was significantly downregulated in the nuclei,while the the SETDB1-SiRNA-NC was significantly increased;(3)The results of Western-Blot showed that the expression of β-catenin in the nuclei of SETDB1-SiRNA-1 and SETDB1-SiRNA-2 transfected groups showed a downward trend,while in the SETDB1-SiRNA-NC the β-catenin protein expression increased.Conclusion1.Silencing SETDB1 can inhibit colon cancer Lovo cells migration and invasion capabilities in vitro;2.SETDB1 may induce epithelial-mesenchymal transition and promote its migration and invasion capabilities in vitro by regulating the Wnt/β-catenin signaling pathway. |
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