| Objective: Colon cancer is the third most common malignant tumor in the world,with the fourth highest mortality rate.Every year,180,000 peopledie from colon cancer in China,which poses a serious threat to people’s health.Metastasis is the main cause of death in patients with colon cancer,and further research on the mechanism of colon cancer metastasis is of great significance for improving the prognosis of patients.Previous studies have found that Lysine-specific demethylase 1(LSD1)and its interacting proteins play an important role in tumorigenesis and development.In order to further investigate the role of LSD1 interacting protein in colon cancer,this article investigate LSD1 interacting protein X-ray repair cross complementary protein 5(XRCC5&Ku80)through colon cancer tissue,colon cancer cells and nude mice)And Forkhead-Related Transcription Factor 2(FOXF2)function and molecular mechanism in colon cancer.Methods: 1.Investigate the expression of LSD1 and Ku80 in the colon cancer cells LOVO,SW480,HT-29 and HCT116 by real-time quantitative PCR(q RT-PCR)and western blot(western blot),combined with immunoprecipitation,Mass spectrometry screening to identify LSD1 interacting proteins.Visualize the network relationship,molecular function and the pathways involved with LSD1 interacting proteins through online databases.Immunofluorescence experiments,immunoprecipitation,and western blot were used to detect the cellular location and interaction of Ku80 and LSD1.effect.2.Using q RT-PCR and western blot methods to detect the m RNA and protein expression levels of LSD1,Ku80,and FOXF2 in 4 colon cancer cells with different invasion andmigration capabilities,and analyze the correlation among the three and the invasion,migration of colon cancer cells Relevance of capabilities.3.Immunohistochemical technique was used to detect the expression of Ku80 and FOXF2 protein in colon cancer tissues,and to analyze the relationship between it and the clinicopathological features of colon cancer.At the same time,the expression of Ku80,LSD1,FOXF2 in the colon cancer data set of the Cancer Genome Atlas(TCGA)database and the expression correlation between the three were analyzed by bioinformatics.4.Using sh RNA to interfere with the expression of Ku80 in HCT116 cells,the monoamine oxidase inhibitor RN-1 inhibits the activity of LSD1,western blot to detect the expression level of FOXF2,chromatin immunoprecipitation combined with PCR(CHIP-PCR)to verify that Ku80 binds to the promoter region of FOXF2 gene Sequence,and the methylation level of H3K4 in the promoter region,through the Transwell invasion test to detect cell invasion ability,scratch test to detect cell migration ability,flow cytometry to detect cell apoptosis,MTT,EDU to detect cell proliferation,cell cloning test And nude mice tumor formation experiment to observe the cell tumor formation ability.In the HCT116 cells,the sh RNA interference technology was used to interfere with Ku80 expression.Western bolt detection of the downstream target genes LGR5,AXIN2,MYC,and CD44 in the wnt/β-catenin pathway was performed.Results: 1.LSD1 and Ku80 were expressed in 4 colon cancer cells,and the expression was positively correlated with the expression of LSD1;immunoprecipitation combined with mass spectrometry screened and identified 364 proteins interacting with LSD1,using online database to visualize the mutuality of LSD1 Interacting networks of interacting proteins and their intracellular metabolic processes,participating pathways and molecular functions.Ku80 is one of the interacting proteins of LSD1.Further immunofluorescence detection revealed that the expression of Ku80 and LSD1 was mainly located in the nucleus.IP combined with western blot detection confirmed the interaction between Ku80 and LSD1.2.Among the 4 colon cancer cells included,HCT116 cells had the highest Ku80 protein and m RNA expression levels;on the contrary,FOXF2 protein and m RNA expression levels were the lowest;correlation analysis found that Ku80 was negatively correlatedwith FOXF2 protein and m RNA levels;Among the 4 colon cancer cells included,HCT116 cells had the strongest invasion and migration ability.The linear correlation analysis showed that the protein level and m RNA level of Ku80 were positively correlated with the invasion and migration ability of colon cancer cells,while the FOXF2 protein and m RNA levels were correlated with the colon.The invasion and migration ability of cancer cells is negatively correlated.3.Ku80 and FOXF2 are expressed in varying degrees in colon cancer tissues and normal control colon tissues.It is found that Ku80 is stained in the nucleus and FOXF2 protein is stained in the cytoplasm;Ku80 is highly expressed in 76.93% of the 108 colon cancer samples.In the control group,58.33% of normal colon tissues were highly expressed.The difference between Ku80 in different TNM stages(Ⅲ/Ⅳ and Ⅰ/Ⅱ)and whether there was lymph node metastasis was statistically significant.In FOXF2,in 108 colon cancer samples,72.22% of the cases showed low expression status,while in the control group,66.67% of normal colon tissue showed high expression status.Further analysis revealed that there was a difference between different TNM stages(I/Ⅱ andⅢ/Ⅳ)and whether there was lymph node metastasis.Bioinformatics analysis of LSD1,Ku80,FOXF2 expression,pan-cancer analysis found that LSD1,Ku80 is highly expressed in a variety of tumors,while FOXF2 is low expression.The transcription of LSD1 and Ku80 in the primary tumor was significantly higher than that of the normal control group,while FOXF2 was significantly lower than that of the normal control group.Ku80 was positively correlated with LSD1 expression in the colon cancer dataset,while both were negatively correlated with FOXF2 expression.4.Using RNAi technology successfully knocked down the expression of Ku80 in colon cancer cell HCT116.Knockdown of Ku80 and inhibition of LSD1 activity can reduce the invasion,migration,proliferation and clonal formation of colon cancer cells HCT116,and induce the apoptosis of colon cancer cells;the ability of subcutaneous allogeneic tumor formation in nude mice is significantly inhibited,and combined knockdown with LSD1 inhibitors Low Ku80 expression has more significant inhibitory effect on the above phenotypes;Western blot detection found that interference with Ku80 expression,inhibition of LSD1 activity or combinedapplication can increase FOXF2 protein expression;CHIP-PCR experiments found that LSD1 interacts with Ku80 and binds to FOXF2 The promoter region of the gene is 687-887 bp,and up-regulates the methylation level of H3K4me2 in the promoter region to promote the transcriptional activation of FOXF2 gene,and down-regulates the expression of downstream target genes LGR5,AXIN2,CD44,MYC via Wnt/β-catenin signaling pathway.Malignant phenotype of colon cancer cells.Conclusion: 1.Ku80 and LSD1 interact in colon cancer cells,the main expression sites of the two are located in the nucleus,and the expression levels are positively correlated.2.The expression level of Ku80 is positively correlated with the invasion and migration ability of colon cancer cells,while the expression level of FOXF2 is negatively correlated with the invasion and migration ability of colon cancer cells.3.Ku80 showed high expression in colon cancer tissues with late TNM stage(stage Ⅲ,Ⅳ)and lymph node metastasis,while FOXF2 showed a significantly low expression state,which was related to TNM stage of colon cancer and the presence or absence of lymph node metastasis.4.Ku80 interacts with LSD1 and binds to the promoter region of FOXF2 gene at 687-887bp;knockdown of Ku80 combined with inhibition of LSD1 can better inhibit colon cancer cell invasion,migration,proliferation and allogeneic tumorigenesis,and induce it Transmigration.The mechanism is to significantly up-regulate the methylation level of FOXF2 promoter region H3K4me2 to promote the transcriptional activation of FOXF2 gene,and down-regulate the expression of downstream target genes LGR5,AXIN2,CD44,MYC via Wnt/β-catenin signaling pathway to inhibit the colon cancer cells.Malignant phenotype. |
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