Study On The Mechanism Of RAD6B Regulating Snail1 Transcription And Colon Cancer Metastasis Through Histone Modification

Objective: Colorectal cancer(CRC)is the third most common malignant tumor in the world and the second leading cause of cancer-related death.There were more than1.8 million new cases and approximately 881 000 deaths in 2018 worldwide.Most patients with colorectal cancer diagnosed at an early stage can be cured surgically.Unfortunately,colorectal cancer is found mostly in middle and advanced stages,at which point,cancer cells metastasize to lymph nodes or distant organs,resulting in a poor prognosis.Metastasis is one of the most important causes of treatment failure.RAD6 B maintains genomic stability through DNA damage response,while RAD6 B deficiency results in failure of DNA damage response,and even lead to tumours.However,RAD6 B is aberrantly expressed in a variety of tumors including colon cancer,and its role in proliferation as well as metastasis of tumors remains to be elucidated.Through this project,we intend to elucidate the specific mechanisms of RAD6 B regulating colon cancer metastasis and provide new targets and theoretical basis for colon cancer treatment,so as to promote the development of precision medicine in colon cancer.Methods: To investigate changes in cellular m RNA expression levels following RAD6 A or / and RAD6 B knockdown,we first knocked out the RAD6 A or / and RAD6 B genes utilizing CRISPR-Cas9 technology in 293 T cells.After screening stable strains,differentially expressed genes were detected with Gene Chip.Significantly altered pathways resulting from RAD6 A or / and RAD6 B knockdown were selected by integrative pathway analysis.Transcript analysis of colon cancer cases as well as normal control samples from The Cancer Genome Atlas(TCGA)database was performed to clarify the expression levels of RAD6 B in cases with different ages,gender,body weight,and TNM stages.The expression level of RAD6 B was further detected by immunohistochemical staining on tissue microarrays containing 45 cases of colon cancer samples as well as corresponding adjacent normal tissues.Colon cancer cell lines with downregulated or upregulated RAD6 B expression levels were generated using si RNA lentiviral vectors targeting the RAD6 B gene as well as overexpression plasmids,respectively.The effect of RAD6 B on colon cancer cell proliferation was examined by CCK-8 assay as well as flow cytometry,while the effect of RAD6 B on migration as well as invasion of colon cancer cell lines was investigated by wound healing assay and Transwell assay.We further established different animal models to fully verify the promoting effect of RAD6 B on colon cancer growth and metastasis.To investigate the detailed mechanism,we investigated the role of RAD6 B on EMT of colon cancer cells by cell morphology observation,immunofluorescence,and Wes.In addition,we predicted the binding sites of NF?B on the RAD6 B promoter using JASPAR,and the RAD6 B promoter was cloned into a luciferase reporter plasmid and co transfected into HCT116 cells with an empty vector(mock)or NF?B-containing plasmid to verify the regulation of RAD6 B expression by the transcription factor NF?B and further verified by Ch IP assay.Through the Cistrome database and Wes as well as Ch IP experiments,the contribution of RAD6 B mediated histone post-translational modifications to Snail1 transcription and the cooperation between RAD6 B and NF?B in the regulation of Snail1 expression were investigated.Results: The transcript analysis of 293 T cells with stable knockdown of RAD6 A or / and RAD6 B showed that the differential genes caused by RAD6 B knockdown were mainly associated with cell cycle,cell survival,colorectal cancer and gene expression.Importantly,RAD6 B deficiency negatively correlates with colon cancer.Transcriptomic analysis of colon cancer cases from the TCGA database revealed that colon cancer cases at TNM stage IV expressed increased levels of RAD6 B.Immunohistochemical staining also demonstrated increased expression of RAD6 B in advanced colon cancer.For the study of cell proliferation,RAD6 B could promote cell cycle progression and RAD6 B knockdown obviously inhibited colon cancer cell proliferation.In wound healing assay as well as Transwell assays,RAD6 B was essential for metastasis of colon cancer cells,and knockdown of RAD6 B inhibited colon cancer migration and invasion,as indicated by slowed wound closure as well as reduced number of migrated and invaded cells,whereas overexpression of RAD6 B significantly promoted colon cancer cell line migration as well as invasion.The promotion of colon cancer growth and metastasis by RAD6 B gene was further verified by animal model,which showed that knockdown of RAD6 B obviously inhibited colon cancer growth and metastasis.Mechanistic studies revealed that RAD6 B overexpression induced the morphology of SW480 and HCT116 cells to change from cobblestone like to fibroblast like.QPCR assays showed that when RAD6 B was downregulated,the transcriptional level of E-cadherin was significantly increased,while the level of vimentin was slightly inhibited.Further immunofluorescence and Wes assays also indicated the involvement of RAD6 B in epithelial mesenchymal transition of colon cancer cells,and elevated expression of RAD6 B could cause a decrease in the expression of epidermal marker E-cadherin.Dual luciferase assay as well as Ch IP experiments showed that NF?B can bind to the promoter of RAD6 B and regulate its transcription in colon cancer.In Wes experiments,knockdown of RAD6 B caused a decrease in H2BK120 ub as well as H3K4me3 and inhibited the transcriptional activation of Snail1 by NF?B.In the Ch IP-q PCR method,silencing the expression of RAD6 B not only caused the decrease of H3K4me3 level in Snail1 promoter region,but more importantly,the binding of NF?B on Snail1 promoter was also significantly restricted.Conclusion: The expression of RAD6 B was significantly up-regulated in advanced colon cancer,and the increased expression of RAD6 B could promote the proliferation,migration and invasion of colon cancer cells.The knockdown of RAD6 B could inhibit the growth and metastasis of colon cancer.RAD6 B promotes colon cancer metastasis by participating in EMT.And RAD6 B is a direct transcriptional target of NF?B in colon cancer,increased RAD6 B level facilitates the activation of Snail1 transcription by NF?B through post-translational modification of histones,further inhibits E-cadherin and enhances EMT and tumor metastasis,providing novel insights into the involvement of RAD6 B in tumor progression,as well as the mechanism research and treatment of colon cancer.