| Background and purpose:Colorectal cancer is one of the leading causes of cancer-related deaths worldwide.The disease is asymptomatic in its early stage and metastatic in its late stage,posing a serious threat to human health.However,the effect of various treatments for colorectal cancer is limited.Therefore,further study of the factors affecting the malignant behavior of colorectal cancer is helpful to the search for new therapeutic targets for colorectal cancer.ADP ribosylation is an important post-translational modification.At present,the studies on ADP ribosylation are mainly realized by investigating ADP-ribosyltransferases.There are few studies focus on the effect of specific sites of ADP ribosylation modification on tumors.In our previous studies,it was shown that arginine-117 of histone H3(H3R117)in LoVo cells with low differentiation degree was modified by mono-ADP-ribosylation,which was closely related to the invasion of colorectal cancer.Epithelial-mesenchymal transition(EMT)plays an important role in tumor invasion.On the basis of previous studies,we further investigated the effect of histone H3 arginine mono-ADP-ribosylation on epithelial-mesenchymal transition in LoVo cells.Besides,transcriptome sequencing was used to analyze the expression of differentially expressed genes between the untransfected LoVo cells and H3R117 A LoVo cells.Functional analysis and pathway enrichment of differentially expressed genes were performed by bioinformatics,so as to explore the possible molecular mechanism of the effect of histone H3 arginine mono-ADP-ribosylation on epithelial-mesenchymal transition in colorectal cancer cells.Our research could provide theoretical and experimental basis for the discovery of new therapeutic target of colorectal cancer.Methods:The study is divided into two parts 1.Effects of histone H3 arginine mono-ADP-ribosylation on EMT in LoVo cells(1)Group of Experiments: LoVo cells with a point mutation of arginine at residue 117 of H3 to alanine or lysine have been successfully constructed(H3R117A,H3R117K).Control group: Untransfected LoVo cells and LV-control LoVo cells,Experimental group: H3R117 A LoVo cells and H3R117 K LoVo cells.(2)Transwell assay was performed to examine the invasion ability of all groups of LoVo cells.(3)Western Blot was performed to evaluate the expression levels of E-cadherin,N-cadherin,Vimentin and Snail.(4)According to the principle that Phalloidin specifically binds to F-actin.Immunofluorescence staining of LoVo cells was performed with Phalloidin.The changes of cytoskeletal protein F-actin were observed by laser confocal microscopy.2.Molecular mechanism of the effect of histone H3 arginine mono-ADP-ribosylation on EMT(1)The differentially expressed genes between untransfected LoVo cells and H3R117 A LoVo cells were screened by transcriptome sequencing,and the functional analysis and pathway enrichment of the differentially expressed genes were analyzed by bioinformatics analysis.(2)In the KEGG pathway enrichment analysis,the Glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate pathway which is closely related to EMT was selected as the research object,and the sequencing results of key genes of the pathway were verified by RT-qPCR.(3)Western Blot was used to detect the expression level of β-catenin which is the key factor of the Wnt pathway.Results: 1.Effects of histone H3 arginine mono-ADP-ribosylation on EMT in LoVo cells(1)Transwell assay was performed and the results demonstrated that compared with control groups,the invasion capacity of H3R117 A LoVo cells and H3R117 K LoVo cells were reduced(P > 0.05).There was no significant difference between the Untransfected LoVo cells and the LV-control LoVo cells(P > 0.05),and no significant difference between the H3R117 A LoVo cells and the H3R117 K LoVo cells(P > 0.05).(2)The results of western blot demonstrated that compared with control groups,the expression of N-cadherin,Vimentin,Snail were decreased significantly in the H3R117 A LoVo cells and H3R117 K LoVo cells(P<0.05),the expression of E-cadherin was elevated(P<0.05).There was no significant difference between the Untransfected LoVo cells and the LV-control LoVo cells(P > 0.05),and no significant difference between the H3R117 A LoVo cells and the H3R117 K LoVo cells(P > 0.05).(3)Laser confocal analysis showed that compared with control groups,the fluorescence intensity of F-actin was decreased significantly in the H3R117 A LoVo cells and H3R117 K LoVo cells(P<0.05).There was no significant difference between the Untransfected LoVo cells and the LV-control LoVo cells(P > 0.05),and no significant difference between the H3R117 A LoVo cells and the H3R117 K LoVo cells(P > 0.05).2.Molecular mechanism of the effect of histone H3 arginine mono-ADP-ribosylation on EMT(1)Transcriptome sequencing were performed to identified 58,174 genes,among these,2,324 genes were significantly different(q-value <0.05;|fold change >2|).GO functional analysis showed that these differentially expressed genes were significantly enriched in metabolic process,such as,organelle,binding and catalytic activity.The KOG functional analysis showed that the differentially expressed genes were most significantly enriched in chromatin structure and dynamics.The KEGG pathway enrichment analysis results showed that the most significant enrichment pathway was Glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate.(2)In the pathway of Glycosaminoglycan biosynthesis-chondroitin sulfate / dermatan sulfate.A total of 7 differentially expressed genes were enriched into this pathway.RT-qPCR was used to confirm the changes in the top three genes based on the log2 FoldChange values.These were B3GALT6,CHST12,and CHST15.Compared with untransfected loVo cells,the expression of B3GALT6 and CHST12 in H3R117 A loVo cells were increased,the expression of CHST15 were decreased.The RT-qPCR results showed that the trend of gene expression was consistent with the sequencing results.(3)The results of Western Blot showed that compared with untransfected LoVo cells,the expression of β-catenin in the H3R117 A LoVo cells was significantly reduced(P < 0.05).Conclusion: Histone H3 arginine mono-ADP-ribosylation can affect the epithelial-mesenchymal transition in LoVo cells,and then regulate the invasion ability of colorectal cancer cells.The mechanism may be related to the effect of histone H3 arginine mono-ADP-ribosylation on the expression of the enzymes B3GALT6,CHST12 and CHST15 associated with glycosaminoglycan synthesis,as well as the expression of β-catenin,which is the key factor of Wnt pathway.The detailed mechanism needs further study. |
PDF Full Text Request