| Objective:Colorectal cancer(CRC)is the most common human cancers,and its incidence is increasing year by year.Most colorectal cancer patients are detected at an advanced stage and their long-term survival rates are usually low.Research the structure and function of the key genome of colorectal cancer will help us to further inquire the molecular mechanisms of colorectal cancer and may provide new biomarkers for clinical diagnosis,drug detection and prognostic analysis.Transcriptome sequencing is a high-throughput technique designed to provide a comprehensive view of the entire transcriptome,helping to identify novel genes,gene-specific expression,and differential gene expression among different groups.In this study,the transcriptional analysis of colorectal cancer cell lines with stable overexpression and knockdown of SETDB1 was conducted to screen out the most significant differential gene,C5AR1,and preliminarily explore its function and molecular mechanism in colorectal cancer.Methods:1.Use sequencing technology,the differentially expressed gene C5AR1 was obtained after SETDB1 overexpression and knockdown;2.Use q RT-PCR and Western Blot test to inquire the expression of C5 a R1 in 5 different human colorectal cancer cell lines.3.HCT116 and SW620 cells with knockdown of C5 a R1 and RKO cells with overexpression of C5 a R1 were constructed by small interfering RNA and plasmid transfection,respectively.4.Use CCK8 assay,Plate clonality assay,Wound Healing assay and Transwell assay to detecte the effects of the proliferation,migration and invasion of colorectal cancer cells in vitro by the knockdown or overexpression of C5 a R1.5.Western Blot was used to detect the effect of knockdown and overexpression of C5 a R1 on epithelial mesenchymal transformation of colorectal cancer cell lines.8.Western Blot was used to detect the effects of C5 a R1 knockdown and overexpression on the expression of key proteins in EMT-related Wnt/β-catenin pathway in colorectal cancer cell lines.Results:1.Through sequencing data analysis,a total of 298 genes were found to be differentially expressed between SETDB1 overexpression group and control group,among which 81 genes were up-regulated after SETDB1 overexpression.There were 535 differentially expressed genes in the SETDB1 knockdown group and the control group,among which 237 genes were also down-regulated after the SETDB1 knockdown,and the gene with the most significant difference was C5 a R1.2.Western Blot and RT-q PCR results showed that in HCT116,SW620,SW480,RKO and Lo Vo cells,the expression of C5 a R1protein in HCT116 and SW620 was the highest,and that in RKO was the lowest.3.Effects of C5 a R1 on biological behavior of colorectal cancer cells in vitro:Knockdown of C5 a R1 inhibits proliferation,migration and invasion of HCT116 and SW620 cells in vitro;In vitro overexpression of C5 a R1 can enhance the proliferation,migration and invasion of RKO cells.4.Related effects of C5 a R1in colorectal cancer cells on EMT process: In HCT116 and SW620,the expression of E-cadherin was observably up-regulated and on the other hand,the expression of Vimentin was obviously down-regulated after C5 a R1 knockdown;After overexpression C5 a R1,E-cadherin expression was down-regulated and inversely,the expression of Vimentin was up-regulated in RKO cells.5.Related effects of C5 a R1 on key proteins of Wnt/β-catenin pathway in colorectal cancer cells: C5 a R1 overexpression,β-catenin expression up-regulated,β-catenin expression down-regulated after C5 a R1 knockdown.Conclusions:1.After overexpression and knockdown of SETDB1,the pro-cancer and anti-cancer effects of SETDB1 on colorectal cancer cells caused the most significant changes in C5AR1 gene.2.C5 a R1 enhances the proliferation,migration and invasion of colorectal cancer cells in vitro.3.C5 a R1 may accelerate the EMT process of colorectal cancer cells by regulating the Wnt/β-catenin pathway. |
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