Study Of Candidate Target Genes Of FOXF2 Acting On Wnt/β-catenin Signaling Pathway In Colon Cancer Cells

Objective:To screen the candidate target genes of FOXF2 acting on Wnt/β-catenin signaling pathway in colon cancer cells and analyze their functions,and to provide a basis for further study of target genes related to FOXF2 regulation of Wnt/β-catenin signaling pathway activity.Methods:1.Three target sequences of FOXF2 interference were designed to construct lentivirus-mediated FOXF2 gene silencing sh RNA vectors,respectively.The obtained lentivirus-FOXF2-sh RNA-1,2 and 3 were transfected into human colon cancer HT29 cells,respectively,and the resistance was screened by purinomycin.RT-q PCR and Western blot were used to verify the effect of FOXF2 silencing by lentivirus in each group.HT29 cell line transfected with optimal interference lentivirus was selected as the experimental group,and HT29 cell line transfected with empty vector lentivirus was selected as the control group.2.RNA-Seq was performed on three samples from the control group and the experimental group,respectively.Quantitative analysis was performed on the known genes and new genes,and the m RNA with significantly up-regulated or down-regulated expression was screened.Hierarchical clustering,GO and KEGG enrichment analysis were performed on the differentially expressed genes obtained by RNA-Seq.Candidate target genes in the Wnt/β-catenin signaling pathway were screened and their possible biological processes were analyzed.3.RT-q PCR and Western blot were used for relative quantitative detection of candidate target genes to verify the reliability of transcriptome sequencing.4.Expression analysis of FOXF2 and related candidate target genes was performed using the colon cancer and normal colon tissue datasets from the GEPIA database,and correlation analysis was performed between the expression of FOXF2 and related candidate target genes.Results:1.Interfering lentiviral vectors were successfully constructed;compared with empty vector lentivirus,interfering lentivirus FOXF2-sh RNA-1,2 and 3 could significantly reduce the m RNA and protein levels of FOXF2 in HT29 cells,with FOXF2-sh RNA-3having the most significant silencing effect.2.RNA-Seq uncovered 2210 new genes,with 979 and 409 with GO and KEGG functional annotations,respectively;there were 389 differentially expressed genes,containing 221 up-regulated genes and 168 down-regulated genes;there was variability between control and experimental groups,and genes in the same class of samples could cluster to appear in the same cluster,and genes clustered in the same cluster had the same or similar biological properties.The differentially expressed genes were significantly enriched in the functional groups of immune effect process,extracellular region,adenylyl transferase activity,etc.The differentially expressed genes were mainly involved in Epstein-Barr virus infection,an influenza A flu and Wnt signaling pathway,etc.There were7 differentially expressed genes in the Wnt/β-catenin signaling pathway,among which BAMBI,LGR5,LGR6 and MSTRG.14182(new genes)were annotated,which were mainly involved in the positive regulation of Wnt signaling pathway,mitochondrial calcium ion homeostasis,cell proliferation and migration.As MSTRG.14182 is a new gene,relevant information was not available in domestic and foreign literature,so BAMBI,LGR5 and LGR6 were selected as candidate target genes of Wnt/β-catenin signaling pathway.3.Compared with the control group,m RNA and protein expression levels of BAMBI,LGR5 and LGR6 in the experimental group were significantly up-regulated,with statistically significant differences(P < 0.05).4.The analysis of GEPIA database showed that compared with normal colon tissue,expression of FOXF2 was significantly down-regulated in colon cancer tissue,while expression of BAMBI,LGR5 and LGR6 were significantly up-regulated(P < 0.05).In colon cancer,the expression of FOXF2 was negatively correlated with BAMBI and LGR5(R=-0.12,P=0.035;R=-0.12,P =0.029),and there was a negative correlation between FOXF2 and LGR6 expression,but it did not reach statistical significance(R =-0.071,P =0.21).Conclusion:1.There are many differentially expressed genes in HT29 cells silenced FOXF2 gene,and candidate target genes of Wnt/β-catenin signaling pathway may be involved in the positive regulation of Wnt signaling pathway,cell proliferation and migration.2.BAMBI,LGR5 and LGR6 genes are candidate target genes of FOXF2 action on Wnt/β-catenin signaling pathway in colon cancer celles;Bioassay predicted that FOXF2 was negatively correlated with BAMBI and LGR5 expression in colon cancer,and had a negatively correlated trend with LGR6 expression.