Dysregulation Of MiR-6868-5p/FOXM1 Circuit Contributes To Colon Cancer Angiogenesis

Background:Colon cancer is the third most common cancer and the third leading cause of cancer-related death in the world.The treatment of colon cancer includes surgery,radiotherapy,chemotherapy and targeted therapy,but the prognosis is still not optimistic.It is imperative to understand the molecular mechanisms of colon cancer progression and identify novel therapeutic targets.Transcription factor FOXM1(Forkhead box M1)belongs to the FOX super family.Our previous studies have shown that FOXM1 expression predicted poor prognosis of colon cancer patients,and FOXM1 promoted the metastasis and chemoresistance of colon cancer cells.However,little is known about the underlying mechanisms responsible for the elevated FOXM1 expression in colon cancer.MicroRNAs(miRNAs)are small non-coding RNAs,and negatively regulate gene expression by targeting the 3'-untranslated region(3'-UTR)of mRNAs.Over the past decades,studies have revealed that miRNA deregulation is implicated in tumor development and progression.Recent advancement in miRNA-based therapy has made it a promising way for cancer treatment.Through bioinformatic screening,we identified miR-6868-5p as a potential mechanism for increased FOXM1 expression and may affect tumor angiogenesis.Therefore,in this study,we aim to clarify the role and mechanism of miR-6868-5p/FOXM1 in colon cancer angiogenesis.Methods:1.Identify potential miRNAs targeting FOXM1 through multiple prediction tools and qRT-PCR validation.2.The luciferase report assay and western blot was used to confirm the inhibition of FOXM1 by miR-6868-5p.3.The culture medium(CM)of colon cancer cells transfected with miR-6868-5p mimics or inhibitor were used to incubate human umbilical vein endothelial cells(HUVEC).Then the proliferation,migration and tube formation ability of endothelial cells were detected.4.The CM of colon cancer cells transfected with FOXM1 overexpression plasmid or interference plasmid were used to incubate HUVEC,and the proliferation,migration andtube formation ability of endothelial cells were detected.5.For rescue assay,the CM of colon cancer cells co-transfected with miR-6868-5p mimics and FOXM1 overexpression plasmid were used to incubate HUVEC,and the proliferation,migration and tube formation ability of endothelial cells were detected.6.Bioinformatics and qRT-PCR were utilized to identify the angiogenesis factor regulated by FOXM1.ChIP assay was usedto verify the binding of FOXM1 on the promoter of IL-8.7.The ago-miR-6868-5p mimics were injected into the xenografted tumor in nude mice,and tumor size and tumor vascular density were measured to analyse the inhibitory role of miR-6868-5p on tumor angiogenesis in vivo.8.qRT-PCR and ChIP assay were used to analysis the inhibition of pri-miR-6868 transcription by FOXM1 through EZH2.9.The clinical significance of miR-6868-5p was determined using clinical specimens of colon cancer.Results:Here,we identified FOXM1 as the direct downstream target of miR-6868-5p.Overexpression of miR-6868-5p in colon cancer cells inhibited the angiogenic properties of endothelial cells,whereas silencing of miR-6868-5p had opposite effects.Rescue of FOXM1 reversed the effect of miR-6868-5p in tumor angiogenesis.Further mechanistic study showed that FOXM1 promoted the expression of IL-8,which was responsible for the miR-6868-5p/FOXMl axis-regulated angiogenesis.Moreover,in vivo delivery of miR-6868-5p blocked tumor angiogenesis in nude mice,resulting in tumor growth inhibition.Reciprocally,overexpression of FOXM1 led to a reduction of miR-6868-5p,attributing to EZH2-mediated H3K27me3 on miR-6868-5p promoter.Clinically,miR-6868-5p expression was significantly downregulated in colon cancer tissues.The levels of miR-6868-5p were inversely correlated with microvessel density in tumor specimens.Conclusion:This study identified a novel miRNA,miR-6868-5p,in the regulation of FOXM1 expression,and established a miR-6868-5p/FOXM1 circuit inthe regulation of colon cancer angiogenesis,highlighting the potential of miR-6868-5p as a target for colon cancer treatment.