The Biological Function And Mechanism Of Long Non-coding RNA BVES-AS1 In Colorectal Cancer

Objective: To investigate the biological function and mechanism of long non-coding RNA BVES-AS1 in colorectal cancer.Methods:(1)The differentially expressed long non-coding RNA(lnc RNA)in colorectal cancer tissues were screened by the TCGA database and cluster analysis was performed.The lnc RNA BVES-AS1 with large differential expression was selected for the study.The expression level of BVES-AS1 in colorectal cancer and adjacent tissues was detected by q RT-PCR and the relationship between BVES-AS1 expression and clinical and pathological characteristics of colorectal cancer patients was analyzed.(2)Colorectal cancer cells stably overexpressing BVES-AS1 were constructed by lentivirus,whereas colorectal cancer cells with down-regulated BVES-AS1 expression were constructed by si RNA.The effects of BVES-AS1 on the proliferation,migration,invasion,epithelial-mesenchymal transformation(EMT)and apoptosis of colorectal cancer cells were verified by CCK-8 assay,Ed U assay,wound healing assay,Transwell assay,Western blot assay and flow cytometry apoptosis detection assay.(3)A nude mouse xenograft tumor model was constructed by stably overexpressing BVES-AS1 and the control vector colorectal cancer cells.The effects of BVES-AS1 on the growing volume,weight and Ki-67 expression of xenograft tumors were observed.(4)The localization of BVES-AS1 in colorectal cancer cells was verified by subcellular separation and fluorescence in situ hybridization.Bioinformatics analysis predicted the miRNA with binding sites with BVES-AS1.The physical binding of BVES-AS1 with predicted miR-1269 a and miR-1269 b was confirmed by the dual luciferase reporter gene test,RNA protein immunoprecipitation test and miRNA pull-down test.(5)The effect of co-transfection of BVES-AS1 plasmid with miR-1269 a mimics and miR-1269 b mimics on the proliferation,migration,invasion,EMT and apoptosis of colorectal cancer cells was verified through CCK-8 assay,Ed U assay,wound healing assay,Transwell assay,Western blot assay and flow cytometry apoptosis detection assay,(6)The common target gene of miR-1269 a and miR-1269 b was predicted by RNA sequencing and bioinformatics methods.q RT-PCR and Western blot experiments verified the relationship between BVES-AS1-miR-1269a/miR-1269b-SVEP1.The dual luciferase reporter gene assay verified whether miR-1269 a and miR-1269 b combined with SVEP1.(7)The expression of SVEP1 in colorectal cancer cells was knocked down by si RNA.The effects of SVEP1 on proliferation,migration,invasion,EMT and apoptosis in colorectal cancer cells were examined by CCK-8 assay,Ed U assay,wound healing assay,Transwell assay,Western blot assay and flow cytometry apoptosis detection assay.Western blot assay was performed to verify the effects of BVES-AS1,miR-1269 a.miR-1269 b and SVEP1 on PI3K/AKT signaling pathway.(8)The transcription factor FOXO1,which can bind to the BVES-AS1 promoter,was predicted by bioinformatic analysis.q RT-PCR detected the changes in BVES-AS1 m RNA expression after the up-or-down-regulation of FOXO1.Promoter dual luciferase reporter gene assay and chromatin immunoprecipitation assay were performed to verify the presence of binding of transcription factor FOXO1 to the BVES-AS1 promoter.(9)Colorectal cancer cells were treated with PI3K/AKT pathway inhibitor LY294002.The changes in FOXO1 protein expression and BVES-AS1 m RNA expression were detected by q RT-PCR and Western blot assays.Results:(1)The lnc RNA BVES-AS1,which is down-regulated in colorectal cancer and has not been reported,was screened by the TCGA database.q RT-PCR confirmed that BVES-AS1 expression was significantly reduced in colorectal cancer tissues compared with normal colorectal tissues and found that the low expression of BVES-AS1 was associated with tumor infiltration depth and lymph node metastasis.(2)Overexpression of BVES-AS1 inhibited proliferation,migration,invasion and EMT and promoted apoptosis of HT-29 and SW480 cells compared with controls.Downregulation of BVES-AS1 promoted proliferation,migration,invasion and EMT and inhibited apoptosis of HT-29 and SW480 cells.(3)In a nude mouse xenograft tumor model,overexpression of BVES-AS1 reduced the growth volume and mass of subcutaneous xenograft tumors and inhibited the expression level of Ki-67 compared to the control group.(4)BVES-AS1 was distributed in both the cytoplasm and nucleus of HT-29 and SW480 cells,but it was mainly distributed in the cytoplasm.Bioinformatics analysis revealed that miR-1269 a and miR-1269 b had binding sites with BVES-AS1,and miR-1269 a and miR-1269 b had the same seed sequence.Dual luciferase reporter gene assay,miRNA pull-down assay,and RNA protein immunoprecipitation assay confirmed the physical binding between BVES-AS1 and miR-1269 a and miR-1269 b.(5)The expression of miR-1269 a and miR-1269 b was significantly upregulated in colorectal cancer tissues compared with para-cancerous tissues.Overexpression of miR-1269 a and miR-1269 b promoted proliferation,migration,invasion and EMT of HT-29 and SW480 cells.The functional recuse assays showed that BVES-AS1 could partially reverse the proliferation,migration,invasion and EMT of HT-29 and SW480 cells caused by miR-1269 a and miR-1269 b.(6)RNA sequencing and bioinformatics analysis showed that SVEP1 can bind to miR-1269 a and miR-1269 b.SVEP1 expression was downregulated in colorectal cancer tissues compared with para-cancerous tissues.Overexpression of BVES-AS1 upregulated SVEP1 m RNA and protein expression levels.Overexpression of miR-1269 a and miR-1269 b inhibited SVEP1 expression.Dual luciferase reporter gene assay confirmed SVEP1 binding to miR-1269 a and miR-1269 b.(7)The downregulation of SVEP1 promoted proliferation,migration,invasion and EMT of HT-29 and SW480 cells compared to the control group;Western blot assays showed that overexpression of BVES-AS1 inhibited phosphorylated PI3K(p-PI3K)and phosphorylated AKT(p-AKT)expression in colorectal cancer cells;overexpression of miR-1269 a and miR-1269 b or knockdown of SVEP1 promoted p-PI3 K and p-AKT expression of HT-29 and SW480 cells.(8)Bioinformatics analysis showed that there was a binding site between FOXO1 and the BVES-AS1 promoter.The expression of FOXO1 in colorectal cancer is downregulated and positively correlated with BVES-AS1.Knocking down or overexpressing FOXO1 can reduce or increase the expression level of BVES-AS1 m RNA.The promoter dual luciferase reporter gene assay and chromatin immunoprecipitation assay confirmed the binding of FOXO1 to the BVES-AS1 promoter.(9)Overexpression of BVES-AS1 increased the m RNA and protein levels of FOXO1.Inhibition of the PI3K/AKT pathway promoted the expression of FOXO1 and BVES-AS1.Conclusion: This study demonstrated that BVES-AS1 could suppress the colorectal cancer progression via the SVEP1-PI3K/AKT-FOXO1 positive feedback loop,which may become a new target for the treatment of colorectal cancer.