Circ＿0008726 Regulates Malignant Progression Of ESCC Cells Through MiR-206/HOXA13 Pathway

BackgroundEsophageal cancer is a common malignant tumor of digestive tract in the world.The incidence of esophageal cancer has obvious regional differences.China is a region with high incidence,accounting for more than 50%of the new cases in the world,ranking sixth in the incidence of malignant tumors in China and fourth in the mortality rate of malignant tumors in China.Pathological types of Esophageal cancer include squamous cell carcinoma,adenocarcinoma and neuroendocrine carcinoma.More than 90%of Esophageal squamous cell carcinoma in China is squamous cell carcinoma.The main treatment methods for esophageal squamous cell carcinoma（ESCC）include surgery,radiotherapy and chemotherapy.However,the overall treatment effect is not ideal,and the 5-year survival rate of advanced esophageal cancer is still low.circular RNA（circ RNA）is a kind of endogenous non-coding RNA,which plays a very important role in the process of proliferation,apoptosis,invasion and metastasis of tumor cells,and may become a new target for tumor diagnosis and treatment.Data from the public GEO database show that circ₀008726 is one of the up-regulated circRNAs in ESCC.The mechanism of action of circ₀008726 in ESCC is unclear.The aim of this study was to elucidate the role and molecular mechanism of circ₀008726 in the process of proliferation,apoptosis,invasion and metastasis of ESCC.To explore the molecular mechanism of miR-206/HOXA13 axis in regulating ESCC cell function;The molecular mechanism related to the promotion of malignant biological behavior of ESCC by circ₀008726 through the miR-206/HOXA13 axis was revealed,which provided an experimental theoretical basis for further elucidating the pathogenesis of ESCC and screened out possible targets for the diagnosis and treatment of ESCC.Methods（1）Firstly,the expression level of circ₀008726 in ESCC tissues and normal tissues was analyzed by GSE131969 database.The expression of circ₀008726 in ESCC tissues and ESCC cell lines was detected by RT-qPCR.The correlation of circ₀008726 expression level with tumor size,tumor stage and lymph node metastasis was analyzed by single factor analysis.Kaplan-Meier survival curve was used to analyze the effect of circ₀008726 expression on the overall survival time of ESCC patients.（2）Down-regulated circ₀008726 expression in ESCC cell lines KYSE30 and KYSE150,and confirmed the down-regulated effect by RT-qPCR;After circ₀008726 was down-regulated,cell proliferation activity was detected by CCK-8 method and EdU analysis,cell apoptosis was detected by flow cytometry,cell invasion ability was detected by Transwell assay,and cell angiogenesis ability was detected by angiogenesis assay.Protein expression levels of Cleaved caspase 3,snail 1 and VEGFA were detected by Western Blot.（3）Bioinformatics was used to screen miRNAs bound to circ₀008726,and RT-qPCR was used to detect the expression levels of candidate mirnas in ESCC cells after circ₀008726 knockdown.circ₀008726 was down-regulated by cell transfection with miR-206 mimics,and luciferase reporting assay was performed to detect the luciferase activity of cirC₀008726-WT and cirC₀008726-MUT.RNA pull down confirmed the binding relationship between circ₀00872 and miR-206.（4）The expression of miR-206 in ESCC tissues and ESCC cell lines was detected by RT-qPCR.Down-regulated the expression of miR-206 in ESCC cell lines KYSE30 and KYSE150,and confirmed the down-regulated effect by RT-qPCR.After down-regulating miR-206,cell proliferation activity was detected by CCK-8 method and EdU analysis,cell apoptosis was detected by flow cytometry,cell invasion ability was detected by Transwell assay,and cell angiogenesis ability was detected by angiogenesis assay.Protein expression levels of Cleaved caspase 3,snail 1 and VEGFA were detected by Western Blot.（5）Biological information was used to analyze the binding relationship between miR-206 and HOXA13;miR-206 mimics were transfected and luciferase reporting assay was performed to detect the luciferase activity of HOXA13-WT and HOXA13-Mut.The expression of HOXA13 in ESCC tissues was detected by RT-qPCR.Western Blot was used to detect HOXA13 protein expression levels in ESCC cells after overexpression of miR-206 and knockdown of miR-206.HOXA13 was up-regulated by transfection of pcDNA-HOXA13,and the overexpression of HOXA13 was verified by RT-qPCR.miR-206 was up-regulated while HOXA13 was overexpressed.Cell proliferation activity was detected by CCK-8 and EdU analysis,cell apoptosis was detected by flow cytometry,cell invasion ability was detected by Transwell assay,and cell angiogenesis ability was detected by angiogenesis assay.Protein expression levels of Cleaved caspase 3,snail 1 and VEGFA were detected by Western Blot.（6）Xenograft mouse model was established to study the effect of circ₀008726 knockout in vivo;The protein expression levels of Ki-67 and HOXA13 in mouse ESCC were detected by immunohistochemical staining.The expression of circ₀008726,miR-206 and HOXA13 in mouse ESCC tissues was detected by RT-qPCR.Protein expression levels of Cleaved caspase 3,snail 1 and VEGFA were detected by Western Blot.Results（1）GSE131969 database analysis showed that circ₀008726 was highly expressed in esophageal squamous cell carcinoma compared with normal tissues.The expression of circ₀008726 in esophageal squamous cell carcinoma was higher than that in adjacent normal tissues（P<0.0001）.Compared with normal esophageal epithelial cells,the expression of circ₀008726 in esophageal squamous cell lines KYSE30 and KYSE150 was significantly increased（P<0.0001）.The high expression of circ₀008726 was associated with large tumor size,late TNM stage and positive lymph node metastasis.Kaplan-Meier survival curve confirmed that the survival rate of ESCC patients in the low circ₀008726 group was higher than that in the high circ₀008726 group.（2）The elimination of circ₀008726 inhibited the proliferation,invasion and angiogenesis of ESCC cells,and promoted apoptosis（P<0.0001）.Knockdown of circ₀008726 increased the expression of Cleaved caspase 3 and decreased the expression of snail 1 and VEDFA in ESCC cells.（3）The expression level of miR-206 was significantly increased in ESCC cells with circ₀008726 knockdown（P<0.0001）.The results of double luciferase reporter gene experiment showed that overexpression of miR-206 significantly decreased luciferase activity in circ₀008726-WT group（P<0.0001）.（4）Compared with paracancer normal esophageal tissue,the expression level of miR-206 in ESCC was significantly decreased（P<0.0001）.Compared with normal esophageal epithelial cells,the expression of miR-206 in esophageal squamous cell lines KYSE30,KYSE150 and medium was significantly decreased（P<0.0001）.Down-regulation of miR-206 partially reversed the decrease in proliferation,invasion,and angiogenesis of ESCC cells by circ₀008726,and the increase in apoptosis rate of ESCC cells by knockout of circ₀008726.（5）HOXA13 is a downstream target gene of miR-206.When miR-206 was up-regulated,HOXA13 expression was decreased,and luciferase activity was significantly decreased in HOxa13-WT group（P<0.0001）.The expression of HOXA13 in esophageal squamous cell carcinoma was significantly higher than that in adjacent normal esophageal tissues（P<0.0001）.Overexpression of HOXA13 partially reversed the decrease of cell proliferation,invasion,angiogenesis and increase of apoptosis rate caused by up-regulation of miR-206.（6）Elimination of circ₀008726 can significantly reduce the volume and quality of ESCC xenografts.circ₀008726 significantly reduced KI67 and HOXA13 protein levels in ESCC tissue.Conclusion（1）circ₀008726 is highly expressed in ESCC tissues and ESCC cell lines,and down-regulated circ₀008726 can inhibit the proliferation,invasion and angiogenesis of ESCC cells,and promote apoptosis.Knockdown of circ₀008726 increased the expression of Cleaved caspase 3 and decreased the expression of snail 1 and VEDFA in ESCC cells.（2）circ₀008726 plays a carcinogenic role through the miR-206/HOXA13 axis,regulating cell proliferation,migration,invasion and apoptosis,and is an important regulatory factor in the occurrence and development of esophageal squamous cell carcinoma.