Study On The Mechanism Of Hsa＿circ＿0004771 Regulating The Proliferation,Migration,Invasion And Angiogenesis Of Colorectal Cancer Cells

Objective:As a novel endogenous non-coding RNA,circular RNA(circRNA)is expressed differently in a variety of tumor tissues,suggesting that circRNA may be closely related to the pathogenesis of cancer.Hsa＿circ＿0004771(circ＿0004771)is low expressed in colorectal cancer(CRC),but the precise functions and mechanism in CRC have not been elucidated.In this study,we aim to comprehensively investigate the role of circ＿0004771 on the malignant biological function of CRC cells in vitro and in vivo.Then,bioinformatics methods were used to analyze and construct the circ＿0004771-hsa-miR-590-3p-SSPN/SLC8A1 regulatory network.This provides a theoretical foundation for the research of circ＿0004771 in CRC.Methods:We first collected tissue samples,including 38 normal colorectal tissues and CRC tissues,and detected the levels of circ＿0004771 expression by quantitative reverse transcription polymerase chain reaction(qPCR).The backsplice junction of circ＿0004771 was verified by Sanger sequencing.RNase R test was performed to assess the stability of this circRNA.Agarose gel electrophoresis experiment was used to certify that circ＿0004771 has a circular closed structure.Then,circ＿0004771overexpression lentivirus vectors were constructed and successfully transfected into DLD1 and SW480 cells.The effects of circ＿0004771 overexpression on the proliferation,migration,invasion and angiogenesis of CRC cell lines were verified by CCK8,colony formation assay,transwell assay,wounding assay and tube formation assay.The effect of circ＿0004771 on CRC tumor proliferation in vivo was confirmed by nude mice subcutaneous tumor formation experiments.Nuclear plasma separation experiments were used to determine the location of circ＿0004771 in CRC cells.Then,we construct the circRNA-miRNA-m RNA function regulation network by target miRNA prediction,expression profile analysis,target gene prediction and correlation analysis.Results:Circ＿0004771 was significantly downregulated in human CRC samples compared with paracancer tissues by qPCR.The back-splice junction of circ＿0004771 was confirmed by the Sanger sequencing experiment,which was coincident with the results in circ Base.RNase R test shows it has good stability.Agarose gel electrophoresis experiment certifies circ＿0004771 have a cyclic structure.Cell function experiments on DLD1 and SW480 overexpressing circ＿0004771revealed that it can inhibit the proliferation,migration,invasion and angiogenesis of DLD1 and SW480 cells.Nude mice subcutaneous tumor formation experiments showed that circ＿0004771 could inhibit tumor proliferation in vivo.Circ＿0004771was localized in the cytoplasm of CRC cells by nuclear plasma separation assay.In addition,bioinformatics analyses suggest that circ＿0004771 may play the role of ce RNA through miRNA sponges.Finally,through a series of bioinformatics analyses,a potential circ＿0004771-hsa-miR-590-3p-SSPN/SLC8A1 regulatory axis linked to CRC carcinogenesis and progression was established.Conclusion:The expression of circ＿0004771 is significantly down-regulated in the CRC tissues.Overexpression of circ＿0004771 can inhibit proliferation,migration,invasion and angiogenesis of CRC cells,and inhibit tumorigenesis in vivo.Circ＿0004771 may serve as a novel tumor-suppressor in CRC through the hsa-miR-590-3p-SSPN/SLC8A1 axis.