Regulation Of MiR-223-3p On FOXO3 In Colorectal Cancer

[Objective](1)The RT-PCR technique was used to compare and analyze the differential expression of miR-223-3p in colorectal cancer mucosal tissueand adjacent normal colorectalmucosal tissue;Western blot was used to compare and analyze the difference of FOX03 expression in mucosal tissues of colorectal cancer and adjacent normal colorectal mucosal tissues.(2)The target gene of miR-223-3p was predicted to be FOXO3 by online target gene software,and then the FOXO3 gene 3’UTR was cloned,and miR-223-3p was targeted to FOXO3 by site-directed mutagenesis and dual luciferase experiments.(3)In Caco2 cells,FOXO3 protein expression was increased by knocking down miR-223-3p;overexpression of miR-223-3p inhibited FOXO3 protein expression,demonstrating that miR-223-3p plays an importantrole in the development of colorectal cancer by regulating FOXO3,The important molecular biomarkers and therapeutic targets for the diagnosis and treatment of colorectal cancer are to explore the mechanism of miR-223-3p regulating the expression of FOXO3 at the post-transcriptional level,and provide a scientific basis for the study of the occurrence and development of colorectal cancer.[Methods](1)10 groups of colorectal cancermucosal tissues and corresponding adjacent normalcolorectal mucosal tissues were collected and compared by RT-PCR to analyze the difference of miR-223-3p expression in colorectal cancer mucosal tissues and adjacent normal colorectal mucosal tissues;Westem blot was used to compare and analyze the difference of FOXO3 expression in mucosal tissues of colorectal cancer and adjacent normalcolorectal mucosa.The experimental group included the colorectal cancer mucosa tissue group and the adjacent normal colorectal mucosa tissue group.All patients included in the study had signed the informed consent form,,There were no tumor history and surgical history,and there was no history of radiochemotherapy or other immunological treatment.(2)The target gene of miR-223-3p was predicted by online prediction target gene software,and the target gene was initially selected as FOXO3,then the correlation between miR-223-3p and FOXO3 was confirmed by cloning the 3’UTR of FOXO3 gene and by site-directed mutagenesis and dual luciferase assay.(3)In Caco2 cells,the expression level of FOXO3 was detected by knockdown of miR-223-3p and overexpression of miR-223-3p,and the mechanism of expression of FOXO3 by miR-223-3p at the post-transcriptional level was preliminarily investigated.[Result](1)The results of RT-PCR showed that the expression level of miR-223-3p was significantly increased in the mucosa of colorectal cancer compared with the corresponding adjacent normal colorectal mucosal tissues,and the difference was statistically significant(P<0.01).(2)The results of western blot showed that the expression level of FOXO3 was significantly lower in the mucosal tissues of colorectal ’cancer than in the corresponding adjacent normal colorectal mucosa tissues,and the difference was statistically significant(P<0.05).(3)The target gene of miR-223-3p was predicted to be FOXO3 by online target gene software,and then the result of cloning FOXO3 gene 3’UTR,site-directed mutagenesis and dual luciferase showed that miR-223-3p can significantly down-regulate wild type FOXO3 reports the luciferase activity of the gene plasmid,but does not down-regulate the luciferase activity of the mutant FOXO3 reporter plasmid.The above results further indicate that miR-223-3p targets the FOXO3 gene.(4)Overexpression of miR-223-3p in Caco2 cells significantly increased miR-223-3p levels,and miR-223-3p had a tendency to reduce FOXO3 protein levels compared to transfected miR-NC;knockdown miR-223-3p significantly reduced the level of miR-223-3p.Compared with transfected antagomiR-NC,antagomiR-223-3p had a tendency to increase FOXO3 protein levels,but none of them reached significance.[Conclusion](1)The expression of miR-223-3p was significantly up-regulated in colorectal cancer mucosaltissues,and the expression of FOXO3 was significantlydown-regulated in colorectal cancer mucosaltissues;(2)miR-223-3p can participate in the regulation of the development of colorectal cancer by targeting FOXO3 gene;(3)miR-223-3p as a molecular biomarker and a new therapeutic target for the diagnosis and treatment of colorectal cancer Higher value provides a scientific basis for exploring the development of colorectal cancer.