The Study Of The Expression Level Of Has-mir-218 In Colon Cancer And Its Target Genes

Objectives:To observe the expression of miR-218 in colon cancer and define the role of miR-218 in clinicopathological characteristics, and study the target genes.Methods: Hybridization in situ and qRT-PCR were used to detect the miR-218 expression in colon cancer tissue. The bioinformatics software and database were applied to predict and analyze target genes of miR-218. Sp1 3'UTR wild type ( includs miR-218 binding site) and mutation type( includs no binding site)were inserted the plasmid pMIR-REPORT. miR-218 mimics and pMIR-REPORT/Sp1 UTR plasmid were cotransfected into HEK293T cells , and then luciferase activity was assayed. miR-218 mimcs or control was transfected into colon cancer cell SW620. The level of PTEN mRNA was determined by RT-PCR, and the expression level of PTEN protein by Western blot.Results: The positive expression rate of the miR-218 was 15.52% in colon carcinoma lower than the control group (89.66%) by by hybridization in situ. It suggested that the miR-218 expression was associated with advanced clinical stage and with lymph node metastasis , stageâ…¢/â…£higher thanâ… /â…¡tumor, tumors with lymph node metastasis. Similarly, the miR-218 expression level by qRT-PCR was lower in colon cance than the control group (p<0.01). There were 699 target genes predicted by the online soft targetscan and 551 target genes by pictar. Sp1was sole common genes by both targetscan and pictar. Luciferase activity assay suggested mir-218 mimics did not affected the luciferase activiy of vector group and UTR mutation type group, however significaly affected luciferase activiy of the UTR wild type group(reduced by 55%, p<0.01). The data suggested mir-218 could bind to sp1 3'UTR. the level of Sp1 mRNA in sw620 cells transfected with mir-218 mimcs showed no significant difference, while expression level of Sp1 protein decreased significantly, which suggested miR-218 could regulate the expression of Sp1 gene mainly at translation level.Conclusion1. miR-218 expression level was reduced in colon cancer and associated with advanced clinical stage and with lymph node metastasis2. miR-218 could regulate the expression of Sp1 gene mainly at translation level.3. miR-218 could regulate the expression of Sp1 gene mainly at translation level, and define the role of miR-218 in clinicopathological characteristics, and study the target genes.