Dual-Luciferase Reporter Gene Assay

Based on the binding sites between LINC00941 and miR-205-5p, MYC and miR-205-5p, the binding and mutant sequences of LINC00941 and MYC were respectively inserted into pmirGLO vector (Promega) to construct LINC00941-wt (wild-type), LINC00941-mut (mutant-type), MYC-wt (wild-type), and MYC-mut (mutant-type). LINC00941-wt or LINC00941-mut was co-transfected with miR-205-5p mimics or NC mimics into colon cancer cells, and with miR-205-5p inhibitor or NC inhibitor into colon cells. Similarly, MYC-wt or MYC-mut was co-transfected with miR-205-5p mimics or NC mimics. 36 hours after the transfection, the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was applied to test the relative luciferase activity, which was standardized according to Renilla luciferase activity.