Pgl3 luc reporter vector

In this study, we constructed a plasmid that contained the amplified 3′UTR sequence of LIMS1 from genomic DNA, using the specified primers listed in Table S1. Additionally, we also included LIMS1 mutant variants in the plasmid construct. The amplified 3′UTR sequence of LIMS1, along with the mutant variants, was successfully inserted into the pGL3‐LUC reporter vector obtained from Promega, located in Madison, USA. This vector is a widely used tool for luciferase reporter assays, allowing the measurement of gene expression regulatory elements. In order to investigate the impact of RBMS3 binding sites on LIMS1, HCT116 cells were seeded into 24‐well plates. Subsequently, the cells were co‐transfected with the pGL3‐LUC reporter vector containing the 3′UTR sequences of LIMS1 (with wild‐type and mutant variants) and the PRL‐TK Renilla luciferase reporter vector, both obtained from Promega. The co‐transfection was achieved using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).To quantify the activities of Firefly and Renilla luciferase reporters, a Promega GloMax®20/20 Luminometer E5311 was employed. To ensure statistical accuracy and reliability, the quantification process was repeated three times.

The 3′-UTR of the target gene, which contains the binding sites of miR-211, was amplified by PCR from genomic DNA. The primers are presented in Supplementary Table 2. The sequence was inserted into the pGL3-LUC reporter vector (Promega). Briefly, 293TN cells were seeded into 96-well plates at a density of 2 × 10⁴ cells per well. Twenty-four hours later, the cells were cotransfected with pGL3-LUC reporter vectors, pRL-TK Renilla luciferase reporter vectors (Promega) and miR-211 mimics or NC. Transfection was performed using Lipofectamine 2000 (Invitrogen). Forty-eight hours posttransfection, Firefly and Renilla luciferase were detected using Promega GloMax® 20/20 Luminometer E5311. All assays were replicated three times.