Identifying miR-203 Target Gene PRC1

microRNA.org prediction website was used for the analysis of the target gene of miR-203. The bioinformatics was applied to predict the target binding site of miR-203 and PRC1 gene. Synthesized primers were obtained from Shanghai GeneChem (Shanghai, China). The WT vector was constructed as follows: amplified 3′ UTR sequence of PRC1 was cloned and inserted in pmiR-RB-REPORT vector digested by restrictive endonuclease XhoI/NotI. The MUT vector was constructed with WT vector used as a template. MUT primers were designed and two mutation subsections were obtained by PCR, and lastly the products of PCR were purified and cleaved by restrictive endonucleases XhoI/NotI, which were harvested and inserted into pmiR-RB-REPORT vector. The generated PRC1-MT or PRC1-MUT plasmid was co-transfected with miR-203 mimic into SCL-1 cells (Guangzhou Jennio Biotech, Guangdong, China), followed by luciferase activity detection at 560 nm with the use of a Firefly Luciferase Reporter Gene Assay Kit (RG005, Beyotime Biotechnology, Shanghai, China) and a microplate reader (MK3, Thermo Fisher Scientific, Waltham, MA, USA). The miScript target protectors were modified RNA oligonucleotides complementary to specific target sites that could not bind to other sequences.³⁹ (link)