Pmirglo expression vector

The full-length 3' untranslated region (3'UTR) amplification products of the Rho GTPase-activating protein 6 (ARHGAP6) gene containing the binding sites of miR-152-5p predicted by the miRDB database were transferred into a pmiRGLO expression vector (Tsingke Biotechnology) to form ARHGAP6-wild-type (WT). Independently, a site-specific mutation targeting the predicted binding site for miR-152-5p in the ARHGAP6 gene was established, and the resultant mutant sequence was also introduced into a pmiRGLO expression vector to form ARHGAP6-MUT. Then, the H9c2 cardiomyocytes were co-transfected with a reporter plasmid and a miR-152-5p mimic or mimic-NC using Lipofectamine 2000. The ARHGAP6-WT- and ARHGAP6-MUT-transfected cells were each used to establish a blank control, miR-152-5p mimic and mimic-NC group with three duplicate wells in each group. After 48 h of culture, the luciferase activity of the cells was detected using a fluorescence microplate reader (Spark 10M; Tecan Group, Ltd.) according to the instructions of the Dual Luciferase Reporter Gene Assay Kit (RG008, Beyotime). The relative luciferase activity was calculated using the following equation: Relative luciferase activity=firefly luciferase activity value/Renilla luciferase activity value.