Dual luciferase reporter gene assay

The target sites of wild type (WT) of miR-34b and TUFT1 mRNA 3′-UTR region and the sequence after site directed mutagenesis from the WT named mutant type (MUT) were synthesized. Restriction endonuclease was used for detachment based on the PmiR-RB-REPORT™ plasmid (RiboBio Co., Ltd., Guangzhou, China). Then the target gene fragments WT and MUT were inserted into pmiR-RB-REPORT™ vector (RiboBio Co., Ltd., Guangzhou, China), respectively. The empty plasmid was simultaneously transferred as the control group while the correct luciferase reporter gene plasmids WT and MUT were utilized to subsequent transfection. The vectors of MUT and WT were co-transferred to 293T cells with mimic-NC or miR-34b mimic together with oe-NC or oe-HNF1A-AS1, respectively. After 48 h transfection, the cells were collected and lysed, and the culture fluid was obtained by centrifugation at 10,000 rpm, 4 °C for 3 min. Relative lights units (RLU) was detected by luciferase detection kit (RG005, Beyotime Biotechnology Co., Ltd, Shanghai, China). Relative fluorescence value was calculated as RLU value determined by renilla luciferase/the RLU value measured by firefly luciferase.