Lipofectamine 3000 reagent

The human CRC cell lines RKO, HCT116 and HT29, and normal colorectal cells (NCM460) were obtained from the cell resource center of Peking Union Medical College (Beijing, China). Cells were maintained at 37 °C in RPMI-1640 medium containing 10 % fetal bovine serum (FBS; GIBCO, Carlsbad, CA) in a humidified atmosphere containing 5 % CO₂.
Small interfering RNAs (siRNAs) targeting ANP32B (siANP32B#1, 5′- GCUUACCUACUUGGAUGGCUAdTdT-3’; and siANP32B#2, 5′- GAGGGCUUAACAGCUGAAUUUdTdT-3′) and HPF1 (siHPF1, 5′-GTGAAGAACTTGATCCTGAAA-3′) and a negative control (siCtrl, 5′- CAGUACUUUUGUGUAGUACAAA-3′) were designed and synthesized by Hippobio (Huzhou, China) for gene knockdown in RKO cells. For cell transfection, Invitrogen Biotechnology's Lipofectamine 3000 (Cat# L3000001, Thermo Fisher Scientific, Waltham, MA, USA) was used. The concentration of siRNAs used was 100 pmol for cell transfection in 6-well plates. Approximately, after transfected for 48–72 h, the cells were collected for further experiments. For gene overexpression in HCT116 cells, DNA fragments covering the ANP32B (NM_006401.2) and HPF1 (NM_017867.3) coding regions were amplified and cloned into pcDNA3.1 vector (Tianyihuiyuan), yielding pcDNA-ANP32B and pcDNA-HPF1, respectively. Blank vector was used as the negative control (Ctrl). Lipofectamine 3000 reagent (Promega) was used for cell transfection.

Wild-type (WT) RMRP and ATP13A3 3′-UTR fragments containing predicted miR-580-3p binding sites were amplified and inserted into pmirGLO vectors (Promega, USA) to establish the reporters RMRP-WT and ATP13A3-WT. GeneArt™ site-directed mutagenesis PLUS system (A14604; Thermo Fisher Scientific) mutated the putative binding site of miR-580-3p in RMRP and ATP13A3 3' -UTR. The mutant (Mut) RMRP and ATP13A3 3′-UTR were inserted into the pmirGLO vector to construct the reporters RMRP-mut and ATP13A3-mut. KYSE150 cells were co-treated with the reporters and miR-580-3p mimic or mimic NC using Lipofectamine 3000 reagent and collected after 48 h to measure luciferase activity in a dual luciferase reporting system (Promega, Madison, WI, USA) as per protocol.

The wild-type (WT) and mutant (MUT) PUMA 3'-UTR luciferase reporter gene plasmids were produced by Promega (WI, USA). The cells were then cotransfected with miR-NC or miR-221/222 mimics together with the WT or MUT PUMA-3'-UTR reporter plasmid using Lipofectamine 3000 reagent at 37 °C for 48 h. The relative luciferase activity was measured using a dual luciferase assay system (Promega). In addition, the WT and MUT ETS-1 3'-UTR luciferase reporter gene plasmids were produced by Promega. The procedures were similar to those described above.

To validate the direct binding of let‐7d with TGFβRI gene promoter region, the dual‐luciferase reporter assay was carried out using the pmirGLO Dual‐Luciferase miRNA Target Expression Vector System (Promega). The recombinant pmirGLO‐WT (wild‐type) TGFβRI and pmirGLO‐Mut (mutant) TGFβRI plasmids were established separately following the manufacturer's instructions, which were then transfected into the mouse lung microvessel pericytes using the Lipofectamine 3000 reagent (Thermo Fisher Scientific) as instructed by the manufacturer. Subsequently, cells transfected with different recombinant plasmids were transfected with the let‐7d mimic using the Lipofectamine 3000 reagent as well, followed by the detection of the luciferase activities using a GloMax^® 20/20 Luminometer (Promega).