Si sox9

Intestinal epithelial cell lines at the exponential growth phase were first digested and inoculated in a 6-well plate at a density of 1×10⁵ cells/well, and then were incubated for subsequent transfections. The pcDNA3-Flag-SOX9 plasmid (OBiO, Shanghai, China), Si-SOX9, miR-145-5p mimic, miR-145-5p inhibitor, or corresponding negative controls (RiboBio, Guangzhou, China) were applied to cell transfection in accordance with different purposes of the respective experiments. All transfection processes were conducted using Lipofectamine™ 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to relevant protocol. The transfected cells were incubated for 48 h or 72 h for the subsequent analyses. In addition, the transfection efficiency was evaluated via determining the distribution and localisation of the FLAG-llabeled plasmid using immunofluorescence.

To restore miR-605 expression, agomir-605 and its negative control, agomir-NC, was obtained from GenePharma (Shanghai, China). For the knockdown assay, small interfering (si) RNAs targeting the expression of SOX9 (si-SOX9) and its scramble control, siRNA (si-ctrl), were chemically synthesized by Ribobio (Guangzhou, China). The enforced expression plasmid (pcDNA3.1) for SOX9 (pcDNA3.1-SOX9; pc-SOX9) and empty plasmid, pcDNA3.1, were generated by the Chinese Academy of Sciences (Changchun, China). The transient transfection for oligonucleotides, siRNA, or plasmid was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer’s protocols. The Cell Counting Kit-8 (CCK-8) assay, transwell migration and invasion assays, and sub-cutaneous xenograft mouse model were carried out at 24, 48 and 24 h after transfection, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was carried out to measure gene expression at 48 h post-transfection, while Western blotting was conducted in transfected cells after 72 h transfection.