Mir 335 5p inhibitor

GC cells in the logarithmic growth phase were digested and inoculated onto a 6-well culture plate. After cells reached 60–80% confluence, the miR-335-5p-mimics and miR-335-5p-inhibitor (GenePharma, Shanghai, China) were added to the corresponding wells for further culture for 24–48 h.

The mimic NC, miR-335-5p mimic, inhibitor NC, miR-335-5p inhibitor (100 nM), pcDNA3.1, and pcDNA3.1-ATG5 (GenePharma, Shanghai, China) were transfected in ASMCs, with 2.5 µg each. The lipofectamine 2000 transfection kit (Thermo Fisher, US) was used for transfection. All transfection experiments were carried out in strict reference to the lipofectamine 2000 transfection instructions. Successfully transfected cells were cultured in DMEM without serum, and later placed incubated for further proliferation [19 (link)].

The human CRC cell lines HCT116 and SW480, purchased from the American Type Culture Collection (ATCC), were cultured in RPMI-1640 medium (Gibco, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 5% CO₂ at 37° C.
Small interfering RNAs (siRNAs) specifically targeting ZEB1-AS1 or APOC1, negative control siRNA (si-NC), miR-335-5p mimics and miR-335-5p inhibitor were all obtained from GenePharma Corporation (Shanghai, China). A plasmid vector, pcDNA-ZEB1-AS1 or pcDNA-APOC1, which could consistently express ZEB1-AS1 or APOC1, was constructed by GenePharma Corporation. Cell transfection of siRNA, miRNA mimic or inhibitor and plasmid vectors were all conducted with Hieff Trans™ Liposomal Transfection Reagent (Yeason, Shanghai, China) and Opti-MEM (Gibco) for 48h, according to the manufacturer’s protocols.