Mimics control

A549 cells were used in the transfection assays. SiRNA, control SiRNA, miR-let-7d mimics, mimics control, miR-let-7d inhibitor, and inhibitor control was purchased from RiBoBio Co. (RiboBio, Guangzhou, China). Cells were transfected using Lipofectamine® 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. Briefly, 50 nM SiRNA or NC (negative-control, NC), 50 nM miR-let-7d mimics or mimics NC, 100 nM miR-let-7d inhibitor or inhibitor NC was diluted in 125 μl serum-free RPMI-1640 medium, 5 μl Lipofectamine® 3000 was diluted in another 125 μl serum-free RPMI-1640 medium. Then the two medium were mixed and incubated for 5 minutes at room temperature. Transfection medium containing target SiRNA or miRNA mimics/inhibitor was transferred to each well of the culture plates, after incubation at 37 °C for 6 h, the medium was replaced by complete medium and the cells were co-cultured 48 h as previously described. After that, the cells were collected to further analysis.

Hsa-let-7b mimics (mimic, 50 nM), mimics control (mimic NC, 50 nM), hsa-let-7b inhibitor (inhibitor, 100 nM), inhibitor control (inhibitor NC, 100 nM), CTHRC1 small interfering RNAs (Si-CTHRC1, 100 nM), and control groups (Si-NC, 100 nM) were constructed by Ribobio Corporation. PDLSCs were transfected with riboFECT™ CP kit (Ribobio, Guangzhou, China) at 30–50% confluence.

The mouse or human miR-155 mimics, mimics control, miR-155 inhibitor and inhibitor control were purchased from RiboBio Co., Ltd (Guangzhou, China). miRNAs were transiently transfected into cells (including hepa1-6, 7402 and C2C12 cells) at a working concentration of 100nM using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer’s procedure. All RNA oligonucleotides treatments proceeded for 48-72h. The effect of in vitro overexpression of miR-155 by using miRNA mimics and the repression of endogenous miR-155 expression by a miR-155 inhibitor on insulin-stimulated AKT phopshorylation was examined in hepa1-6 cells. After stimulation with 50IU/L human insulin (Novo Nordisk) for 15 min at 37°C (S6 Fig), the medium was removed and the cells were immediately lysed with ice-cold lysis buffer.