Control rnas

LPS, GW4869 and PKH-67 were purchased from Sigma-Aldrich. The following antibodies were used: anti-CD68, anti-CD9, anti-CD63, anti-PTEN, anti-Alix, anti-Akt, anti-p-Akt, goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) (Abcam), anti-p65, and anti-p-p65(Beyotime); miR-92a-3p mimic, mir-92a-3p inhibitor, control RNAs and the primers for miRNAs were all purchased from Guangzhou RiboBio.

To assess tumor formation in nude mice, CD90⁺ and CD90^- cells were sorted and injected (amounts ranging from 1 × 10³ to 5 × 10⁵) subcutaneously into different sides of 6-week-old male nude mice for controlled visualization and comparison. The mice were maintained under standard conditions and were examined for tumor formation for 12 weeks. After the tumors formed, the mice were sacrificed, and xenografts were harvested for IHC and primary culture. The fresh tumor xenografts from the nude mice were cut into small pieces and plated in a cell culture flask, and tumor cells migrated out from these pieces. DMEM containing 15 % FBS was used to initially establish the primary cultures, and DMEM containing 10 % FBS was used for subsequent maintenance.
To assess the effect of miR-589-5p on HCC tumorigenesis, 3 days after 1 × 10⁵ CD90⁺ MHCC97H cells were subcutaneously injected into nude mice, micrON™ agomir-589-5p (25 nmol, 50 μl) or control RNAs (RiboBio, China) were injected into the same site every 3 days within the next 2 weeks. The mice were maintained under standard conditions and were examined for tumor formation for 12 weeks.