| Objective: To investigate the role and mechanisms of IL-12 on autophagy in human hepatocellular carcinoma cell(HCC) line Hep-G2 and SMMC-7721.Methods: The Hep-G2 and SMMC-7721 cells were treated with IL-12 for indicated time. Western blot assay and immunofluorescence was used to detect the expression of LC3 protein, and transmission electron microscopy(TEM) was applied to view the autophagosome formation. Cell counting kit-8(CCK8) assay was used to detect the viability of SMMC-7721 and Hep G2 cells which were subjected with different treatment, trypan blue exclusion assay was used to detect the amount of dead cells. The expression of p-AKT, p-mTOR and p-STAT3 were test by Western blot, then, the STAT3 signaling was inhibited with pharmacological inhibitor stattic or small interfering RNA(siRNA) and the PI3K/Akt/mTOR pathway was activated by IGF-1. After cells were treated with these factors, then the change of autophagy was analyzed by Western blot and immunofluorescence.Results: The expression of LC3 and Beclin-1 were significantly upregulated in a dose- and time-dependent manner after IL-12 treated.Immunofluorescence and TEM showed the autophagosomes accumulation with a punctuated distribution. The CCK-8 assay revealed that IL-12 significantly inhibited the cell growth from the third day, and silencing Beclin1 significantly enhanced IL-12-induced proliferation inhibition. In addition, IL-12 treatment significantly induced HCC cells death and silencing Beclin1 further increased the cell death. IL-12 treatment significantly inhibited the expression of p-AKT, p-mTOR and p-STAT3.After STAT3 signaling was inhibited, the LC3 protein level increased, but activating PI3K/Akt pathway by IGF-1 decreased the LC3 protein level compared to the IL-12 group.Conclusion: IL-12 can induce the autophagy in HCC cells, which is mediated by STAT3 inactivation and inhibition of PI3K/AKT/mTOR signaling pathways. Furthermore, IL-12 induced autophagy may play a prosurvival effect on HCC. |
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