[Regulation mechanism of autophagy-related protein LC3 by c-Jun in methotrexate resistant human choriocarcinoma JEG-3 cells].

ABSTRACT

OBJECTIVE: To explore the regulation mechanism of autophagy- related protein, microtubule- associated protein 1 light chain 3 (LC3), via c-Jun in methotrexate resistant human choriocarcinoma JEG-3 cell lines. METHODS: Human choriocarcinoma JEG-3 cell lines, and methotrexate resistant choriocarcinoma JEG-3 (JEG-3/MTXR) cell lines were used in our present study. Phosphorylation c-Jun (p-c-Jun) was evaluated after exposure to 0.02 ng/ml methotrexate for 72 hours in both cells by western blot. c-Jun gene was knockdown by small interference RNA (siRNA) in JEG-3/MTXR cells, and LC3 was evaluated by western blot and reverse transcription-PCR. The binding of LC3 promoter with c- Jun protein was detected via chromatin immunoprecipitation assay (ChIP) with or without 0.02 ng/ml methotrexate exposure. RESULTS: The results showed that p-c-Jun was up-regulated after methotrexate treatment for 72 hours (1.99 ± 0.20, versus 0.20 ± 0.06 at 0 hour; P < 0.05) by western blot analysis in JEG-3/MTXR cell lines. Further investigation demonstrated that c-Jun-siRNA could inhibit the up-regulation of LC3 formation and after methotrexate exposure (LC3 mRNA 1.24 ± 0.17 versus 3.03 ± 0.43; LC3 protein 0.52 ± 0.07 verus 1.20 ± 0.15; all P < 0.05). The binding of LC3 promoter by c-Jun protein was up-regulated after methotrexate treatment by the method of ChIP in methotrexate resistant JEG-3/MTXR cells [(2.95 ± 0.35) times]. CONCLUSION: Autophagy- related gene LC3 expression regulated by c-Jun protein may be involved in the effect mechanism of the development of methotrexate resistance in choriocarcinoma JEG-3 cells.