JMJD6-STAT3Y705ph axis promotes autophagy in osteosarcoma cancer cells by regulating ATG

Abstract

Background: Osteosarcoma cancer is a malignant tumor with poor outcome. Activation of STAT3 is closely related with tumor development. We intended to study the effects of JMJD6 on phosphorylation of STAT3 at Y705 site.

Methods: Osteosarcoma cancer cell lines (Saos-2, MG-63, and HOS) and clinical specimens were obtained. Interference RNA or JMJD6 mimic was transfected into the cells to silence or mimic JMJD6. Immunoprecipitation assay and glutathione S-transferase (GST) pull down was implemented to investigate whether JMJD6 is associated with STAT3. STAT3-null HOS cells were simultaneously transfected with the plasmids bearing STAT3^WT or STAT3^Y705F and EGFP-tagged LC3 plasmids. Recombinant full-length JMJD6 protein was subjected to in vitro kinase activity assay for testing its ability to phosphorylate STAT3. The severity of autophagy was indicated by the number of autophagosomes, expression of EGFP-LC3, ratio of LC3-II to LC3-I, degradation percentage of long-lived proteins and expression of autophagy associated gene (ATG).

Results: JMJD6 modulated the phosphorylation of STAT3 at Y705 site in osteosarcoma cells. Results from immunoprecipitation and GST pull down assays showed that JMJD6 associated with STAT3 in osteosarcoma cells. JMJD6 silence impeded the formation of autophagosomes, inhibited the accumulation of EGFP-LC3, decreased the ratio of LC3-II to LC3-I, blocked the degradation of long-lived proteins, and repressed the expression of ATG. JMJD6-induced autophagy was impaired by STAT3^Y705F which was not phosphorylated by JMJD6. The JMJD6-STAT3^Y705ph axis was implicated in the transcriptional regulation of ATG.

Conclusion: JMJD6 was included in regulating the phosphorylation of STAT3^Y705 and promoted autophagy of osteosarcoma cells through its kinase activity.

Keywords: ATG; JMJD6; STAT3Y705ph; autophagy; osteosarcoma.