ATG7-mediated autophagy protects human gingival myofibroblasts from irradiation-induced apoptosis

Background: Apoptosis resistance of myofibroblasts is critical in pathology of irradiation-induced fibrosis and osteoradionecrosis of the jaw (ORNJ). However, molecular mechanism of apoptosis resistance induced by irradiation in oral myofibroblasts remains largely obscure.Methods: Matched ORNJ fibroblasts and normal fibroblasts pairs from gingival were primarily cultured, and myofibroblast markers of alpha-SMA and FAP were evaluated by qRT-PCR and western blot. CCK8 assay and flow cytometric analysis were performed to investigate the cell viability and apoptosis under irradiation treatment. Autophagy-related protein LC3 and ATG7, and punctate distribution of LC3 localization were further detected. After inhibition of autophagy with inhibitor CQ and 3-MA, as well as transfected ATG7-siRNA, cell viability and apoptosis of ORNJ and normal fibroblasts were further assessed.Results: Compared with normal fibroblasts, ORNJ fibroblasts exhibited significantly higher alpha-SMA and FAP expression, increased cell, viability and decreased apoptosis under irradiation treatment. LC3-II and ATG7 were up-regulated in ORNJ fibroblasts with irradiation stimulation. After inhibition of irradiation-induced autophagic flux with lysosome inhibitor CQ, LC3-II protein was accumulated and punctate distribution of LC3 localization was increased in ORNJ fibroblasts. Moreover, autophagy inhibitor CQ and 3-MA enhanced the irradiation-induced apoptosis but inhibited viability of ORNJ fibroblasts. Silencing ATG7 with siRNA could obviously weaken irradiation-induced LC3-II expression, and promoted irradiation-induced apoptosis of ORNJ fibroblasts. After knockdown of ATG7, finally, p-AKT(Ser473) and p-mTOR(Ser2448) levels of ORNJ fibroblasts were significantly increased under irradiation.Conclusion: Compared with normal fibroblasts, human gingival myofibroblasts are resistant to irradiation-induced apoptosis via autophagy activation. Silencing ATG7 may evidently inhibit activation of autophagy, and promote apoptosis of gingival myofibroblasts via Akt/mTOR pathway.

Automated summary

Oral myofibroblasts resist apoptosis induced by irradiation through autophagy activation. Silencing ATG7 inhibits autophagy and promotes apoptosis of oral myofibroblasts.