MiR-34a Regulates Autophagy And Chemotherapeutic Response In Retinoblastoma Cells Through Repression Of HMGB1

Purpose:To emphasize the mechanisms governing the regulation of HMGB1expression by microRNA, and their possible contribution to autophagy and drug resistance.Methods:HMGB1is a target of miR-34a in human retinoblastoma cells: After transfected by miR-34a mimic or antagomir-34a, pRT-PCR, Western-blotting and luciferase assays were perfomed on Y79and Weri-RB1cell lines.miR-34a induces apoptosis by modifying HMGB1expression: Y79cells were transfected with miR-34a mimic, HMGB1shRNA or pUNO1-HMGB1by72hours, apoptosis and Caspase3activities were detected, expression of cleaved-cap3and cleaved PARP was also evaluated by Western-blotting.Transfected by miR-34a mimic or HMGB1shRNA72for72hours, Y79cells were cultured in HBSS for1hour. Protein expression of LC3, p62and HMGB1were evaluated by Western-blotting, LC3puncta was analysed by immunofluorescence.Moreover, ultrastructural electron microscopy analysis was performed to detect autophagic vesicles.Inhibition of autophagy enhances chemotherapeutic efficacy in retinoblastoma cells:After treated by VCR, ETO and CBP, expression of LC3and p62was detected by Western-blotting. Cells tranfected with miR-34a mimic,MGB1shRNA,Beclin shRNA and Atg5shRNA were treated by VCR, ETO and CBP for48hours, then LC3puncta and cell viability were monitored.Inhibition of autophagy enhances DNA damage in chemotherapy:After transfected by miR-34a mimic,HMGB1shRNA or Atg5shRNA, Y79cells were treated with ETO and CBP for48hours,y-H2AX expression and apoptosis activity were evaluated.Results:The mRNA levels of HMGB1decreased following miR-34a mimic treatment, whereas antagomir-34a increased HMGB1expression. miR-34a mimic inhibited HMGB1luciferase activities, whereas antagomir-34a increased HMGB1luciferase activities in Y79cells.Consistently, the protein levels of HMGB1decreased following miR-30mimic treatment, whereas antagomir-34a increased HMGB1protein expression in Y79cellsSuppression of HMGB1expression promotes apoptosis by flow cytometric analysis of the percentage of the cells that are Annexin V-positive.Knockdown of HMGB1or miR-34a mimic increased activity of caspase3, cleaved-caspase3, and cleaved-PARP.Furthermore, overexpression of HMGB1by transfection with pUNO1-HMGB1inhibited miR-34a mimic-induced apoptosis, caspase3activity, and cleaved-PARP.Starvation increased mRNA expression of HMGB1at one to two hours, and returned to the baseline levels at six hours when miR-34a expression was increased in the retinoblastoma cell. miR-34a mimic or knockdown of HMGB1inhibited starvation-induced LC3-II expression and LC3puncta number.In contrast, treatment with lysosomal protease inhibitorsE/Prestored LC3-II expression in miR-34a-transfection cells.Moreover, ultrastructural electron microscopy analysis revealed that miR-34a mimic decreased the number of autophagic vesicles after starvation.Vincristine (VCR), etoposide (ETO), and carboplatin (CBP)increased LC3-II expression and decreased p62expression in retinoblastoma cells. miR-34a mimic and knockdown of HMGB1significantly decreased VCR, ETO, and CBP-induced autophagy by monitoring LC3puncta, but enhanced chemotherapeutic efficacy in retinoblastoma cells.Inhibition of autophagy by knockdown of beclin1and ATG5also increased chemotherapeutic efficacy in retinoblastoma cells.Exposure of Y79cells to ETO and CBP resulted in the phosphorylation of histone H2A at Ser139(y-H2AX), a marker for DNA damage. miR-34a mimic, knockdown of HMGB1, and knockdown of ATG5increased chemotherapy drug-induced γ-H2AX expression.miR-34a mimic, knockdown of HMGB1, and knockdown of ATG5increased chemotherapy-induced apoptosis. Moreover, the antioxidant NAC attenuated ETO-and CBP-induced DNA damage and apoptosis in ATG5knockdown cells.Conclusions:miR-34a targets HMGB1mRNA, leading to translational repressionof HMGB1. miR-34a is a potent inhibitor of autophagy, which promotes chemotherapeutic agent-induced DNA damage and apoptosis.