HMGB2负调控STEAP4介导铁载荷抑制铁死亡并增强骨肉瘤化疗耐受的作用研究

铁死亡减少是肿瘤细胞获得化疗耐受的潜在原因。申请人前期证明HMGB2在骨肉瘤化疗耐药肿瘤组织中表达增高，上调HMGB2可减少铁死亡，增强骨肉瘤细胞对顺铂的耐受性。HMGB2在骨肉瘤细胞及肿瘤组织中均与铁代谢相关蛋白STEAP4的表达负相关，且HMGB2可与STEAP4的候选转录抑制因子YY1结合，但三者的具体作用关系及机制未明。本研究计划分析HMGB2及STEAP4的表达与骨肉瘤的临床病理相关性，探讨HMGB2及STEAP4对骨肉瘤细胞铁载荷、铁死亡以及顺铂敏感性的调控，利用激光共聚焦显微镜及Ch-IP等明确HMGB2协同YY1，调控STEAP4转录的作用位点，利用动物模型观察HMGB2对顺铂治疗骨肉瘤效果的影响，证实HMGB2在核内结合YY1，协同抑制下游因子STEAP4转录，降低铁载荷并下调铁死亡的研究设想，揭示HMGB2增强顺铂耐受性的作用机制，为骨肉瘤化疗耐药防治提供新的理论依据。

Reduction of ferroptosis may lead to cisplatin chemo-resistance. Our previous results showed increased HMGB2 expression in osteosarcoma paraffin tissue from chemo-resistant patients.HMGB2 over expression in osteosarcoma cell lines would lead to a reduction of chemo-sensitivity and decreased ferroptosis. The expression of HMGB2 presented negative correlation with iron metabolism-mediated protein STEAP4 expression both in osteosarcoma paraffin tissue and cell lines.YY1 was predicted to be a candidate transcription factor of STEAP4 via RPOMO and JASPAR analysis. PPI analysis and Co-IP demonstrated that HMGB2 may have binding affinity with YY1 in vitro.We hence deduced a scientific hypothesis that HMGB2 might bind with transcription factor YY1 as transcription repressor, to decrease STEAP4 transcription and henceforth affect iron metabolism and ferroptosis. To prove this hypothesis, we will carry our our study under the clinico-pathlogical perspective, in vitro and in vivo investigation. The clinico-pathlogical correlation of HMGB2, YY1 and STEAP4 expression will be analyzed to appraise the predictive value of HMGB2 in chemo-therapy. In vitro, through altering the expression of HMGB2 or STEAP4, and exogenously altering ferropotosis and intra-cellular iron load, we aimed to clarify whether HMGB2 could lower STEAP4 and thereafter induce chemo-resistance with decreased ferroptosis. The exact location of STEAP4 would also be ascertained. Ch-IP, luciferase reporter system and motif mutation technique will be applied to identify the binding site of HMGB2 with YY1 on STEAP4 promoter. Lastly, in vivo experiment will be conducted to verify the effect of HMGB2 on reducing ferroptosis to increase chemo-resistance. This study aims to disclose novel mechanism of HMGB2 in regulating chemo-resistance, which hopefully could provide theoretical basis in prevention and management in osteosarcoma chemo-resistance.