TWEAK调控自噬和凋亡逆转上皮性卵巢癌铂类耐药的研究

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to successful long-term treatment of human epithelial ovarian cancer (EOC). Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that regulates cellular proliferation, metastasis, and apoptosis. However, the effect of TWEAK on cisplatin resistance in EOC remains elusive. It has been demonstrated that two major cell death pathways, autophagy and apoptosis, play important roles in regulating chemoresistance,but the role of TWEAK in regulating autophagy and apoptosis was unknown. Our previous study provided for the first time evidence that TWEAK played a role in restoring cisplatin sensitivity in EOC in vitro and in vivo. We also found that TWEAK excessively activated autophagy and increased apoptosis in A2780/CDDP cell line. In the present study, we detect the expression of TWEAK and its receptor, Fn14, cell autophagy and apoptosis in tumor tissues from patients with primary,recurrent platinum-resistant,or recurrent platinum-sensitive EOC, and in cisplatin resistant and cisplatin sensitive EOC cell lines, thus to analyse the relationship among TWEAK and Fn14, cell autophagy and apoptosis and cisplatin resistance in EOC. We then investigate the roles of autophagy and apoptosis in TWEAK restoring cisplatin sensitivity by controling autophagy, apoptosis, or autophagy and apoptosis pathway of in EOC. Our study would shed light on the detail mechanism of TWEAK restoring cisplatin sensitivity and regulating autophagy and apoptosis, thus to provide theoretical basis for developing new therapy for cisplatin chemoresistance in EOC.

上皮性卵巢癌（EOC）铂类耐药是导致其五年生存率低的主要原因。肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)可调控细胞凋亡、增殖和侵袭等生物学行为，但其在EOC铂类耐药中作用不明。自噬和凋亡作为细胞死亡的两大通路，在肿瘤耐药中起重要作用，但TWEAK对自噬和凋亡是否具调控作用尚不明确。我们前期研究首次发现，TWEAK可逆转EOC铂耐药，TWEAK过度激活EOC细胞自噬和促进其凋亡。本课题将检测EOC患者原发灶、铂敏感/耐药复发灶和EOC顺铂敏感/耐药株中TWEAK及其受体Fn14表达、细胞自噬及凋亡水平，分析TWEAK及其受体、细胞自噬和凋亡与EOC顺铂耐药的相关性；同时在体外通过调节自噬、凋亡、自噬或凋亡相关通路的活化状态探讨自噬和凋亡在TWEAK逆转EOC顺铂耐药中的作用，阐明TWEAK调控自噬和凋亡，逆转EOC细胞顺铂耐药的具体机制，为临床开创逆转EOC铂类耐药的治疗新途径提供理论依据。

上皮性卵巢癌（EOC）铂类耐药和远处转移是导致其五年生存率低的主要原因。肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)及其受体Fn14具有调控细胞凋亡、增殖和侵袭等生物学行为的作用。我们研究发现，TWEAK和Fn14在卵巢恶性肿瘤组织中表达；EOC患者癌组织中sTWEAK的含量高于良性卵巢肿瘤的患者；顺铂敏感组卵巢癌组织中Fn14的表达水平与顺铂耐药卵巢癌组织中Fn14的表达水平强度具显著差异。体外实验发现，TWEAK能诱导自噬，WB结果显示，TWEAK 可诱导LC3 等表达上调；激光共聚焦结果显示，TWEAK 可诱导GFP-LC3致密斑形成。同时，TWEAK能诱导凋亡，显著增加A2780/ DDP 细胞对DDP 的敏感性。流式结果显示,随着TWEAK 剂量增加,A2780/ DDP 细胞的凋亡显著增加。TWEAK 刺激后A2780/ cDDP 细胞中caspase 3 活化体表达增加,细胞核染色质发生形态变化,诱导了细胞的凋亡及凋亡小体形成。进一步研究发现，Fn14也与EOC耐药相关，Fn14过表达质粒转染A2780/DDP细胞后，DDP的IC50值降低；Fn14能增加顺铂诱导的A2780/DDP凋亡细胞数目; WB结果显示, 促凋亡蛋白caspase3表达升高，抗凋亡蛋白Bcl2表达明显下降。同时，TWEAK/Fn14在上皮性卵巢癌转移中发挥重要作用。Fn14过表达质粒转染HO8910-PM细胞后，细胞的迁移和侵袭能力明显降低，同时细胞中EMT相关蛋白表达明显下降。而 TWEAK作用于肿瘤相关巨噬细胞后，后者能抑制HO8910-PM的增殖和侵袭能力。进一步，我们发现TWEAK可以通过外泌体途径介导巨噬细胞抑制EOC转移，Tanswell结果示，巨噬细胞分泌的外泌体可促进EOC细胞株迁移和侵袭能力增高；而TWEAK刺激后，其分泌的外泌体则可降低EOC细胞的侵袭和迁移能力。通过芯片筛选及后续验证，我们发现TWEAK作用于巨噬细胞后，巨噬细胞释放的外泌体可转运mir-7至EOC细胞株。.综上，认为TWEAK/Fn14在上皮性卵巢肿瘤转移和铂类耐药中具有重要作用，为卵巢癌的治疗提供了理论基础。