TNFAIP8调控上皮性卵巢癌细胞自噬参与铂类耐药的机制研究

Platinum resistance is one of the major bottlenecks in epithelial ovarian cancer (EOC) treatment. Our studies have shown that tumor necrosis factor α–induced protein 8 (TNFAIP8) overexpressed in EOC and was related to platinum resistance. Furthermore, we have demonstrated that down-regulation of TNFAIP8 expression would inhibit EOC cells proliferation，enhance the sensitivity of EOC cells to cisplatin and increase the expression of autophagy-related protein Beclin1 and LC3II. However, the mechanism that TNFAIP8 regulates autophagy involved in platinum-resistant EOC has not been reported at home and abroad. With respect to that PI3K/AKT/mTOR signaling pathway involves in resistance to chemotherapy by inhibiting the autophagy of tumor cell, we propose the hypothesis: TNFAIP8 could inhibit EOC cell autophagy to promote platinum resistance by activating PI3K/AKT/mTOR signaling pathway. To confirm this hypothesis, we intend to elaborate the relationship of TNFAIP8 with EOC cell proliferation and with chemosensitivity in vivo and in vitro experiments, respectively. Inducing tumor cell resistance to cisplatin, we further explain the molecular mechanism that TNFAIP8 promote EOC platinum resistance by using inhibitor intervention of autophagy, mTOR and PI3K/AKT. We should provide theoretical basis to achieving reversal of EOC platinum resistance in clinical practice.

铂类耐药是上皮性卵巢癌治疗的主要瓶颈之一。我们研究发现肿瘤坏死因子α诱导蛋白8（TNFAIP8）与上皮性卵巢癌铂类耐药具有相关性，沉默TNFAIP8能有效抑制细胞增殖、增强细胞对顺铂的敏感性、上调该细胞自噬相关蛋白Beclin1、LC3II的表达。然而，TNFAIP8调节自噬参与上皮性卵巢癌铂类耐药的机制，国内外尚未见报道。鉴于PI3K/AKT/mTOR信号通路通过抑制肿瘤细胞自噬参与化疗耐药，我们提出假说：TNFAIP8通过活化PI3K/AKT/mTOR信号通路抑制上皮性卵巢癌细胞自噬从而促进铂类耐药。为证实这一假说，我们拟通过体内外实验来明确TNFAIP8与上皮性卵巢癌细胞增殖、化疗敏感性的相关性。用顺铂诱导肿瘤细胞耐药，通过自噬、mTOR、PI3K/AKT抑制剂干预，进一步说明TNFAIP8促进上皮性卵巢癌细胞铂类耐药的分子机制，为临床中实现逆转上皮性卵巢癌铂类耐药提供理论依据。

铂类耐药是上皮性卵巢癌治疗的主要瓶颈之一。TNFAIP8作为一种癌基因发挥着多种生物学功能，该基因与肿瘤细胞的生长、增殖、迁移、侵袭转移及细胞凋亡均有密切关系，参与人类多种肿瘤的发生发展过程，是很有前景的肿瘤治疗靶点。 本研究通过构建慢病毒载体、筛选稳定转染TNFAIP8基因卵巢癌细胞系，通过体内外实验来明确TNFAIP8与上皮性卵巢癌细胞增殖、转移、化疗敏感性的相关性。检测肿瘤细胞自噬情况，通过应用雷帕霉素抑制mTOR信号通路，检测TNFAIP8基因促进卵巢癌细胞增殖和转移情况。CCK8实验证明TNFAIP8过表达或敲除明显影响卵巢癌肿瘤细胞的增殖；Transwell实验证明TNFAIP8与卵巢癌细胞侵袭具有一定相关性，虽然无统计学意义，需进一步实验证明；CCK8实验证明TNFAIP8过表达增加了肿瘤胞耐药，TNFAIP8敲减增加肿瘤细胞敏感性；LDH实验证明TNFAIP8过表达显著降低了顺铂诱导的细胞死亡，TNFAIP8表达沉默显著增加顺铂诱导的细胞死亡；免疫荧光证实TNFAIP8敲除显著增加自噬相关蛋白LCII的表达；WB检测表明TNFAIP8表达沉默显著增加顺铂诱导的自噬相关蛋白Beclin1和LC3Ⅱ的表达；TNFAIP8过表达显著上调p-mTOR的表达，同时上调mTOR下游磷酸化产物，TNFAIP8沉默显著下调p-mTOR的表达，同时下调mTOR下游磷酸化产物；TNFAIP8通过mTOR信号通路促进卵巢癌细胞增殖和转移；雷帕霉素可抑制TNFAIP8调节的mTOR信号通路相关分子及自噬相关分子的表达。该研究结果揭示了TNFAIP8参与卵巢癌转移、耐药的分子机制，在临床中将为逆转上皮性卵巢癌铂类耐药、探索肿瘤靶基因治疗提供理论依据。