H19阻止ATG7泛素化降解增敏卡麦角林疗效的机制研究

Cabergoline is the first-line therapy of prolactinomas. Our recent study indicates that cabergoline induces autophagy of pituitary tumor cells, and finally results in autophagy-dependent cell death. Knockdown of ATG7 rescues cabergoline-mediated cell death and then increases the resistance of pituitary tumor to cabergoline treatment. In addition, according to the microarray results of sensitive- and resistant- prolactinomas, we find that expression of long non-coding RNA (lncRNA)-H19 in sensitive prolactinomas is higher than that in resistant prolactinomas. Knockdown of H19 obviously increases the resistance of pituitary tumor cells to cabergoline treatment, conversely, overexpression of H19 increases the sensitivity to cabergoline. Furthermore, H19 regulates the protein level of ATG7 without the change of ATG7 mRNA level. Therefore, we put forward our hypothesis that H19 possibly binds ATG7, blocks the ubiquitin degradation of ATG7 protein, and increases the sensitivity of tumor cell to cabergoline treatment. In order to further confirm our hypothesis, we need to investigate: ① the direct combination of H19 and ATG7 using the technique of RNA pull down; ② the possible ATG7-specific E3 ubiquitin ligase and the function of E3 ubiquitin ligase; ③ the in vivo effect of cabergoline treatment using transgenic mice with H19 knockout; ④ the serum H19 level of patients with prolactinoma, whether H19 is a sensitivity biomarker of prolactinomas to cabergoline treatment. This study from the angle of lncRNA blocking ATG protein ubiquitin will give us insight into the novel mechanism of CAB treatment to prolactinomas. If successful, the findings from this study provide us a new avenue for treatment of clinical dopamine-resistant prolactinomas, which has far-reaching clinical significance and academic value.

垂体泌乳素腺瘤首选药物如卡麦角林（CAB）治疗。我们最近对CAB的作用机制研究表明，CAB诱导细胞发生自噬和自噬依赖的细胞死亡，敲低ATG7能增加CAB治疗的耐药性。我们前期还针对敏感和耐药泌乳素腺瘤进行芯片检测，发现长链非编码RNA-H19在敏感中高表达；敲低H19使肿瘤细胞对CAB的耐药性增加，而过表达H19则敏感性增加。进一步发现，H19能调控ATG7的蛋白降解，而对mRNA水平没有影响。由此提出设想：H19绑定ATG7，阻止其蛋白的泛素化降解，从而增加药物治疗的敏感性。本课题后续将采用RNA pull down等技术，证明两者直接结合；同时寻找ATG7特定的E3泛素化连接酶，进而验证其功能；构建条件性H19敲除的转基因鼠模型，在体水平验证药物治疗效果；最后检测患者血液中H19水平作为判断药物治疗敏感性的生物标志物。本课题将从lncRNA的角度全新阐释泌乳素腺瘤药物治疗敏感性的机制。

垂体瘤约占所有颅内肿瘤的25%，其中泌乳素腺瘤约占40%，多巴胺受体激动剂为首选治疗。但临床上仍有10%～20%的泌乳素腺瘤对药物治疗耐药，阐述耐药机制及如何逆转耐药是临床治疗的挑战。长链非编码RNA-H19在泌乳素瘤发生发展和药物治疗中的作用不详。该项目通过系列研究，主要成果如下：①H19在垂体腺瘤中包括泌乳素瘤中低表达，而过表达H19能够有效抑制垂体腺瘤生长；其机理主要通过抑制mTORC1底物4E-BP1的磷酸化来发挥作用（Nature communications，2018）；②垂体腺瘤患者血浆外泌体中H19的表达和患者的耐药临床表现相关，外泌体中H19能协同卡麦角林对垂体腺瘤的抑制起到增敏作用（J Clin Endocrinol Metab，2019）；③H19通过“海绵”吸附效应，抑制miRNA-93的表达，后者靶向ATG7从而促进ATG7的表达，增强多巴胺受体激动剂的药物治疗敏感性（Mol Cell Endocrinol，2020）。综上所述，H19在垂体腺瘤的临床诊断、耐药评估以及逆转耐药治疗中具有重要的作用，有望成为垂体腺瘤治疗的新靶标。目前为止，该项目标注发表的文章11篇，其中SCI文章9篇，累计IF超过50分，并参与发表专著一部；累计培养硕士研究生3人，博士研究生1人；授权国家发明专利一项；作为第二完成单位和完成人，项目《垂体腺瘤发病机制及诊疗关键技术研究》获2018年重庆市科技进步一等奖。