H19阻止miRNA-93a对自噬的抑制而增敏DRD2受体激动剂疗效的机制研究

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. However, the miRNA-93a is up-regulated in drug resistant prolacinomas. Meanwhile, we did some preliminary research and found that miRNA-93a could compromise the CAB effects in vitro and in vivo. Mechanically, overexpression of miRNA-93a decreased the LC3-II level,a classical biomaker of autophay, which induced by serum starvation in GH3 and MMQ cells. A another bioinformatics analysis indicates that H19 directly binds to miRNA-93a in some nucleotide sequences. Above all, we consider that H19 could enhance the effects of CAB by promoting autophagic cell death through decreasing miRNA-93a level. In order to further confirm our hypothesis, we need to investigate: ① the direct combination of H19 and miRNA-93a using the technique of RNA pull down and northern blot experiment; ②mechanism studies to demonstrate H19 promotes autophagy through decreasing miRNA-93a level in GH3 and MMQ cells; ③ the in vivo effect of cabergoline treatment using F344 rats with H19 knockdown;④ the serum H19 level of patients with prolactinoma, whether H19 is a sensitivity biomarker of prolactinomas to cabergoline treatment. This study from the axis of lncRNA H19-miRNA-93a-autophagy 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治疗，如卡麦角林。我们对CAB的研究表明，CAB诱导细胞发生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，而miRNA-93a表达水平下降。体内外i实验验证miRAN-93a能够降低CAB的药物敏感性。进一步研究发现miRNA-93a能够抑制细胞的自噬水平和ATG7蛋白表达，并且生物信息学分析miRNA-93a和H19存在靶向结合位点。我们由此提出设想H19能够通过抑制miRNA-93a的表达，促进自噬水平，增加药物敏感性。本课题后续将采用RNA pull down等技术，证明H19和miRNA-93a直接结合；构建大鼠原位瘤模型体内验证机制；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。本课题从H19角度全新阐释泌乳素瘤耐药机制。

垂体腺瘤主要包括生长激素细胞腺瘤、泌乳素细胞腺瘤、促甲状腺激素细胞腺瘤、促性腺激素细胞腺瘤、混合型腺瘤等，而泌乳素腺瘤是最常见的垂体腺瘤，其约占所有垂体腺瘤的百分之40%~45%。泌乳素腺瘤首选药物治疗，目前临床多选择多巴胺受体激动剂为一线药物，常用的有溴隐亭和卡麦角林，以上两种药物能够使80%至90%的病人获益，包括恢复正常的体内泌乳素水平、明显减小泌乳素腺瘤肿瘤体积而缓解肿瘤压迫产生的症状。但是，剩余的10%至20%的病人对药物治疗不敏感甚至无效，此类病人称为耐药泌乳素腺瘤，此类病人往往手术治疗后仍然难以恢复正常的泌乳素水平。因此，研究泌乳素腺瘤如何形成耐药机制以及如何解决泌乳素腺瘤耐药难题成为此类研究热点。在本项目获得基金资助以后，着手开展从长链非编码RNA-H19的角度切入展开泌乳素瘤的耐药机制研究：卡麦角林增加H19的表达，并与外泌体分泌的H19对垂体瘤起协同治疗作用。外泌体H19抑制mTORC1底物4E-BP1的磷酸化。同时，所有亚型垂体瘤患者外泌体H19的表达水平均明显低于健康人群。血浆外泌体H19水平的变化可能与患者的预后或药物反应有关；另外，我们报道了H19与多巴胺受体激动剂治疗泌乳素瘤在体内外均有协同作用，H19可以通过抑制miR-93a的表达从而促进垂体肿瘤细胞ATG7的表达。此外，miR-93a和H19之间存在一个潜在的结合位点，miR-93在多巴胺受体激动剂耐药的泌乳素瘤中也出现低表达对情况，最终我们证明H-19-miR-93a通过靶向ATG7 mRNA调控ATG7的表达从而影响药物敏感性。总之，我们的研究已经确定了H19-miR-93-ATG7轴在多巴胺受体激动剂治疗泌乳素瘤中的作用，这可能是一个潜在的治疗人类催乳素瘤的靶点；并且，血浆外泌体H19可能作为预测泌乳素瘤患者医疗反应的重要生物标志物。