HOXB7-BAP1-D2R信号轴参与泌乳素瘤溴隐亭耐药发生的分子机制研究

Dopamine agonist, Bromocriptine, is the first-line treatment of prolactinomas, yet the drug resistance followed is inevitable. HOX genes are developmental genes, which code for proteins that function as critical master regulatory transcription factors during embryogenesis. We have found that HOXB7 was remarkedly increased in bromocriptine-resistance pituitary PRL-secreting adenoma, compared with in primary prolactinoma. In rat MMQ pituitary cells, forced expression of HOXB7 could duplicate the phenotype of bromocriptine resistance. Moreover, HOXB7 could inhibit the expression of D2 dopamine receptor. We further performed PCR array of control and HOXB7 overexpression MMQ cells, very interestingly, we detected that the deubiquitinating enzyme, BAP1, might be the underlying molecular target of HOXB7. These results strongly indicated HOXB7 involved in the development of bromocriptine resistance. We thus hypothesize that HOXB7 may promote the degradation of D2R by inhibiting BAP1 expression. In this project, we will establish bromocriptine resistant mouse model and apply primary cell isolation as well as other molecular techniques to further investigate the role of HOXB7 in bromocriptine resistance in vitro and in vivo, and the role of HOXB7 in regulating D2R expression, thereby elucidating the molecular mechanism of HOXB7 in the development of drug resistance. This study will provide a novel candidate involved in the pathogenesis of bromocriptine resistance.

溴隐亭是泌乳素治疗的一线用药，然而服用溴隐亭所带来的耐药问题一直困扰着临床医师。HOX家族是一类胚胎发育的主控基因，研究发现在多种肿瘤中其表达水平异常。我们前期研究发现，HOXB7在溴隐亭耐药泌乳素瘤患者的垂体肿瘤组织中表达明显上调。在大鼠垂体瘤MMQ细胞系中，过表达HOXB7能模拟溴隐亭耐药表型，且HOXB7可下调多巴胺D2受体（D2R）的表达水平。PCR array分析显示去泛素化酶BAP1可能作为HOXB7的下游靶分子；Co-IP实验结果显示BAP1和D2R在体内外条件下均存在相互作用，以上结果提示HOXB7可能通过调控BAP1影响D2R降解参与溴隐亭耐药发生。本课题拟从细胞及动物水平，利用细胞分子生物学技术，详细探究HOXB7在溴隐亭耐药发生中的分子机制，为泌乳素瘤耐药治疗和药物筛选提供新的靶标。

溴隐亭是泌乳素瘤治疗的一线用药，然而服用溴隐亭所带来的耐药问题一直困扰着临床医师。为了研究泌乳素瘤耐药的发生及分子机制，我们用CAB处理MMQ细胞后进行转录组测序分析，以鉴定参与催乳素瘤进展和多巴胺受体激动剂（DA）敏感性的基因。根据LogFC为2，P<0.05的筛选标本，共筛选到253个上调基因及335个下调的基因。其中HOXB7和NEK2表达显著上调，进一步我们用qPCR技术验证发现HOXB7和NEK2在PRL瘤中表达上调。分析既往垂体瘤芯片GSE119063数据和我们测序的数据一致，免疫组化分析也证实NEK2的上调，提示NEK2可能参与PRL瘤的发生。进一步我们分别在细胞系水平验证HOXB7和NEK2的功能，我们分别构建了HOXB7和NEK2的过表达及敲除慢病毒。在垂体瘤细胞系GH3和MMQ中过表达NEK2，能促进细胞的增殖及克隆形成；裸鼠实验也证实过表达NEK2对肿瘤生长有促进作用。另外，NEK2过表达能显著降低垂体瘤GH3和MMQ细胞对CAB的增殖，而NEK2下调能促进细胞对CAB的敏感性。机制上，我们发现NEK2通过调控wnt信号通路调控细胞增殖；免疫共沉淀法证实USP7能与NEK2结合通过调控泛素化水平增加其稳定性。我们的结果表明NEK2是泌乳素瘤治疗的潜在靶点。我们同样构建了HOXB7的过表达及敲除慢病毒稳转株。可以看到过表达HOXB7能显著抑制细胞的凋亡。反之，在垂体瘤细胞系GH3和MMQ细胞中敲除HOXB7后，能显著促进垂体瘤细胞系GH3细胞及MMQ细胞的凋亡；裸鼠实验也证实敲除HOXB7能抑制肿瘤生长。另外，过表达HOXB7能降低垂体瘤细胞对多巴胺受体激动剂的药物敏感性，而敲除HOXB7能增加垂体瘤细胞对CAB及麦角新碱的敏感性。目前HOXB7的作用机制尚在进一步研究中。

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