兰瑞肽治疗垂体生长激素腺瘤耐药的分子机制

Acromegaly is caused by growth hormone (GH)-secreting pituitary adenomas,and accompanied signs and severity symptoms due to excessive growth hormone-releasing hormone secretion and an expanding tumor mass with suprasellar expansion and parasellar invasion. The elevated GH and insulin like growth factor 1 (IGF-1) levels lead to a wide range of cardiovascular, respiratory, and metabolic morbidities, and effect quality of life in acromegalic patients. In our previously study, we found that a part of acromegalic patients were lanreotide nonresponders with SSA lanreotide-treated. MiR-524-5p were differentially expressed between lanreotide responders and lanreotide nonresponders in GH-secreting tumor tissue samples. MiR-524-5p were involved in mitogen activated protein kinase (MAPK) signal pathway. Based on the previously outcome,we will investigate the expression of IGF-1, insulin-like growth factor-binding protein 3 (IGFBP3) and insulin-like growth factor-binding protein complex acid labile subunit (ALS) at transcriptive and translational level in GH tumor tissue samples, primary culture cell of GH-secreting pituitary adenomas, and the rat pituitary adenoma GH3 cell line with lanreotide-treated in this study. We also investigate whether the cell proliferation and apoptosis in primary culture cell of GH-secreting pituitary adenomas and GH3 cell are involved in the altered miRNAs expression of miR-524-5p, to investigate the relationship between the altered expression of miR-524 and the altered expression of IGF-1, IGFBP3 and ALS. Then, we investigate whether miR-524-5p are involved in regulate MAPK signal pathway through IGF-1, IGFBP3 and ALS or not. Finally, we investigate whether MAPK signal pathway activated apoptosis pathway through extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) or not. In a word, our study aims at the mechanisms of lanreotide nonresponders in acromegalic patients with lanreotide-treatment, and provides the mechanisms of SSA treatment for acromegalic patients with GH-secreting pituitary adenomas.

垂体源性肢端肥大症除激素分泌过多和肿瘤压迫引起损害外，易产生心血管、呼吸和代谢系统并发症，严重影响生存质量。我们前期研究发现部分垂体生长激素腺瘤病人对兰瑞肽治疗不敏感，治疗敏感与不敏感的GH腺瘤间存在miR-524-5p的差异表达，miR-524-5p参与调节MAPK通路。本研究拟观察兰瑞肽处理后GH腺瘤组织、GH腺瘤原代培养细胞和GH3细胞系中IGF1、IGFBP3和ALS在转录和翻译水平的变化；观察miR-524-5p水平变化对原代培养细胞和GH3细胞系细胞增殖、凋亡的影响；明确IGF1、IGFBP3和ALS表达变化与miR-524-5p变化之间的关系；明确miR-524-5p是否通过IGF1、IGFBP3和ALS调节MAPK活性并通过何种通路（ERK?/p38?/JNK?）激活凋亡通路；旨在阐明兰瑞肽治疗不敏感的分子机制，为GH腺瘤治疗提理论依据。

GH腺瘤因其伴随的不同系统的并发症，其死亡率较其他类型的垂体腺瘤高，其治疗方式包括手术治疗，药物治疗及放射治疗。兰瑞肽是目前治疗垂体腺瘤的一线用药，但存在部分GH腺瘤对药物不敏感。本课题组前期研究发现敏感GH腺瘤与耐药GH腺瘤间存在microRNA表达差异，且microRNA是否通过MAPK通路调节肿瘤耐药尚未得到阐明，研究microRNA与MAPK通路之间的潜在调节关系对于耐药性GH腺瘤的治疗具有重大意义。.经过研究实验，本课题组已通过Transwell共培养方法成功建立体外GH-IGF-1分泌轴，这对于肢端肥大症的体外研究具有重大意义；通过构建miR-524-5p质粒载体，最终包装目的基因的病毒，成功转染到GH原代细胞中，为后续的实验打下坚固基础；我们使用转染了miR-524-5p的原代细胞与肝细胞共培养，在兰瑞肽作用下，检测其对肝细胞所产生的下游蛋白（IGF-1、IGF-BP3、IGF-ALS）的影响，进而比较未转染目的基因的原代细胞对肝细胞分泌的下游蛋白的作用，以此明确miR-524-5p的过表达对于肿瘤细胞的耐药性的影响；实验结果显示，在添加了兰瑞肽的条件下，对比未转染的GH原代细胞，转染了目的基因的细胞对肝细胞所产生的下游蛋白的促进作用并未明显减弱，说明了miR-524-5p的过表达使得肿瘤细胞产生了一定的耐药性。进一步用转染了目的基因的原代细胞与未转染的原代细胞作对比，qRTF-PCR结果显示，实验组的ERK1及ERK2的转录水平较对照组的上调，这说明了miR-524-5p的过表达可能通过激活MAPK/ERK通路使得肿瘤细胞获得耐药特性。.对ERK蛋白的研究需要补充相关的信号通路抑制实验及其他相关靶向蛋白的分析研究，深入探讨miR-524-5p与MAPK/ERK通路的潜在调节关系，为兰瑞肽治疗耐药性GH腺瘤提供新的理论依据。