ALK阳性间变大细胞淋巴瘤 IGF-IR调控机制的研究

ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is an aggressive type of malignant lymphoma. It is currently based on the CHOP combination chemotherapy regimen. After an initial response to CHOP, around 40% of the patients develop resistance and have a relapse. At the molecular level, NPM-ALK+ ALCL is characterized by the expression of the chimeric tyrosine kinase NPM-ALK, which induces significant oncogenic effects through interactions with downstream molecules that promote lymphoma cell survival. We have demonstrated that IGF-1R play an important roles in promoting the survival of the ALK+ ALCL cells by interaction with NPM-ALK. However, the mechanisms that contribute to the aberrant expression of IGF-1R in ALK+ALCL are not completely understood. Here, we focus on post-transcriptional regulation of IGF-1R. RNA-binding proteins (RBPs) and microRNAs (miRNAs) are potent post-transcriptional regulators of gene expression, acting primarily through 5’ and 3’ untranslated regions (UTRs). Our specific aims are to investigate the regulating mechanism of IGF-1R expression by hsa-let-7b and HuR; To investigate the clinicopathologic correlation of hsa-let-7b and HuR and to identify the in vitro and in vivo preclinical effects of has-let-7 and HuR siRNA in ALK+ ALCL. Our project will provide insight into the regulation of IGF-1R expression by hsa-let-7b and HuR in in ALK+ ALCL. Our final aim is to understand IGF-IR-dependent signaling in this aggressive lymphoma in order to develop novel, targeted and safer therapeutic strategies.

ALK阳性间变大细胞淋巴瘤（ALK+ ALCL）以化疗为主，虽然缓解率高，但仍有40%患者复发。在分子学水平，这类淋巴瘤存在NPM-ALK融合蛋白。近年来研究提示ALK+ ALCL细胞IGF-IR异常高表达，且和NPM-ALK相互作用，在肿瘤进展中起着非常重要的作用。但IGF-IR表达上调的机制尚未阐明。本项目旨在从转录后水平探索IGF-IR的调控机制，MicroRNAs和RNA结合蛋白是两类重要的转录后调节因子。研究目标：1 探索Hsa-let-7b和HuR(RNA结合蛋白)竞争性调控IGF-IR的表达；2分析淋巴瘤标本IGF-IR和Hsa-let-7b 的mRNA水平，分析与临床及生存时间的关系；3从体内外实验研究Hsa-let-7b和HuR siRNA抗肿瘤活性。本项目旨在探索该类淋巴瘤IGF-IR的调控机制，并进一步揭示其与淋巴瘤疾病进展和预后的关系，为开拓分子靶向干预提供思路。

目的：探讨miRNA-let-7b在ALK阳性间变大细胞淋巴瘤（ALK+ ALCL）细胞株中的表达及对细胞株生物学功能的影响；揭示miRNA-let-7b对ALK+ ALCL细胞株IGF-1R的调节作用；探讨miRNA-let-7b对ALK+ ALCL发生发展相关信号通路的调节作用。.方法：1.miRNA靶向数据库和293T细胞中的双荧光素酶报告实验探索miRNA-let-7b与IGF-1R的关系；2.慢病毒转染ALK+ ALCL细胞株，并用免疫荧光法和qPCR检测转染效率；3.慢病毒转染ALK+ ALCL细胞株，MTS法、软琼脂克隆形成实验、细胞流式凋亡实验检测细胞功能学改变；4.慢病毒转染ALK+ ALCL细胞株，应用Western blot检测转染细胞株前后凋亡相关蛋白（caspase、cleaved caspase-3等）及STAT、AKT信号通路蛋白的表达。最后通过小鼠移植瘤模型研究了 miRNA-let-7b在体内的抗淋巴瘤活性。.结果：1.miRNA靶向数据库和双荧光素酶报告实验验证miRNA-let-7b可与IGF-1R的3’UTR端靶向结合；2.慢病毒转染miRNA-let-7b效率大约40-50%；3.在ALK+ ALCL细胞株中过表达miRNA-let-7b后，MTS法、软琼脂克隆形成实验表明，淋巴瘤细胞的增殖被抑制；流式检测细胞凋亡比例增加；4.在ALK+ ALCL细胞株中过表达miRNA-let-7b后，cleaved caspase-3和cleaved PARP表达增加；ALK、pALK、IGF-1R、p-IGF-1R蛋白表达减少；STAT、AKT信号通路相关蛋白表达减少；5. 转染miRNA-let-7b的小鼠肿瘤体积明显低于对照组，从而证实了miRNA-let-7b体内抗肿瘤活性。.结论：1.miRNA-let-7b可以与IGF-1R的3’UTR端靶向结合；2.过表达miRNA-let-7b可抑制ALK+ ALCL细胞增殖；3.过表达miRNA-let-7b可促进ALK+ ALCL的凋亡；4.miRNA-let-7b可通过STAT、AKT信号通路调节ALK+ ALCL中IGF-1R的表达;5. miRNA-let-7b可抑制移植小鼠瘤生长。