piRNA-823通过调控自噬途径促进多发性骨髓瘤疾病进展的研究

Until now, multiple myeloma (MM) is an incurable disease. It is very important to explore a new mechanism of refractory disease progression of MM. piRNA is a newly-discovered small non-coding RNA, which can be self-amplified through specificly combining with PIWI protein. piRNA exhibits high tissue specificity which is not expressed in body cells. Recently, piRNA attracted much attention because it was found to be related with tumorigenesis and bad prognosis of malignancies. This year, we reported piRNA-823 was highly expressed in myeloma cells in leukemia journal. It was found to promote the proliferation and inhibit the apoptosis of myeloma cells. In addition, we found piRNA-823 level correlated positively with ISS stage of MM patients, which suggested piRNA-823 might be a new mechanism of MM progression. Autophagy is an important pathway which was newly found involved in MM progression and drug resistance. According to the results from pathway enrichment analysis of DNA microarray data and literature reviews, it can be inferred that autophagy pathway might be the critical pathway by which piRNA-823 modulated MM cell abnormality. Therefore, in this study, we endeavoured to systematically verify the important role of piRNA-823 in promoting MM progression by clinical, cell and mouse model experiments, and simultaneouly determine if piRNA-823 promoted MM progression by modulating autophagy pathway. In addition, we tried to find new methods to regulate piRNA-823 based on PIWI protein. Thus this study aims to provide a novel potential target for disease stratification and treatment of MM patients.

目前多发性骨髓瘤（MM）仍难以治愈，探索其新的顽固性进展机制非常重要。piRNA是一种新型非编码小RNA，通过与PIWI蛋白特异性结合来自我扩增。piRNA特异性极强，在体细胞中不表达，近期被发现与肿瘤发生、预后不良关系密切而备受关注。我们今年在Leukemia上报道了piRNA-823在MM细胞中高表达，促进MM细胞增殖、抑制其凋亡，并与MM患者ISS分期呈正相关，提示piRNA-823可能是促进MM进展的新机制。自噬是近期发现的参与MM进展、耐药的重要途径，结合基因芯片的生物学信息分析结果和文献报道，推测piRNA-823调控MM异常的关键通路极有可能是自噬途径。因此，本课题拟从临床、细胞、小鼠三个水平，系统验证piRNA-823在促MM进展中的重要地位，同时研究它是否通过自噬途径发挥作用，并从PIWI蛋白着手试图找到调控piRNA的新方法，以期为MM疾病分层和治疗提供一个新的靶标。

piRNA是一种新型非编码小RNA，可与PIWI蛋白特异性结合自我扩增。研究发现piRNA与肿瘤发生发展密切相关。本课题组之前在Leukemia上报道了piRNA-823在MM细胞中高表达，并与MM患者分期呈正相关，提示piRNA-823可能是促进MM进展的新机制。本研究中，我们分别对MM细胞系ARH77或RPMI8226转染agomir-823，agomir-823-NC，antagomir-823，antagomir-823-NC，改变MM细胞中的piRNA-823的表达水平，探讨piRNA-823在MM中的作用机制。研究结果显示：上调piRNA-823促进MM细胞增殖，抑制细胞凋亡，且提高细胞自噬水平，表现为自噬小体数目增加，同时下调了p-mTOR、P62蛋白的表达，上调了p-AKT，LC3-II蛋白水平。在小鼠皮下移植瘤模型中上调piRNA-823组小鼠显示出明显的肿瘤生长，且小鼠皮下瘤组化显示Ki-67、p-AKT、Bcl-2水平明显升高，p-mTOR、p62水平明显下降。而下调piRNA-823组与其阴性对照成现相反的结果。PIWIL1作为Argonaute蛋白家族的重要成员，在肿瘤细胞的生存和侵袭中起着关键作用。在本研究中，我们发现PIWIL1在MM细胞系和MM患者中高表达。然而，PIWIL1在MM中的确切功能和机制仍不清楚。我们利用慢病毒转染人MM细胞系RPMI8226和NCL-H929，改变MM细胞中的PIWIL1的表达水平，来探讨PIWIL1在MM中的作用机制。结果显示：上调PIWIL1表达可促进MM细胞增殖，增强对化疗药物的耐药性，此外，上调PIWIL1的标达增强自噬体，尤其是线粒体自噬的形成，同时下调了p-mTOR、P62蛋白的表达，并上调了LC3-II与线粒体自噬相关的蛋白Parkin1、optineurin和p-tbk1的表达。在小鼠体内皮下移植瘤模型中，注射过表达PIWIL1的细胞的小鼠显示出明显的肿瘤生长。过表达PIWIL1后MM细胞中侧群细胞比例增加。而下调PIWIL1表达后以上实验结果呈现相反的趋势。综上所述，piRNA-823通过调节自噬和凋亡双通道影响MM进展；干扰PIWIL1可通过抑制线粒体自噬和靶向MM干细胞群促进细胞死亡来克服耐药。piRNA-823和PIWIL1可望作为靶点来开发新的MM治疗策略