MiR-214-PHLDA2-PI3K/AKT通路介导骨肉瘤起始细胞辐射抵抗的机制研究

Osteosarcoma tumor-initiating cells(OTICs) may be one of the main factors of radioresistance, and also restrict the improvement of therapeutic effect in osteosarcoma. However, the molecular mechanism of OTICs’ radioresistance has not been fully understood. Our previous study has shown that the expression of miR-214 was negatively associated with the response to radiation, and knockdown miR-214 significantly enhanced the apoptosis induced by X-ray in osteosarcoma cells. Furthermore, we confirmed that the expression of miR-214 was upregulated in OTICs. and predicted that PHLDA2 may be the direct target of miR-214 by bioinformatic softwares. Meanwhile, it has been reported that PHLDA2 was downregulated in OTICs, which could modulate the activation of PI3K/AKT pathway by PH domain .In this research, the direct relationship between miR-214 and PHLDA2 was confirmed firstly. Then OTICs, isolated and cultured in vitro, were combined with X-ray interference. Some experimental methods were used, such as proliferation, apoptosis, migration, model of nude mice, clinical verification experiments between phenotype and function, and transcriptome analysis, to clarify the mechanism how miR-214 inhibited PHLDA2 expression, activated PI3K/AKT pathway, and ultimately affected the radioresistance of OTICs. The aim of this study was to overcome the radioresistance of OTICs and offer a new target to improve the therapeutic effect of osteosarcoma.

骨肉瘤起始细胞(OTICs)的存在是骨肉瘤辐射抵抗并制约其疗效的主要因素之一，但目前对参与调控OTICs辐射抵抗的分子机制尚不清楚。我们前期研究发现，miR-214与骨肉瘤放疗疗效负相关，抑制其表达能显著增加X射线诱导的骨肉瘤细胞凋亡；OTICs高表达miR-214，预测显示PHLDA2基因是其直接作用靶点。文献报道，OTICs中PHLDA2表达降低，并可能通过PH结构域影响PI3K/AKT通路的活性。本项目拟首先确认PHLDA2与miR-214的直接作用关系；体外分离培养OTICs，结合X射线干扰等试验因素，利用细胞增殖、凋亡和迁移、裸鼠成瘤、临床大样本表型与功能验证以及转录组学分析等实验方法，研究miR-214是如何通过抑制PHLDA2的表达，从而激活PI3K/AKT信号通络，并最终影响OTICs辐射抵抗的功能与分子机制，以期为提高OTICs的放疗敏感性及骨肉瘤的治疗效果提供新的靶点。

骨肉瘤是严重危害青少年健康的常见骨恶性肿瘤，传统治疗以手术切除为主，但单纯手术后的五年生存率仅为20%左右。辅以手术的放射治疗，不仅降低了肿瘤负荷，使手术时肿瘤播散的风险下降；同时能大大增加保肢率，改善患者生活质量。但骨肉瘤细胞对放射治疗的反应性差，治疗后常有肿瘤细胞残留，导致了肿瘤的复发和放疗抵抗，成为制约疗效提高的“瓶颈”。因此深入探讨残留恶性细胞的生物学特性及放疗抵抗的分子机制，对提高骨肉瘤患者的生存获益具有非常重要的临床意义。越来越多的证据表明，骨肉瘤起始细胞(osteosarcoma tumor initiating cells, OTICs)是骨肉瘤治疗后复发的根源，对放射治疗高度抵抗。课题组前期研究发现，骨肉瘤组织中miR-214表达与放疗疗效负相关，上调miR-214能显著减少X射线诱导的骨肉瘤细胞凋亡，并进一步在OTICs中证实miR-214表达较瘤细胞明显增高(详见前期工作基础,图1-6)。我们推测，miR-214表达上调可能是OTICs放疗异质性产生的重要原因。本项目拟首先确认与miR-214直接作用的下游靶标基因，并利用体外分离培养的OTICs，结合细胞增殖、凋亡和迁移等试验、裸鼠成瘤体内功能试验以及临床大样本表型与功能验证试验，研究miR-214调控OTICs抵抗辐射的功能与分子机制，研究结果包括：.1.miR-214通过靶向PHLDA2 3’-UTR抑制PHLDA2基因表达；.2.骨肉瘤组织中miR-214表达较癌旁组织明显增高，其与PHLDA2低表达和骨肉瘤肺转移显著相关；.3.通过体内和体外实验证实下调miR-214表达显著增加骨肉瘤细胞及骨肉瘤起始细胞的放疗敏感性；.4.下调miR-214表达显著增加放射性诱导的骨肉瘤起始细胞凋亡；.5.通过外源性表达PHLDA2和加入PIK3CA抑制剂证实PHLDA2/AKT通路介导了miR-214相关的骨肉瘤起始细胞的放疗抵抗效应。.本研究从miRNAs层面研究PHLDA2表达的上游调控机制，并进一步从肿瘤起始细胞水平深入探讨miRNAs/PHLDA2通路对辐射抵抗的影响和作用机制，相关研究成果为提高骨肉瘤起始细胞放疗敏感性及骨肉瘤的治疗疗效提供了新的靶点。