长非编码RNA RPS24P1调控鼻咽癌侵袭和转移的分子机制研究

Recent studies have showed that long non-coding （lnc） RNA plays an important role in the regulation of tumor invasion and metastasis, but the relevant studies are rarely reported in nasopharyngeal carcinoma (NPC). A high-throughput array was performed to compare the lnc RNA expression levels between metastatic tumor and primary tumor of NPC in our previous study, and RPS24P1 was found differentially expressed, which was also confirmed in the clinical tissue samples. Up-regulation of RPS24P1 enhanced invasion and migration of NPC cells in vivo. Bioinformatics analysis shows that there is a conserved SOX12 binding site located in the promoter region of RPS24P1. Also, down-regulation of SOX12 decreased the expression of RPS24P1 was also proved in NPC cells. Furthermore, we detected the expression level of RPS24P1 in NPC patients and the results showed that high expression level of RPS24P1 was related with poor prognosis. The combination of above results suggests that RPS24P1 plays an important role in the development of invasion and metastasis in NPC. In this study, we are going to thoroughly investigate the underlying molecular mechanisms of RPS24P1 regulating invasion and metastasis of NPC using NPC cell lines, metastatic animal models and clinical NPC samples based on our previous study, including: (1) To confirm the function of RPS24P1 in the enhancement of NPC invasion and metastasis in vitro and in vivo. (2) To clarify the regulatory mechanism of SOX12 to the expression of RPS24P1. (3) To identify the downstream targets of RPS24P1. (4) To investigate the metastatic regulation mode and the pathway of RPS24P1. (5) To explore the prevention and therapeutic effects of metastasis in NPC using adenovirus-bored RPS24P1-shRNA in vivo.

近年研究发现长非编码RNA在调控肿瘤转移中至关重要，但其与鼻咽癌发生发展及转移的相关报道罕见。申请人前期应用高通量芯片筛选出长非编码RNA RPS24P1在鼻咽癌患者远处转移灶与原发灶之间存在差异表达，并在临床样本中得到验证；体外研究证实其高表达促进鼻咽癌细胞的迁移和侵袭能力；生物信息分析表明其启动子区域存在保守的SOX12结合位点，敲减SOX12可下调其表达；临床样本也进一步证实RPS24P1高表达者预后差。由此我们推测RPS24P1在调控鼻咽癌转移中有着重要功能。本项目拟在前期工作基础上通过细胞、转移动物模型及临床标本全面深入探讨RPS24P1调控鼻咽癌侵袭转移的分子机制：①体内外功能实验明确其促进鼻咽癌侵袭、迁移和转移的作用；②探索潜在转录因子SOX12对其调控机制；③鉴定其下游作用靶标；④探索其调控转移的作用模式及通路；⑤探讨运载RPS24P1shRNA腺病毒对鼻咽癌转移防治作用。

鼻咽癌（NPC）是一种少见的头颈部肿瘤，在南部和西方国家均有较高的转移潜能。寻找新的肿瘤转移标志物对鼻咽癌的诊断和治疗具有重要意义。比较鼻咽癌和癌旁组织的基因组表达谱，发现长非编码RNA uc002xyu（NKILA）是鼻咽癌中一个显著下调的基因。与正常鼻咽上皮组织相比，鼻咽癌组织中NKILA的表达降低。NKILA在高转移潜能鼻咽癌细胞中的过表达抑制了其在体内外的运动行为和转移能力。相反，RNAi介导的NKILA的耗竭增加了转移潜能较低的细胞的侵袭运动性。另外，我们发现NKILA可通过NF-κB途径调节鼻咽癌的转移。此外，我们通过原位杂交分析了107例鼻咽癌标本中NKILA的预后价值。我们发现NKILA表达增加与NPC患者不良的临床病理特征和不良的生存结局（包括总生存率、无病生存率、无远处转移生存率和局部无复发生存率）相关。在校正潜在的混杂因素后，NKILA的过度表达是一个独立的预后因素，与不良的生存结局相关相应成果已正式发表在PLoS Genet。.另外，与正常鼻咽上皮组织相比，鼻咽癌组织中长非编码RNA LOC440173的表达升高。LOC440173在高转移潜能鼻咽癌细胞中的过表达抑制了其在体内外的运动行为和转移能力。相反，RNAi介导的LOC440173的耗竭降低了转移潜能。另外，我们发现LOC440173可通过结合ACO2蛋白从而影响Hypoxia信号途径调节鼻咽癌的转移。此外，我们通过分析了106例鼻咽癌标本中LOC440173的预后价值。我们发现LOC440173表达增加与NPC患者不良的临床病理特征和不良的生存结局（包括总生存率、无病生存率、无远处转移生存率和局部无复发生存率）相关。.我们选取了5对CAFs和NFs,对其上清中分离的得到的外泌体进行了miRNAs测序。我们对差异的miRNAs进行了筛选，我们选取了miR-148a-3p作为后续研究的miRNA。结果发现，miR-148a-3p在高转移潜能鼻咽癌细胞中的过表达抑制了其在体外的增殖能力和转移能力。另外，我们发现miR-148a-3p可通过EMT和TGF-β途径调节鼻咽癌的转移。