miR-1207-5p/SKA3调控AKT2介导糖酵解代谢重塑促进喉鳞癌侵袭转移的机制与临床意义研究

Laryngeal squamous cell carcinoma (LSCC) has the malignant behaviors of invasion and metastasis which lead to the recurrence and poor prognosis after treatment. Glycolysis, the special metabolic model in tumor, is related with invasion and metastasis. Previous studies have shown that AKT2 play an important role in tumor invasion and metastasis promoted by glycolysis. PPP2R2B could play a role in inhibition of AKT phosphorylation. However, the upstream regulatory mechanism has not been fully understood. We found that SKA3 and AKT2 were significantly upregulated in the metastatic laryngeal squamous carcinoma through genetic microarray. Immunohistochemistry showed that SKA3 and AKT2 were co-expressed and positively related to the characteristics of metastasis. Furthermore, si-SKA3 could inhibit laryngeal squamous cancer cell invasion and metastasis obviously. We conjecture that the accumulation of SKA3 could combine PPP2R2B and might inhibit dephosphorylation of AKT2 from PPP2R2B. We have confirmed SKA3 is a target gene of unique down-regulating molecular Has-miR-1207-5p in metastatic laryngeal squamous carcinoma. Meanwhile, the key molecules in glycolysis might be the target genes of Hsa-miR-1207-5p. In this study, we will illuminate the absorbingly mechanism of regulation between Hsa-miR-1207-5p/SKA3 with glycolysis and invasion by the means of CoIP, pulldown, report gene analysis in vivo and vitro respectively. Our finding would provide new targets and theoretical basis for molecular targeted therapy in LSCC.

喉鳞癌易侵袭转移的恶性行为导致治疗后复发及预后不良。糖酵解是肿瘤特有的代谢模式，与侵袭转移密切相关。研究表明AKT2在糖酵解助力侵袭转移中发挥重要介导作用，而PPP2R2B磷酸酶可抑制AKT激活，但上游调控机制不明。课题组通过芯片发现SKA3与AKT2在转移性喉鳞癌中上调，免疫组化表明两者共表达且与转移正相关，敲减SKA3可明显抑制喉鳞癌细胞系增殖及侵袭转移能力。文献报道SKA3与PPP2R2B相互作用，推测SKA3堆积可能阻遏PPP2R2B对下游AKT2去磷酸化；我们已证实SKA3是转移喉鳞癌独有下调分子Hsa-miR-1207-5p靶基因，同时预测到糖酵解关键分子也为其靶基因。本项目拟利用CoIP/pulldown/报告基因分析并结合体内外实验，揭示Hsa-miR-1207-5p/SKA3异常是影响糖酵解代谢重塑进而助力喉鳞癌侵袭转移的重要分子调控机制，以期为喉鳞癌生物治疗提供新靶标。

喉鳞癌是第二大呼吸道高发的恶性肿瘤。罹患喉鳞癌后，患者发声、呼吸及吞咽等重要生理功能发生严重损害，生存质量低。易侵袭转移及化疗抵抗的恶性行为是导致喉鳞癌治疗后复发及预后不良的主要因素。但是，喉鳞癌侵袭转移及化疗抵抗的机制尚不清楚。糖酵解是肿瘤偏好的代谢模式，与侵袭、转移、化疗抵抗密切相关。研究表明miRNA参与肿瘤发生发展及放化疗敏感性的调控。本项目通过表达谱芯片和高通量测序技术发现，喉鳞癌组织中miR-1207-5p表达显著下调，而SKA3表达显著上调。统计学分析表明SKA3与喉鳞癌T分期、N分期和临床分期正相关，与喉鳞癌分化程度负相关。更重要的是，SKA3高表达与喉鳞癌不良预后显著相关。功能研究表明miR-1207-5p抑制喉鳞癌细胞增殖、迁移、侵袭和细胞周期进展。SKA3促进喉鳞癌细胞增殖、迁移、侵袭及细胞周期进展等恶性表型。进一步研究发现miR-1207-5p通过结合SKA3 3' UTR抑制其表达水平，从而发挥抑癌功能。本研究还发现转录因子c-Myc在SKA3基因启动子区结合，反式激活SKA3表达。最重要的是，我们的研究揭示SKA3与PLK1蛋白结合，增强PLK1蛋白稳定性。PLK1累积进一步促进AKT磷酸化、激活AKT信号通路，进而上调糖酵解途径关键基因HK2、PFKFB3和PDK1表达，导致喉鳞癌细胞糖酵解水平升高的分子机制。此外，我们发现SKA3蛋白第360位苏氨酸（Thr360）的磷酸化对其介导的喉鳞癌恶性表型发挥重要作用，将该位点替换为非磷酸化丙氨酸（T360A）的SKA3突变体对喉鳞癌恶性表型的促进作用显著降低。最后，我们通过细胞和动物模型实验证实SKA3促进喉鳞癌化疗抵抗，敲降SKA3增强喉鳞癌细胞对化疗药物的敏感性。总之，本项目研究揭示了miR-1207-5p/SKA3异常是影响糖酵解代谢重塑进而促进喉鳞癌侵袭、转移及化疗抵抗的分子机制，对理解喉鳞癌发生和发展机理具有重要意义。本研究结果不仅为喉鳞癌诊断和预后评估提供了重要的潜在生物标志物，还为喉鳞癌分子靶向治疗提供了新的靶标。