环状RNA(circ_0127646)竞争性结合miR-22调节骨肉瘤化疗耐药的分子机制研究

Osteosarcoma is the most common primary malignant bone tumor clinically, and failed to cure mainly due to chemotherapy resistant. Circular RNA is a potential target for the prevention or reversal of tumor resistance, based on its stable and non-coding characteristics. In pre-experiment, the increased circ_0127646 levels had been confirmed in cisplatin-resistant osteosarcoma cell lines. Inhibition of circ_0127646 could significantly enhanced chemosensitivity of cisplatin-resistant cell lines. Combined with the competitive endogenous RNA mechanism of circular RNA and bioinformatics analysis results, we proposed the hypothesis that “circ_0127646 regulated chemotherapy resistant of osteosarcoma by competitively binding to miR-22”. We intend to determine the correlation between circ_0127646 expressions and efficacy and prognosis of osteosarcoma at clinical level respectively, and investigate the role and mechanism of circ_0127646 on osteosarcoma chemotherapy resistant, and explore the feasibility of circ_0127646 as a potential therapeutic target for osteosarcoma chemotherapy resistant. The results would contribute to the understanding of the pathogenesis of chemotherapy resistant, and provide a new theoretical basis for diagnosis, treatment and prognosis of osteosarcoma chemotherapy.

骨肉瘤（Osteosarcoma）是临床上最常见的原发恶性骨肿瘤，化疗耐药是其治疗失败的主要原因。环状RNA具有稳定及非编码的特性，是预防或逆转肿瘤耐药的潜在靶点。我们前期在顺铂耐药的骨肉瘤细胞株中发现circ_0127646水平明显升高；抑制其表达显著增强耐药细胞株顺铂的化疗敏感性。基于环状RNA的竞争性内源RNA机制和生物信息学分析结果，我们提出“circ_0127646通过竞争性结合miR-22，引起骨肉瘤化疗耐药”的科学假设。我们拟分别在临床水平确定circ_0127646表达与骨肉瘤化疗效果与预后的相关性；在细胞和分子水平研究circ_0127646对骨肉瘤化疗耐药的作用和机制；在动物水平探索circ_0127646作为预防或逆转骨肉瘤化疗耐药潜在靶点的可行性。研究结果将丰富骨肉瘤化疗耐药的机制网络，为骨肉瘤的化疗诊治及预后提供新的研究方向。

骨肉瘤（Osteosarcoma）是临床上最常见的原发恶性骨肿瘤，化疗耐药是其治疗失败的主要原因。环状RNA具有稳定及非编码的特性，是预防或逆转肿瘤耐药的潜在靶点。本项目通过细胞、分子和临床样本等层次的实验，研究circ_0127646表达与骨肉瘤化疗效果与预后的相关性；circ_0127646对骨肉瘤化疗耐药的作用和机制；circ_0127646作为预防或逆转骨肉瘤化疗耐药潜在靶点的可行性。.项目执行期间，发现circ_0127646表达水平在骨肉瘤组织表达明显升高。在耐药的骨肉瘤组织和细胞系中，circ_0127646的表达水平明显高于不耐药癌组。通过qRT-PCR证实，circ_0127646在不同的骨肉瘤细胞系中表达不一致，高表达于MG63和OS9901细胞系，而相对低表达于HOS和U2OS细胞系。在顺铂处理后，四种细胞系中circ_0127646的表达均明显升高。进一步以MG63和OS9901细胞系作为靶细胞研究circ_0127646在骨肉瘤顺铂敏感性中的作用和机制。敲低circ_0127646的骨肉瘤细胞在顺铂处理24h后表现出低生长活力、高凋亡率和较低的克隆形成数量。通过生物信息学、报告基因系统以及拯救实验，发现circ_0127646可作为miR-22的竞争性内源性RNA，其通过结合miR-22参与骨肉瘤的顺铂耐药机制。敲低circ_0127646增强了miR-22对其靶基因KAT6B表达抑制的调节作用。PI3K/Akt/mTOR通路的激活与肿瘤细胞对化疗药物的耐药性密切相关，敲低circ_0127646显著降低了骨肉瘤细胞中p-PI3K、p-Akt和p-mTOR的蛋白表达，也降低了细胞自噬的活性。.此外，项目组还发现一种五碱基端粒酶产物触发的CRISPR-Cas12a增强滚环扩增的骨肉瘤诊断方法，分析了骨肉瘤相关的手术疗效、临床病例以及相关研究进展的文献综述。共发表SCI收录论文2篇，核心期刊收录论文6篇。协助培养硕士研究生1名。