环状RNA ZNRF2调控miR-32-5p及miR-92a-3p在骨肉瘤增殖侵袭中的作用

Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. Due to the malignant degree, aggressive and lack of effective molecular treatment of OS, we need to find the new molecular targets for OS pathogenesis and diagnosis. Base on our previous research, the adrenomedullin (ADM) expression in human OS tissue is strongly positive and reduced expression of ADM significantly inhibites the OS cells proliferation. But the mechanism of the abnormal increasing ADM activity in OS is unclear yet. Circular RNAs represent the special form of non-coding RNAs and play their function as microRNAs sponges. In our previous research, we also find circRNA ZNRF2(circZNRF2) expression is usually high in OS compared to the peritumoral tissue and its expression is correlation with ADM. Base on above results , we will continue to explore circZNRF2 function in the malignant and transformation mechanism of OS in this study.By using the in vivo osteosarcoma nude mouse models and in vitro MG-63、U-2 OS、Saos-2 OS cell lines, we will firstly detect the circZNRF2、miR-32-5p、miR-92a-3p and ADM、Drp1 levels and function by analyzing the mRNA、protein expression and the binding and inhibition activity by bioinformatics and Luciferase assay.Then, we will investigate the coordinate effect and its mechanisms of circZNRF2 expression.In OS nude mouse models and OS cells interfered by over expression or down regulation of circZNRF2 via lentivirus, we will count the apoptosis cells by MTT、TUNEL method, test the cell scratch-wound assay 、Matrigel invasion and migration assay， analyze the cell cycle by flow cytometry， evaluate the tumour malignancy degree by measuring tumour volume, detect the level of bone alkaline phosphatase (BALP)、calculate the tumourmicrovessel density(MVD) and observe the mitochondral dysfuncitons. Finally, we will approve the expression of circZNRF2、miR-32-5p、 miR-92a-3p and ADM、Drp1 in human OS tissue combined with clinical data. Our studies will identify circZNRF2 as the potent factor whose effectiveness relies on its ability to effectively regulate the ADM activity through the circZNRF2-miRNAs-ADM/Drp1 pathway against OS malignant biological behavior in vitro and in vivo. Collectedly, this study will help to identify the pathway included of Circular RNAs (circZNRF2) regulate the function of key tumour protein (ADM/Drp1) in pathogenic mechanism of osteosarcoma,which is also a new therapeutic target or procedure for OS therapy.

针对严重危害青少年健康的骨肉瘤恶性度高、侵袭性强, 目前临床缺乏有效分子治疗靶点的难题，基于项目组前期发现下调异常高表达的肾上腺髓质素(ADM)显著抑制骨肉瘤增殖侵袭，环状RNA ZNRF2(circRNA ZNRF2)参与调控ADM表达的研究基础，本项目将继续探讨circRNA ZNRF2在骨肉瘤增殖侵袭中的作用机制：⑴基于骨肉瘤细胞和裸鼠模型，明确circRNA ZNRF2、miR-32-5p和miR-92a-3p的表达与骨肉瘤增殖侵袭的关系；⑵确定骨肉瘤患者组织标本及血浆中circRNA ZNRF2表达与骨肉瘤临床分期的相关性；⑶阐明circRNA ZNRF2--miR-32-5p/miR-92a-3p--ADM/Drp1作用轴在骨肉瘤增殖侵袭中的作用和分子调控机制。本项目将circRNA ZNRF2机制引入骨肉瘤研究领域，将为骨肉瘤发病机制研究、临床诊断和治疗提供新思路和分子靶点。

骨肉瘤恶性度高、侵袭性强，严重危害青少年健康，且目前临床缺乏有效分子治疗靶点。项目组前期研究发现下调异常高表达的肾上腺髓质素(ADM)显著抑制骨肉瘤增殖侵袭，环状RNA ZNRF2(circRNA ZNRF2)参与调控ADM的表达；同时实验过程中发现miR-4295介导的非受体酪氨酸磷酸酶14（PTPN14）能够抑制骨肉瘤细胞的增殖，但circRNA ZNRF2和PTPN14在骨肉瘤增殖侵袭中的作用机制仍不清楚。通过开展如下的研究：⑴基于骨肉瘤细胞和裸鼠模型，明确circRNA ZNRF2、miR-32-5p和miR-4295的表达与骨肉瘤增殖侵袭的关系；⑵阐明circRNA ZNRF2/miR-32-5p/ADM和miR-4295/PTPN14作用轴在骨肉瘤增殖侵袭中的作用和分子调控机制，本项目阐明了circRNA ZNRF2、miR-32-5p、PTPN14和miR-4295在骨肉瘤增殖侵袭中的作用机制，将为骨肉瘤发病机制研究、临床诊断和治疗提供新思路和分子靶点。