分类号：密级：
U D C：学号：416523217571 南昌大学专业学位硕士研究生
学位论文
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Expression and mechanism of LncRNA in osteosarcoma
巨承
培养单位（院、系）：南昌大学第三附属医院
mvkkk
指导教师姓名、职称：张志平副教授
专业学位种类：临床医学硕士
专业领域名称：外科学
论文答辩日期：2020年06月04日
答辩委员会主席：杨凤云
评阅人：徐聪
郝亮
2020年6 月2日
声明和授权书
I
摘要
型钢摘要
目的：电脑视保屏
shlr已发现长链非编码RNA(LncRNA) SNHG5、Gclnc1在肿瘤中起重要作用。然而，lncRNA SNHG5和Gclnc1在骨肉瘤中的作用和机制尚不清楚。本研究的目的是探讨SNHG5与Gclnc1在骨肉瘤中的具体功能及调控机制
纸币识别器
方法：
通过对于在线数据库GEO的骨肉瘤的芯片进行生信分析，预测lncRNA SNHG5在骨肉瘤中的表达与预后情况。收集骨肉瘤组织标本20例，采用实时荧光定量PCR（RT-PCR）技术检测lncRNA Gclnc1在骨肉瘤标本的表达情况。选取143B、MG63（基因敲低）和U2OS、U2R(过表达)细胞株进行SNHG5的功能研究。选取MG63(基因敲低)和U2OS (过表达)细胞株进行Gclnc1的功能研究。在对SNHG5进行敲低和过表达后，通过MTT法、克隆形成实验、流式细胞仪、Transwell实验、细胞划痕实验探讨SNHG5、miR-212-3p和SGK3对骨肉瘤细胞生长的影响。RT-PCR、蛋白免疫印迹法（Western blot）分析和荧光素酶报告实验等方法检测lncRNA SNHG5与miR-212-3p之间的相互作用。在对Gclnc1进行敲低和过表达后，使用MTT法检测细胞活力。克隆形成实验检测细胞增殖情况。流式细胞仪检测细胞凋亡、Transwell实验检测细胞的迁移及侵袭。RIP实验检测Gclnc1与NONO结合，Western blot检测NONO的表达量。体内动物实验验证敲低Gclnc1在体内抑制骨肉瘤增长。 结果：
在本研究中，敲低lncRNA SNHG5与Gclnc1抑制了骨肉瘤细胞的生长和转移，而过表达则产生了相反的结果。在机制上，lncRNA SNHG5作为海绵吸附miR-212-3p，抑制miR-212-3p/SGK3信号通路。miR-212-3p的过表达和抑制分别可逆转SNHG5已发挥的促癌和抑癌作用。SNHG5的功能可以被miR-
212-3p 所拯救并通过miR-212-3p的下游靶点SGK3来调节骨肉瘤细胞的生长和转移。另一方面，我们通过RT-PCR实验对于收集的骨肉瘤标本进行检测后发现与癌旁组织相比，Gclnc1在骨肉瘤组织中显著表达。机制上，lncRNA Gclnc1可以与RNA结合蛋白NONO相结合从而调控骨肉瘤的进展。另外，我们的结果表明在裸鼠实验中与对照组相比，敲低Gclnc1组中裸鼠肿瘤的体积及重量显著减少。
II
摘要
结论：
综上所述，本研究证实了lncRNA SNHG5可能通过miR-212-3p/SGK3轴调控骨肉瘤细胞的增殖和转移。而lncRNA Gclnc1 在骨肉瘤中表达显著增高，且高表达的lncRNA Gclnc1通过调控NONO促进了骨肉瘤细胞的增殖和转移。这些发现可能为未来的临床提供新的靶点。
关键词：骨肉瘤，lncRNA SNHG5，lncRNA Gclnc1，增殖，转移，凋亡
III
ABSTRACT
ABSTRACT
Objective:
It has been found that long non-coding RNA (LncRNA) SNHG5 and Gclnc1 play an important role in tumors.However, the role and mechanism of lncRNA SNHG5 and Gclnc1 in osteosarcoma is not clear.The purpose of this study is to explore the expression and regulation mechanism of SNHG5 and Gclcn1 in osteosarcoma. Methods:
The bioinformatics analysis of osteosarcoma chip based on online database GEO was used to predict the expression and prognosis of lncRNA SNHG5 in osteosarcoma.20 osteosarcoma tissue samples from the third affiliated Hospital of Nanchang University and Jiangxi traditional Chinese Medicine Hospital were collected, and the expression of lncRNA Gclnc1 in osteosarcoma samples was detected by real-time fluorescence quantitative PCR (RT-PCR).143B, MG63 (gene knockout) and U2OS, U2R (overexpression) cell lines were selected to study the function of SNHG5.MG63 (gene knockout) and U2OS (overexpression) cell lines were selected to study the function of Gclnc1.After knockdown and overexpression of SNHG5, the effects of SNHG5, miR-212-3p and SGK3 on the growth of osteosarcoma cells were investigated by MTT method, clone formation test, f
low cytometry, Transwell test and cell scratch test.The interaction between lncRNA SNHG5 and miR-212-3p was detected by RT-PCR, Western blotting (Western blot) analysis and luciferase report test.After knockdown and overexpression of Gclnc1, the cell viability was detected by MTT method.Colony formation assay was used to detect cell proliferation.Apoptosis was detected by flow cytometry and cell migration and invasion were detected by Transwell assay.RIP assay was used to detect the expression of Gclnc1 and NONO combined with, Western blot to detect the expression of NONO.Animal experiments in vivo confirmed that Gclnc1 inhibited the growth of osteosarcoma in vivo. Results:
In this study, knockout of lncRNA SNHG5 and Gclcn1 inhibited the growth and metastasis of osteosarcoma cells, while overexpression produced the opposite result.In
IV
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