长链非编码RNA GAS5通过miR-135b-5p/APC轴抑制胶质瘤进展  

LncRNA GAS5 inhibits glioma progression through miR-135b-5p/APC axis

作　　者：张吉东 王雨涛 冀建文 ZHANG Jidong;WANG Yutao;JI Jianwen(Neurological Diseases Center,the Third Affiliated Hospital of Chongqing Medical University,Chongqing,China)

机构地区：[1]重庆医科大学附属第三医院神经疾病中心,重庆

出　　处：《陆军军医大学学报》2025年第3期243-254,共12页Journal of Army Medical University

基　　金：重庆市渝北区科技计划项目[2022(农社)24]。

摘　　要：目的探讨长链非编码RNA生长停滞特异性5(growth arrest-specific 5,GAS5)与miR-135b-5p和腺瘤性结肠息肉病基因(adenomatous polyposis coli,APC)轴之间的相互作用,以阐明其在胶质瘤增殖、转移、侵袭中的生物学功能。方法通过CGGA数据库分析GAS5表达水平与胶质瘤患者的生存率的关系;通过qRT-PCR检测GAS5在胶质瘤组织和细胞系中的表达水平;人类神经胶质瘤T98和A172细胞系分别用于过表达和敲低实验。通过转染来过表达或者沉默GAS5、miR-135b-5p和APC;通过Western blot与qRT‑PCR技术检测GAS5、miR-135b-5p、APC的表达水平;利用CCK-8实验检测胶质瘤细胞活力,通过Transwell和划痕实验分析肿瘤细胞运动活性;通过双荧光素酶报告基因实验验证miR-135b-5p与GAS5或APC的靶向关系。结果CGGA数据库分析显示GAS5低表达的胶质瘤患者的生存率显著降低(P<0.001)。Western blot与qRT‑PCR检测结果显示,分别与癌旁组织和正常人星形胶质瘤细胞系比较,GAS5在胶质瘤组织和胶质瘤细胞系中的表达下调(P<0.01);CCK-8、Transwell和划痕实验分析表明,与vector组比较,GAS5过表达抑制了胶质瘤细胞活力、侵袭和迁移(P<0.01);双荧光素酶报告基因实验证明,GAS5和miR-135b-5p之间存在结合位点,且二者mRNA表达水平呈负相关(P=0.019)。CCK-8、Transwell和划痕实验显示,GAS5对胶质瘤细胞生物学功能的影响是通过靶向miR-135b-5p完成的,APC过表达逆转了miR-135b-5p对胶质瘤细胞的促进作用(P<0.01);Western blot结果显示,与mimic_NC比较,miR-135b-5p升高能够抑制APC的表达(P<0.001),GAS5通过靶向miR-135b-5p上调APC的表达(P<0.01)。结论GAS5通过miR-135b-5p/APC轴共同抑制肿瘤发展,揭示了GAS5/miR-135b-5p/APC在胶质瘤发展中的分子调控机制。Objective To explore the interaction between long non-coding RNA growth arrestspecific 5(GAS5)and miR-135b-5p/adenomatous polyposis coli(APC)axis,and to elucidate its biological function in the proliferation,metastasis and invasion of glioma.Methods The relationship of GAS5 expression level with survival rate of glioma patients was analyzed based on CGGA database.qRT-PCR was used to detect the expression level of GAS5 in glioma tissues and cell lines.Human glioma T98 and A172 cell lines were subjected for overexpression and knockdown of GAS5,miR-135b-5p and APC by transfection.Western blotting and qRT-PCR were applied to measure the expression of related genes in glioma tissues or cell lines.CCK-8,Transwell and wound healing assays was conducted to determine the effect of GAS5 on the viability and motility of glioma cells.Moreover,dual luciferase reporter analysis were utilized to elucidate the regulatory mechanisms of GAS5 and miR-135b-5p/APC.Results Analysis on CGGA database showed that the survival rate of glioma patients with low GAS5 expression was significantly decreased(P<0.001).Western blotting and qRT-PCR indicated that GAS5 was down-regulated in the glioma cell lines and tissues than human normal astrocytes and para-cancer tissues(P<0.01).The results of CCK-8,Transwell and wound healing assays revealed that overexpression of GAS5 significantly inhibited the viability,invasion and migration abilities in T98 cells when compared with the cells of the vector group(P<0.01).Dual luciferase reporter analysis displayed that there were binding sites between GAS5 and miR-135b-5p,and the expression of the two was negatively correlated(P=0.019).Further studies suggested that the effect of GAS5 on biological function in glioma cells was through targeting miR-135b-5p,and overexpression of APC reversed the promoting effect of miR-135b-5p on glioma cells(P<0.01).Western blotting displayed that enhancing miR-135b-5p expression inhibited the expression of APC(P<0.001),and GAS5 upregulated the expression of APC by targeti

关 键 词：长链非编码RNA生长停滞特异性5 神经胶质瘤 miR-135b-5p 腺瘤性结肠息肉病基因 

分 类 号：R394.3[医药卫生—医学遗传学] R73-362[医药卫生—基础医学] R730.264