LINC00665靶向miR-761调控Wnt/β-catenin信号通路影响胶质瘤细胞增殖、细胞周期分布和凋亡  

LINC00665 affects the proliferation,apoptosis and cell cycle distribution of glioma cells by targeting miR-761 to regulate Wnt/β-catenin signal pathway

作　　者：陈宁军 刘志刚 赵军 李剑 CHEN Ning-jun;LIU Zhi-gang;ZHAO Jun;无(Department of Neurosurgery,Affiliated Baoji Hospital of Xi’an Medical University,Baoji 72106,China;Department of Neurosurgery,the Fourth People’s Hospital of Shaanxi,Xi'an 710043,China;Department of Neurosurgery,the Second People’s Hospital of Jiangyou,Jiangyou 621701,China;Department of Neurosurgery,Xijing Hospital,the First Affiliated Hospital of Air Force Military Medical University,Xi'an 710032,China)

机构地区：[1]西安医学院附属宝鸡医院神经外科,陕西宝鸡721006 [2]陕西省第四人民医院神经外科,陕西西安710043 [3]江油市第二人民医院神经外科,四川江油621701 [4]空军军医大学第一附属医院西京医院神经外科,陕西西安710032

出　　处：《中国实验诊断学》2020年第11期1869-1875,共7页Chinese Journal of Laboratory Diagnosis

摘　　要：目的探讨LINC00665对胶质瘤细胞增殖、细胞周期和凋亡的影响和分子机制。方法实时荧光定量PCR(RT-qPCR)检测人脑正常胶质细胞HEB和3种胶质瘤细胞(U-251MG、A172、SHG139)中LINC00665和miR-761的表达水平。利用脂质体转染法将LINC00665小干扰RNA、miR-761模拟物分别转染A172细胞,细胞计数试剂盒(CCK-8)检测细胞活力;流式细胞术检测细胞凋亡以及细胞周期的分布情况;蛋白质印记(Western Blot)检测Ki-67、细胞周期素D1(CyclinD1)和活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)蛋白的表达水平。双荧光素酶报告基因实验和RT-qPCR验证LINC00665对miR-761的靶向调控关系。Western Blot检测LINC00665靶向miR-761对Wnt/β-catenin信号通路的影响。结果与HEB比较,3种胶质瘤细胞中LINC00665的表达水平显著升高,miR-761的表达水平显著降低(P<0.05)。沉默LINC00665或过表达miR-761均可降低A172细胞Ki-67和CyclinD1的表达水平,升高Cleaved caspase-3的表达水平,抑制细胞增殖,引起细胞周期G0-G1期阻滞,诱导细胞凋亡(P<0.05)。miR-761是LINC00665的靶基因,LINC00665负性调控miR-761的表达水平。抑制miR-761表达部分逆转沉默LINC00665对A172细胞增殖、凋亡以及细胞周期分布的影响(P<0.05)。沉默LINC00665可抑制β-catenin和c-myc表达,抑制Wnt/β-catenin信号通路活化,抑制miR-761表达可逆转沉默LINC00665对Wnt/β-catenin信号通路的影响(P<0.05)。结论沉默LINC00665通过上调miR-761抑制Wnt/β-catenin信号通路可引起胶质瘤细胞G0-G1期阻滞,抑制细胞增殖,诱导细胞凋亡。Objective To investigate the effect of LINC00665 on proliferation,cell cycle distribution and apoptosis of glioma cells and its molecular mechanism.Methods Real-time quantitative PCR(RT-qPCR)was used to detect the expression levels of LINC00665 and miR-761 in human brain normal glial cells HEB and three glioma cells(U-251 MG,A172,SHG139).LINC00665 small interfering RNA and miR-761 mimics were transfected into A172 cells by lipofection,respectively.CNK-8 assay detected cell viability.Flow cytometry detected apoptosis and cell cycle distribution.Western Blot detected the expression levels of Ki-67,CyclinD1 and Cleaved caspase-3 proteins.Dual luciferase reporter assay and RT-qPCR verified the targeting regulation of LINC00665 on miR-761.Western Blot detected the effect of LINC00665 on Wnt/β-catenin signaling pathway by targeting miR-761.Results Compared with HEB,the expression level of LINC00665 was significantly increased in the three glioma cells,while the expression level of miR-761 was significantly decreased(P<0.05).Silencing LINC00665 or overexpressing miR-761 decreased the expression levels of Ki-67 and CyclinD1 in A172 cells,increased the expression level of Cleaved caspase-3,inhibited cell proliferation,caused cell cycle G0-G1 arrest,and induced apoptosis(P<0.05).miR-761 is the target gene of LINC00665,and LINC00665 negatively regulates the expression level of miR-761.Inhibition of miR-761 expression partially reversed the effect of silencing LINC00665 on proliferation,apoptosis and cell cycle distribution of A172 cells(P<0.05).Silencing LINC00665 inhibited the expression ofβ-catenin and c-myc,inhibited the activation of Wnt/β-catenin signaling pathway,and inhibiting miR-761 reversed the effect of silencing LINC00665 on Wnt/β-catenin signaling pathway(P<0.05).Conclusion Silencing LINC00665 caused cell cycle G0-G1 arrest,inhibited cell proliferation and induce apoptosis of glioma cells by up-regulating miR-761 to inhibit Wnt/β-catenin signal pathway.

关 键 词：LINC00665 miR-761 胶质瘤 Wnt/β-catenin信号通路 细胞周期 增殖 凋亡 

分 类 号：R739.4[医药卫生—肿瘤]