hsa_circ_0006948靶向miR-490-3p调控自噬相关蛋白7对骨肉瘤细胞增殖迁移和侵袭的影响

Hsa_circ_0006948 regulates the proliferation, migration and invasion in osteosarcoma by regulation of the expression of miR-490-3p target ATG7

目的探讨hsa_circ_0006948(circ_0006948)对骨肉瘤细胞增殖、迁移和侵袭的影响及其作用机制。方法120例骨肉瘤组织和40例癌旁正常组织标本来源于2009—2015年就诊于商丘市第一人民医院的骨肉瘤患者。采用微阵列分析筛选骨肉瘤细胞Saos-2中差异表达的circRNA,实时荧光定量聚合酶链反应检测骨肉瘤细胞Saos-2和正常成骨细胞NHOst、骨肉瘤组织及癌旁组织中circ_0006948、miR-490-3p和自噬相关蛋白7(ATG7)mRNA的表达水平,细胞克隆形成实验检测细胞的增殖能力,Transwell实验检测细胞的侵袭能力,细胞划痕实验检测细胞的迁移能力,双荧光素酶报告基因实验检测circ_0006948与miR-490-3p、miR-490-3p与ATG7之间的相互作用,TargetScan数据库分析miR-490-3p与ATG7的相关性,Western blot法检测细胞中Bcl-2和Bax蛋白的表达水平。结果Saos-2细胞中circ_0006948、miR-490-3p和ATG7 mRNA的表达水平与NHOst细胞差异均有统计学意义(均P<0.01),骨肉瘤组织及癌旁正常组织中circ_0006948、miR-490-3p和ATG7 mRNA的表达水平差异均有统计学意义(均P<0.01)。circ_0006948-siRNA组细胞的克隆数目、迁移数目和迁移率分别为(32.78±1.76)个、(37.58±1.82)个和(36.93±1.45)%,均低于siRNA NC组[分别为(65.72±1.45)个、(78.63±1.93)个和(65.32±1.74)%,均P<0.01]。miR-490-3p mimics组细胞的克隆数目、迁移数目和迁移率分别为(20.08±1.54)个、(30.24±1.78)个和(21.15±1.68)%,均低于mimics NC组[分别为(60.36±1.83)个、(76.93±1.64)个和(40.56±1.27)%,均P<0.01]。miR-490-3p inhibitor+siRNA NC组细胞的克隆数目、迁移数目和迁移率分别为(90.34±1.72)个、(120.89±2.34)个和(70.83±1.93)%,均高于inhibitor NC+siRNA NC组[分别为(61.27±1.73)个、(75.82±1.82)个和(42.38±1.74)%,均P<0.01]。circ_0006948 siRNA+miR-490-3p inhibitor组细胞的克隆数、迁移数和迁移率分别为(58.74±1.98)个、(73.46±1.04)个和(40.35±1.72)%,均低于miR-490-3p inhibitor+siRNA NC组[分别为(90.34±1.72)个、(120.89±2.34)个和(70.83±1.93)%,均P<0.01]。ATG7-siRNA组细胞的克隆数目、迁移数目和迁移率分别为(20.56±1.87)个、(40.36±1.76)个和(20.96±1.73)%,均低于siRNA NC组[分别为(65.46±1.74)个、(90.87±2.32)个和(40.87±2.03)%,均P<0.01]。miR-490-3p mimics+pcDNA-ATG7组细胞的吸光度值为0.54±0.11,高于miR-490-3p mimics组(0.36±0.08,P<0.05);miR-490-3p mimics组细胞中Bax和Bcl-2蛋白的表达水平与mimics NC组比较,差异均有统计学意义(均P<0.01)。miR-490-3p mimics+pcDNA-ATG7组细胞中Bax和Bcl-2蛋白的表达水平与miR-490-3p mimics组比较,差异均有统计学意义(均P<0.01)。结论circ_0006948通过miR-490-3p调控ATG7蛋白的表达,从而调控骨肉瘤细胞的增殖、迁移和侵袭。

Objective To investigate the effect of hsa_circ_0006948(circ_0006948)on the proliferation,migration and invasion of osteosarcoma cells and the underlying mechanism.Methods A total of 120 osteosarcoma tissues and 40 adjacent normal tissue samples were collected from patients admitted to the First People′s Hospital of Shangqiu City from 2009 to 2015.Microarray analysis was performed to detect the differential expressions of circRNA in Saos-2 cell.The mRNA expressions of circ_0006948,microRNA(miR)-490-3p and autophagy-related protein 7(ATG7)in osteosarcoma cells,NHOst cells,osteosarcoma tissues and adjacent tissues were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR).Cell clone formation assay was used to detect cell proliferation ability,Transwell assay was used to detect cell invasion ability,and cell scratch assay was used to detect cell migration ability.The interactions between circ_0006948 and miR-490-3p,miR-490-3p and ATG7 were detected by dual luciferase reporter gene assay.The correlation between miR-490-3p and ATG7 was analyzed by TargetScan database,and the expression levels of Bcl-2 and Bax proteins in cells were detected by western blot.Results The mRNA expression levels of circ_0006948,miR-490-3p and ATG7 in SAOS-2 cells were significantly different from NHOst cells(P<0.01).The mRNA expression levels of circ_0006948,miR-490-3p and ATG7 in osteosarcoma tissues were significantly different from adjacent tissues(P<0.01).The numbers of cell clone,migration and mobility in circ_0006948-siRNA group were(32.78±1.76),(37.58±1.82)and(36.93±1.45)%,respectively,lower than(65.72±1.45),(78.63±1.93)and(65.32±1.74)%in the siRNA NC group(all P<0.01).The numbers of cell clone,migration and mobility in the miR-490-3p mimics group were(20.08±1.54),(30.24±1.78)and(21.15±1.68)%,respectively,lower than(60.36±1.83),(76.93±1.64)and(40.56±1.27)%in the mimics NC group(all P<0.01).The numbers of cell clone,migration and mobility in the miR-490-3p inhibitor+siRNA NC group were(90.34±1.72),(120.89±2.34)and(70.83±1.93)%,respectively,higher than(61.27±1.73),(75.82±1.82)and(42.38±1.74)%in the inhibitor NC+siRNA NC group(P<0.01).The numbers of cell clone,migration and mobility in the circ_0006948 siRNA+miR-490-3p inhibitor group were(58.74±1.98),(73.46±1.04)and(40.35±1.72)%,respectively,lower than(90.34±1.72),(120.89±2.34)and(70.83±1.93)%in the miR-490-3p inhibitor+siRNA NC group(P<0.01).The numbers of cell clone,migration and mobility in the ATG7 siRNA group were(20.56±1.87),(40.36±1.76)and(20.96±1.73)%,lower than(65.46±1.74),(90.87±2.32)and(40.87±2.03)%in the siRNA NC group(P<0.01).The absorbance of miR-490-3p mimics+pcDNA-ATG7 group was 0.54±0.11,higher than(0.36±0.08)of miR-490-3p mimics group(P<0.05).The expression levels of Bax and Bcl-2 protein in Saos-2 cells of miR-490-3p mimics group were significantly different from mimics NC group(P<0.01).The protein expression levels of Bax and Bcl-2 in Saos-2 cells of miR-490-3p mimics+pcDNA-ATG7 group were significantly different from miR-490-3p mimics group(P<0.01).Conclusion Circ_0006948 regulates ATG7 expression through miR-490-3p,therefore regulates the proliferation,migration and invasion of osteosarcoma cells.