Dai W, Liu H, Zhang P
Department of Joint Surgery, the First People's Hospital of Shangqiu City, Shangqiu 476000, China.
Department of General Medicine, the First People's Hospital of Shangqiu City, Shangqiu 476000, China.
Zhonghua Zhong Liu Za Zhi. 2021 Apr 23;43(4):457-465. doi: 10.3760/cma.j.cn112152-20200303-00164.
To investigate the effect of hsa_circ_0006948 (circ_0006948) on the proliferation, migration and invasion of osteosarcoma cells and the underlying mechanism. 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. The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in SAOS-2 cells were significantly different from NHOst cells (<0.01). The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in osteosarcoma tissues were significantly different from adjacent tissues (<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 <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 <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 (<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 (<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 (<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 (<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 (<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 (<0.01). Circ_0006948 regulates ATG7 expression through miR-490-3p, therefore regulates the proliferation, migration and invasion of osteosarcoma cells.
探讨人源环状RNA hsa_circ_0006948（circ_0006948）对骨肉瘤细胞增殖、迁移和侵袭的影响及其潜在机制。收集2009年至2015年商丘市第一人民医院收治患者的120例骨肉瘤组织和40例癌旁正常组织样本。进行基因芯片分析以检测Saos-2细胞中环状RNA的差异表达。采用实时定量逆转录聚合酶链反应（RT-qPCR）检测骨肉瘤细胞、NHOst细胞、骨肉瘤组织及癌旁组织中circ_0006948、微小RNA（miR）-490-3p和自噬相关蛋白7（ATG7）的mRNA表达水平。采用细胞克隆形成实验检测细胞增殖能力，Transwell实验检测细胞侵袭能力，细胞划痕实验检测细胞迁移能力。采用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。通过TargetScan数据库分析miR-490-3p与ATG7的相关性，采用蛋白质免疫印迹法检测细胞中Bcl-2和Bax蛋白的表达水平。SAOS-2细胞中circ_0006948、miR-490-3p和ATG7的mRNA表达水平与NHOst细胞相比差异有统计学意义（<0.01）。骨肉瘤组织中circ_0006948、miR-490-3p和ATG7的mRNA表达水平与癌旁组织相比差异有统计学意义（< .01）。circ_0006948-siRNA组的细胞克隆数、迁移数和迁移率分别为（32.78±1.76）%、（37.58±1.82）%和（36.93±1.45）%，低于siRNA NC组的（65.72±1.45）%、（78.63±1.93）%和（65.32±1.74）%（均<0.01）。miR-490-3p模拟物组的细胞克隆数、迁移数和迁移率分别为（20.08±1.54）%、（30.24±1.78）%和（21.15±1.68）%，低于模拟物NC组的（60.36±1.83）%、（76.93±1.64）%和（40.56±1.27）%（均<0.01）。miR-490-3p抑制剂+siRNA NC组的细胞克隆数、迁移数和迁移率分别为（90.34±1.72）%、（120.89±2.34）%和（70.83±1.93）%，高于抑制剂NC+siRNA NC组的（61.27±1.73）%、（75.82±1.82）%和（42.38±1.74）%（<0.01）。circ_0006948 siRNA+miR-490-3p抑制剂组的细胞克隆数、迁移数和迁移率分别为（58.74±1.98）%、（73.46±1.04）%和（40.35±1.72）%，低于miR-490-3p抑制剂+siRNA NC组的（90.34±1.72）%、（120.89±2.34）%和（70.83±1.93）%（<0.01）。ATG7 siRNA组的细胞克隆数、迁移数和迁移率分别为（20.56±1.87）%、（40.36±1.76）%和（20.96±1.73）%，低于siRNA NC组的（65.46±1.74）%、（90.87±2.32）%和（40.87±2.03）%（<0.01）。miR-490-3p模拟物+pcDNA-ATG7组的吸光度为0.54±0.11，高于miR-490-3p模拟物组的（0.36±0.08）（<0.05）。miR-490-3p模拟物组Saos-2细胞中Bax和Bcl-2蛋白的表达水平与模拟物NC组相比差异有统计学意义（<0.01）。miR-490-3p模拟物+pcDNA-ATG7组Saos-2细胞中Bax和Bcl-2的蛋白表达水平与miR-490-3p模拟物组相比差异有统计学意义（<0.01）。Circ_0006948通过miR-490-3p调控ATG7表达，进而调控骨肉瘤细胞的增殖、迁移和侵袭。
