摘要
目的探讨LncRNA-DUXAP8在骨肉瘤细胞增殖、侵袭与凋亡过程中的作用及潜在的分子生物学机制.方法将转染后的对数生长期细胞分为si-DUXAP8组、si-NC组、miR-635 mimic组、miR-NC组、miR-635 inhibitor组和miR-635 inhibitor-NC组.应用qRT-PCR检测DUXAP8、TOP2A和miR-635的表达水平,应用细胞计数试剂盒-8(CCK-8)检测细胞活性;通过细胞划痕实验检测细胞的迁移能力,通过Transwell实验检测细胞的侵袭能力,通过集落形成试验检测细胞的增殖能力;应用双荧光素酶报告基因实验验证LncRNA-DUXAP8与微小RNA-635的靶向关系,应用Western blotting法检测各种蛋白表达水平.结果与hFOB 1.19细胞系相比,lncRNA DUXAP8在5个OS细胞系中的表达明显增加;调低lncRNA DUXAP8可导致细胞增殖的显著下降,骨肉瘤细胞的迁移能力显著降低,同时还可抑制细胞的侵袭.集落形成实验进一步表明:调低lncRNA DUXAP8可降低肿瘤细胞的集落数量;在骨肉瘤细胞中,miR-635的表达显著下调.此外,在DUXAP8调低的U2OS和SaOS-2细胞中,miR-635的表达增加;调低DUXAP8可显著降低TOP2A的mRNA和蛋白表达,过表达miR-635可降低TOP2A的表达,并消除DUXAP8诱导的TOP2A上调.结论抑制LncRNA-DUXAP8、上调miR-635可下调TOP2A的表达,进而抑制骨肉瘤细胞的增殖,促进肿瘤细胞凋亡.
Objective To investigate the role and potential molecular biological mechanism of LncRNA-DUXAP8 in the proliferation,invasion and apoptosis of osteosarcoma cells.Method The experimental groups were si-DUXAP8 group,si-NC group,miR-635 mimic group,miR-NC group,miR-635 inhibitor group and miR-635 intertior-NC group.The expression levels of DUXAP8,TOP2A and miR-635 were detected by QRT-PCR.Cell Counting Kit-8(CCK-8)to measure cell activity;Cell migration ability was detected by cell scratch test.The invasiveness of cells was detected by Transwell assay.Colony formation test was used to detect the proliferation ability of cells.The targeting relationship between LncRNA-DUXAP8 and microRNA-635 was verified by dual luciferase reporter assay.Western blotting was used to detect the expression levels of various proteins.Results Compared with hFOB 1.19 cell line,the expression of LncRNA-DUXAP8 was significantly increased in five OS cell lines.LncRNA DUXAP8 knockdown significantly reduced cell proliferation,migration and invasion of osteosarcoma cells.Colony formation assay further showed that LncRNA DUXAP8 knockdown reduced the number of tumor cell colonies.miR-635 expression was significantly down-regulated in osteosarcoma cells.In addition,miR-635 expression was increased in DUXAP8-knockdown U2OS and SaOS-2 cells.DUXAP8 knockdown significantly reduced the mRNA and protein expression of TOP2A,while overexpression of miR-635 reduced the expression of TOP2A and eliminated the upregulation of TOP2A induced by DUXAP8.Conclusion Inhibition of LncRNA-DUXAP8 can inhibit the proliferation of osteosarcoma cells and promote the apoptosis of tumor cells by up-regulating miR-635 and down-regulating the expression of TOP2A.
作者
马越
朴香花
李凡
段显亮
MA Yue;PIAO Xianghua;LI Fan;DUAN Xianliang(Affiliated Hospital of Beihua University,Jilin 132011,China;Jilin City Central Hospital,Jilin 132011,China)
出处
《北华大学学报（自然科学版）》
CAS
2022年第2期194-199,共6页
Journal of Beihua University（Natural Science）
基金
吉林省卫生健康技术创新项目(2019J045)
吉林省教育厅科学技术研究项目(JJKH20200070KJ)
北华大学校级重点教学项目(XJZD2019042).
