Wu Dajiang, Zhang Hao, Ji Fang, Ding Wenbin
Department of Traumatic Orthopedics, Changhai Hospital, Shanghai, 200433, China.
Department of Traumatic Orthopedics, Changhai Hospital, Shanghai, 200433, China.
Pathol Res Pract. 2019 Jan;215(1):115-120. doi: 10.1016/j.prp.2018.10.012. Epub 2018 Oct 22.
MicroRNAs (miRNAs) are abnormally expressed in numerous diseases, which are intimately associated with cell proliferation, migration and invasion. Recent study indicated that miR-17 may be involved in regulating osteosarcoma (OS) occurrence and development, but its function and mechanism have not been reported. In this study, quantitative real-time PCR (qRT-PCR) was used to measure the expression of miR-17, and Western blotting assay was performed to measure the expressions of SAM and SH3 domain containing 1 (SASH1), phosphoinoinositide-3 kinase (PI3K), protein kinase B (AKT), Caspase3, Bcl-2 gene family (Bcl-2, Bax) and matrix metalloprotein (MMP-2, MMP-9) in MG-63 cells. Luciferase reporter assay was conducted to confirm the target of SASH1 by miR-17. Cell proliferation, migration, invasion and apoptosis assay was performed to investigate the role of miR-17 in OS cells. We found that the expression of miR-17 was significantly up-regulated in OS cell lines. MiR-17 inhibitor inhibited the proliferation ability, and induced apoptosis of OS cells. Besides, miR-17 inhibitor prevented the migration and invasion of OS cells. Further, we identified that SASH1 was a target gene of miR-17. In addition, knockdown of miR-17 increased the protein expression of SASH1, and regulate related genes of cell proliferation, invasion and anti-apoptosis in the downstream of OS cells. These findings indicated that miR-17 was over-expressed and promoted cell proliferation, migration and inhibited cell apoptosis by targeting SASH1 in OS cells.
微小RNA(miRNA)在众多疾病中表达异常,这与细胞增殖、迁移和侵袭密切相关。最近的研究表明,miR-17可能参与调节骨肉瘤(OS)的发生和发展,但其功能和机制尚未见报道。在本研究中,采用定量实时PCR(qRT-PCR)检测miR-17的表达,并通过蛋白质印迹法检测MG-63细胞中含SAM和SH3结构域蛋白1(SASH1)、磷酸肌醇-3激酶(PI3K)、蛋白激酶B(AKT)、半胱天冬酶3、Bcl-2基因家族(Bcl-2、Bax)和基质金属蛋白酶(MMP-2、MMP-9)的表达。进行荧光素酶报告基因检测以证实miR-17对SASH1的靶向作用。进行细胞增殖、迁移、侵袭和凋亡检测以研究miR-17在OS细胞中的作用。我们发现,miR-17在OS细胞系中的表达显著上调。miR-17抑制剂抑制了OS细胞的增殖能力并诱导其凋亡。此外,miR-17抑制剂还阻止了OS细胞的迁移和侵袭。进一步研究发现,SASH1是miR-17的靶基因。此外,敲低miR-17可增加SASH1的蛋白表达,并调节OS细胞下游与细胞增殖、侵袭和抗凋亡相关的基因。这些研究结果表明,miR-17在OS细胞中过表达,并通过靶向SASH1促进细胞增殖和迁移,抑制细胞凋亡。