Liu Min, Wu Lifen, Cai Chunyuan, Liu Liangle, Xu Youjia
Department of Orthopaedics, the Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215101, China.
J BUON. 2020 Jan-Feb;25(1):472-478.
Osteosarcoma is one of the rare but fatal malignancies. The high metastatic rate, late diagnosis, emergence of drug resistance against drugs such as doxorubicin, and the lack of therapeutic targets obstructs the treatment of osteosarcoma. This study was undertaken to investigate the role and therapeutic potential of miR-187 in human osteosarcoma cells.
The WST-1 proliferation assay was used for investigation of cell viability. Transfections were carried out by Lipofectamine 2000 reagent. The qRT-PCR was used for expression analysis. DAPI, acridine orange (AO)/ethidium bromide (EB) and Annexin V/propidium iodide (PI) assay were used for apoptosis. Western blot analysis was used for the determination of protein expression.
The expression of miR-187 was significantly downregulated in human osteosarcoma cells. Out of all osteosarcoma cell lines the SAOS-2 showed the lowest expression of miR-187 and therefore this cell line was selected for further studies. Overexpression of miR-187 caused significant inhibition in the proliferation of SAOS-2 osteosarcoma cells. The miR-187-triggered growth inhibition was found to be mainly due to induction of G2/M phase cell cycle arrest of the SAOS-2 cells. The G2/M cell cycle arrest was also accompanied by depletion of Cyclin-B1 expression. Additionally, miR-187 enhanced the chemosensitivity of the osteosarcoma cells to doxorubicin. The wound healing and transwell assay showed that miR-187 overexpression resulted in the suppression of migration and invasion of the SAOS-2 osteosarcoma cells. In silico analysis showed that miR-187 exerts its effects by inhibiting mitogen activated protein kinase 7 (MAPK7). The expression of MAPK7 was found to be significantly upregulated in osteosarcoma cells and overexpression of MAPK7 could nullify the effects of miR-187 on the proliferation of the osteosarcoma cells.
骨肉瘤是一种罕见但致命的恶性肿瘤。其高转移率、诊断延迟、对阿霉素等药物产生耐药性以及缺乏治疗靶点阻碍了骨肉瘤的治疗。本研究旨在探讨miR-187在人骨肉瘤细胞中的作用及治疗潜力。
采用WST-1增殖试验检测细胞活力。用Lipofectamine 2000试剂进行转染。采用qRT-PCR进行表达分析。用DAPI、吖啶橙（AO）/溴化乙锭（EB）和膜联蛋白V/碘化丙啶（PI）试验检测细胞凋亡。采用蛋白质印迹分析测定蛋白质表达。
miR-187在人骨肉瘤细胞中的表达明显下调。在所有骨肉瘤细胞系中，SAOS-2细胞系中miR-187的表达最低，因此选择该细胞系进行进一步研究。miR-187的过表达导致SAOS-2骨肉瘤细胞的增殖受到显著抑制。发现miR-187引发的生长抑制主要是由于诱导SAOS-2细胞的G2/M期细胞周期阻滞。G2/M期细胞周期阻滞还伴随着细胞周期蛋白B1表达的减少。此外，miR-187增强了骨肉瘤细胞对阿霉素的化疗敏感性。伤口愈合试验和Transwell试验表明，miR-187的过表达导致SAOS-2骨肉瘤细胞的迁移和侵袭受到抑制。计算机分析表明，miR-187通过抑制丝裂原活化蛋白激酶7（MAPK7）发挥作用。发现MAPK7在骨肉瘤细胞中的表达明显上调，MAPK7的过表达可消除miR-187对骨肉瘤细胞增殖的影响。
