Open AccessRoles of microRNA‐22 in Suppressing Proliferation and Promoting Sensitivity of Osteosarcoma Cells via Metadherin‐mediated Autophagy
Author(s) -
Wang Peng,
Zhao Zhenqun,
Guo Shibing,
Yang Tieyi,
Chang Zhiqiang,
Li Daihe,
Zhao Wei,
Wang Yuxin,
Sun Chao,
Wang Yong,
Feng Wei
Publication year - 2019
Publication title -
orthopaedic surgery
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 23
eISSN - 1757-7861
pISSN - 1757-7853
DOI - 10.1111/os.12442
Subject(s) - mtt assay , autophagy , western blot , transfection , cisplatin , flow cytometry , atg5 , microrna , cell growth , microbiology and biotechnology , chemistry , cell culture , cancer research , biology , apoptosis , biochemistry , chemotherapy , gene , genetics
Objective To analyze the effect of microRNA‐22 on autophagy and proliferation and to investigate the underlying molecular mechanism of osteosarcoma cell chemotherapy sensitivity. Methods MG‐63 cells were divided into four groups, including a control group, a negative control (NC) group, a cisplatin group, and a cisplatin + miR‐22 group. Proliferation of MG‐63 cells that had been treated with cisplatin and transfected with miR‐22 mimics was determined using MTT assay and colony formation assay. We assessed the degree of autophagy using flow cytometry through cellular staining of the autophagy lysosomal marker monodansylcadaverine (MDC). The effect of microRNA‐22 on autophagy was observed along with the expression levels of Beclin1, LC3, metadherin (MTDH) and ATG5 by western blot and quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Luciferase reporter assay revealed the targeted binding site between miR‐22 and the 3′‐untranslated region (3′‐UTR) of MTDH mRNA. Western blot and qRT‐PCR were used to explore the level of MTDH in the control group, the NC group, the cisplatin group, and the miR‐22 group for 6, 12, and 24 h. Results In the in vitro study, the MTT results indicated that the MG‐63 cells with overexpression of miR‐22 exhibited a significant decline in the proliferation capacity compared with the control group (0.513 ± 0.001, P < 0.0005). Similar to the MTT results, MG‐63 cells that were transfected with miR‐22 mimic (101.0 ± 10.58) formed fewer colonies compared with the cisplatin group (129.7 ± 4.163). MDC staining revealed that miR‐22‐overexpressing osteosarcoma (OS) cells treated with cisplatin showed a significant decrease in the expression of autophagy (7.747 ± 0.117, P < 0.0001). Our data revealed that miR‐22 could regulate not only autophagy but also proliferation in the chemosensitivity of osteosarcoma cells. We found that miR‐22 sensitized osteosarcoma cells to cisplatin treatment by regulating autophagy‐related genes. In addition, Luciferase Reporter Assay revealed that miR‐22 negatively regulated autophagy through direct targeting of MTDH. We performed western blot analysis to detect the MTDH expression level. The results revealed that the overexpression of miR‐22 obviously decreased the expression of MTDH (1.081 ± 0.023, P < 0.001). Conclusion Inhibition of miR‐22 ameliorated the anticancer drug‐induced cell proliferation decrease in osteosarcoma cells. MTDH was identified as the miR‐22 target in OS cells and MTDH‐triggered autophagy played a key function in the miR‐22‐associated chemotherapy sensitivity.