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The Effects of miR-708-5p on the Apoptosis and Metastasis of Osteosarcoma Cell and Its Mechanism
Feng Tianyu1, Zhu Zhongkai2, Wang Hao1, Mao Xiaohan1, Liu Dan1, Yuan Renjie1, Liu Yuehua1, Zuo Guowei1, Zhang Minghao3*
1The Key Laboratory of Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; 2Department of cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; 3Center for Lab Teaching and Management, Chongqing Medical University, Chongqing 400016, China
Abstract: This article aimed to investigate the expression of microRNA-708-5p (miR-708-5p) in osteosarcoma cell and its effects on cell apoptosis， migration and its mechanism. MiRNA microarray was utilized to screen differential expressed miRNAs. MiR-708-5p expression level in MG63 cell line， normal cells hMSC and HS-5 were detected by quantitative Real-time PCR (qRT-PCR). MiR-708-5p was overexpressed in osteosarcoma cell MG63 using Lipofectamine 2000. Hoechst 33258 staining， flow cytometry (FCM) were utilized to measure cell apoptosis. Wound healing assay and Transwell assay were employed to measure migration ability. The RNA expression of miR-708-5p and ZEB1 (Zinc finger E-box binding homeobox 1) were examined by qRT-PCR. Protein levels of E-cadherin， N-cadherin and ZEB1 were detected by means of Western blot. Targeting relationship between miR-708-5p and ZEB1 was predicted and validated using TargetScan and dual-luciferase reporter assay， respectively. The results indicated that miR-708-5p was lower expressed in MG63 cell compared to normal cells. Restoring miR-708-5p could induce cell apoptosis and inhibit migration. Showed by Western blot， protein levels of E-cadherin increased after miR-708-5p overexpression while N-cadherin and ZEB1 decreased. Indicated by dual-luciferase reporter assay， miR- 708-5p directly targeted ZEB1. What’s more， knocking down ZEB1 can inhibit migration of MG63. These data demonstrated that miR-708-5p could induce osteosarcoma apoptosis and decrease migration ability by targeting ZEB1， resulting in inhibiting cell migration.
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