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Effects of SOST Gene Silencing on Proliferation, Apoptosis, Invasion, and Migration of Human Osteosarcoma Cells Through the Wnt/β-Catenin Signaling Pathway

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Abstract

Our study explored the effects of SOST gene silencing on the proliferation, apoptosis, invasion, and migration of human osteosarcoma cells through Wnt/β-catenin signaling pathway. Fresh tissues were obtained from 108 patients with osteosarcoma and 46 patients with osteochondroma. Human osteosarcoma cells (MG-63, U2-OS, HOS, and Saos-2) and normal osteoblast (hFoB1.19) were selected and cultured. Osteosarcoma cells were grouped randomly into the blank group, the scrambled control group, and the SOST-siRNA group. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis were tested by flow cytometry. Transwell and scratch test were performed to determine cell invasion and migration. The qRT-PCR and Western blotting were used to detect mRNA and protein expression level of sclerostin, Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7. The activity of caspase-3 was assessed by immunocytochemistry. Alkaline phosphatase (ALP) activity was measured using P-nitrophenylphosphate as a substrate. Low SOST mRNA and sclerostin protein expression levels were observed in osteosarcoma tissues and cells. Compared with the blank and scrambled control groups, sclerostin expression, apoptotic cells, ALP activity, and caspase-3 activity were down-regulated, while the proliferation, invasion, and migration abilities of osteosarcoma cells were evidently enhanced in the SOST-siRNA group. After SOST gene silencing, the mRNA and protein expression levels of Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7 in osteosarcoma cells and β-catenin protein expression levels in the nucleus and cytoplasm were significantly elevated. SOST gene silencing promotes the proliferation, invasion, and migration, and inhibits apoptosis of osteosarcoma cells by activating Wnt/β-catenin signaling pathway.

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Acknowledgements

We would like to acknowledge the helpful comments on this paper received from our reviewers.

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Authors and Affiliations

  1. Department of Orthopedics, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, Xuhui District, Shanghai, 200233, People’s Republic of China

    Jian Zou, Wei Zhang & Xiao-Lin Li

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  1. Jian Zou
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  2. Wei Zhang
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  3. Xiao-Lin Li
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Correspondence to Wei Zhang or Xiao-Lin Li.

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Human and Animal Rights and Informed Consent

This study was approved by the Ethics Committee in Shanghai Jiaotong University Affiliated Sixth People’s Hospital, and written informed consents were obtained from all studied subjects.

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Supplementary Figure 1

The transfection efficiency of the PLKO.1-SOST-GFP-siRNA recombinant plasmid in MG-63 cells Notes: A, the identification of the PLKO.1-SOST-GFP-siRNA recombinant plasmid and the negative control plasmid by dual-enzyme digestion and PCR; M, DNA Marker; 1, the identification of the PLKO.1-SOST-GFP-siRNA recombinant plasmid by dual-enzyme digestion of Age I and EcoR I; 2, the identification of the PLKO.1-SOST-GFP-siRNA recombinant plasmid by PCR; 3, the identification of the negative control siRNA plasmid by dual-enzyme digestion. B, comparison of the SOST mRNA expression in MG-63 cells among the blank, scrambled control and SOST-siRNA groups (Mean ± SD, n = 5); C, the sclerostin protein expression in MG-63 cells among the blank, scrambled control and SOST-siRNA groups detected by Western blotting. D, comparison of sclerostin protein expression in MG-63 cells among the blank, scrambled control and SOST-siRNA groups (Mean ± SD, n = 5); PCR, polymerase chain reaction; mRNA, messenger RNA; SD, standard deviation; *, P < 0.05 compared with the blank group Supplementary material (JPEG 196 KB)

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Zou, J., Zhang, W. & Li, XL. Effects of SOST Gene Silencing on Proliferation, Apoptosis, Invasion, and Migration of Human Osteosarcoma Cells Through the Wnt/β-Catenin Signaling Pathway. Calcif Tissue Int 100, 551–564 (2017). https://doi.org/10.1007/s00223-016-0231-6

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