https://orcid.org/0000-0003-3087-4270
Figures & data
Table 1. Primers used for qRT-PCR.
Table 2. The oligonucleotides transfected in this study are listed as follows.
Table 3. Correlation between patients’ clinicopathological features and circ_0078767 expression in osteosarcoma tissues.
Figure 1. Circ_0078767 is downregulated in OS cells and tissues.
(a). The volcano plot was used to show the differentially expressed circRNAs in OS tissues compared with normal tissues in the GSE140256 dataset. CircRNAs with significantly up-regulated expression were marked in red, and the down-regulated circRNAs were marked in green. Grey represented circRNAs that were not significantly differentially expressed. Screening criteria: log2(fold change) > 1 or < −1 and P < 0.05.(b). The expression profile of circ_0078767 in OS tissue (n = 3) and normal adjacent tissue (n = 3).(c&d). Detection of circ_0078767 expression in OS tissues and cells by qRT-PCR.(e). Subcellular distribution analysis of circ_0078767 in Saos2 and MG63 cells.(f). The relative levels of circ_0078767 and GAPDH mRNA in Saos2 and MG63 cells with or without RNase R incubation were detected by qRT-PCR.(g). Saos2 and MG63 cells were treated with actinomycin D, and the relative expression levels of circ_0078767 and GAPDH were examined by qRT-PCR at different times (0, 8, 16 and 24 h).(h). Patients with OS were divided into two groups according to the median level of circ_0078767 expression. Overall survival was analyzed utilizing the Kaplan-Meier method with the log-rank test.(i). Detection via qRT-PCR of circ_0078767 expression in Saos2 cells transfected with circ_0078767 overexpression plasmids and MG63 cells transfected with circ_0078767 siRNAs.All experiments were carried out in triplicate. ***P < 0.001.
Figure 2. Circ_0078767 restrains OS cell growth, migration, invasion, EMT, and promotes apoptosis.
(a). Detection by MTT assay of the impact of circ_0078767 overexpression or knockdown on OS cell proliferation.(b). Detection via scratch healing assay of the impact of circ_0078767 overexpression or knockdown on OS cell migration. Scale bars, 100 μm.(c). Transwell invasion assay was conducted for detecting the impact of circ_0078767 overexpression or knockdown on OS cell invasion. Scale bars, 250 μm.(d). Western blot was used to detect the protein levels of E-cadherin and N-cadherin in OS cells after circ_0078767 overexpression or knockdown.(e). TUNEL assay was used to detect the apoptosis of OS cells after circ_0078767 overexpression or knockdown. Scale bars, 250 μm.Each experiment was conducted in triplicate. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 3. Circ_0078767 targets miR-889.
(a). The binding site of miR-889 to circ_0078767 was predicted employing the Circular RNA Interactome database.(b). The targeted relationship of miR-889 with circ_0078767 was verified through dual-luciferase reporter gene assay.(c&d). The direct interaction between miR-889 and circ_0078767 was verified via RIP assay.(e). RNA pull-down assay explored the interaction between circ_0078767 and miR-889.(f). Detection via qRT-PCR of miR-889 expression in Saos2 and MG63 cells with circ_0078767 overexpression or knockdown.(g&h). Detection of miR-889 expression in OS tissues and cells via qRT-PCR.(i). Pearson correlation analysis of the correlation of circ_0078767 with miR-889 expression in OS tissues.Each experiment was performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4. Circ_0078767 represses OS cell growth, migration and invasion via absorbing miR-889.
(a). The co-transfection efficiency with miR-889 mimics+circ_0078767 overexpression plasmids, and si-circ_0078767#2+ miR-889 inhibitors was determined through qRT-PCR.(b). Detection of MG63 and Saos2 cell proliferation after transfection by MTT assay.(c&d). Detection of MG63 and Saos2 cell migration after transfection via scratch wound healing assay. Scale bars, 100 μm.(e&f). Detection of Saos2 and MG63 cell invasion after transfection by Transwell invasion assay. Scale bars, 250 μm.(g). Western blot was used to detect the protein levels of E-cadherin and N-cadherin in OS cells after transfection.(h). TUNEL assay was used to detect the apoptosis of OS cells after transfection. Scale bars, 250 μm.Each experiment was conducted in triplicate. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 5. Circ_0078767 upregulates KLF9 through targeting miR-889.
(a). The starBase database prediction of the binding site of miR-889 with KLF9 3ʹUTR.(b). The targeted relationship of KLF9 3’-UTR with miR-889 was verified through dual-luciferase assay.(c-f). Detection of KLF9 mRNA and protein expressions in OS cells by Western blot and qRT-PCR after circ_0078767 and miR-899 were selectively modulated. The original Western blot images with marker are shown in Supplementary material 1: Figure S4.(g). Detection of KLF9 mRNA expression in OS tissues via qRT-PCR.(h&i). Pearson correlation analysis of the correlation of KLF9 mRNA expression with circ_0078767 or miR-889 expression in OS tissues.Each experiment was carried out in triplicate. **P < 0.01, ***P < 0.001.
Figure 6. Circ_0078767 inhibits OS progression by regulating KLF9.
(a). Western blot was used to detect the expression level of KLF9 in MG63 cells transfected with KLF9 overexpression plasmid and control plasmid.(b&c). Western blot was used to detect the expressions of KLF9 mRNA and protein in MG63 cells after transfection of si-circ_0078767#2 or/and KLF9 overexpression plasmid.(d). Detection of MG63 cell proliferation after transfection of si-circ_0078767#2 or/and KLF9 overexpression plasmid by MTT assay.(e). Detection of MG63 cell migration after transfection of si-circ_0078767#2 or/and KLF9 overexpression plasmid by scratch wound healing assay. Scale bars, 100 μm.(f). Detection of MG63 cell invasion after transfection of si-circ_0078767#2 or/and KLF9 overexpression plasmid by Transwell invasion assay. Scale bars, 250 μm.(g). Western blot was used to detect the expression levels of E-cadherin and N-cadherin in MG63 cells transfected with si-circ_0078767#2 or/and KLF9 overexpression plasmid.(h). TUNEL assay was used to detect the apoptosis of MG63 cells after transfection of si-circ_0078767#2 or/and KLF9 overexpression plasmid. Scale bars, 250 μm.Each experiment was carried out in triplicate. **P < 0.01, ***P < 0.001.
Supplemental material
Supplemental Material
Download Zip (103 MB)Data availability statement
The data used for supporting the findings of this study are available from the corresponding authors upon request.
