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Circ_0105346 Knockdown Inhibits Osteosarcoma Development via Regulating miR-1182/WNT7B Axis - PubMed

  • ️Fri Jan 01 2021

Circ_0105346 Knockdown Inhibits Osteosarcoma Development via Regulating miR-1182/WNT7B Axis

Jinbao Liu et al. Cancer Manag Res. 2021.

Retraction in

Abstract

Background: Osteosarcoma (OS) is a common bone malignancy in children and adolescents. Circular RNAs (circRNAs) have been reported to affect OS progression. This paper mainly delineated the role of circRNA circ_0105346 in OS development and the potential mechanism.

Methods: Quantitative reverse transcription PCR (qRT-PCR) and Western blot assays were applied to detect the expression of circ_0105346, microRNA (miR)-1182 and wingless-type MMTV integration site family 7B (WNT7B). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was conducted to evaluate cell viability, and flow cytometry was performed to monitor cell apoptosis and cycle. In addition, cell migration and invasion were determined via transwell assay. Wound healing assay was also employed to evaluate the migrated capacity of OS cells. Western blot assay was also employed to examine the levels of protein markers. Additionally, the interaction between miR-1182 and circ_0105346 or WNT7B was confirmed by the dual-luciferase reporter, RNA immunoprecipitation (RIP) and pull-down assays. Mouse xenograft model was constructed to clarify the effect of circ_0105346 on tumor growth in vivo.

Results: Circ_0105346 and WNT7B were upregulated, while miR-1182 was downregulated in OS tissues and cells. Circ_0105346 knockdown suppressed OS cell proliferation, cell cycle, migration, invasion and glycolysis, as well as accelerated apoptosis, which was attenuated by miR-1182 inhibition. Interestingly, circ_0105346 targeted miR-1182, and miR-1182 interacted with WNT7B. Circ_0105346 could upregulate WNT7B by downregulating miR-1182 expression. Furthermore, circ_0105346 knockdown blocked tumor growth in vivo.

Conclusion: Circ_0105346 knockdown repressed OS progression by regulating miR-1182/WNT7B axis, at least in part.

Keywords: WNT7B; apoptosis; circ_0105346; glycolysis; metastasis; miR-1182; osteosarcoma; proliferation.

© 2021 Liu et al.

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Conflict of interest statement

The authors declare that they have no conflicts of interest for this work.

Figures

Figure 1
Figure 1

Circ_0105346 was upregulated in OS tissues. (A) QRT-PCR assay for the expression level of circ_0105346 in OS tissues and normal tissues (N=40). (B) Overall survival rate of OS patients with high expression level and low expression level of circ_0105346. *P<0.05.

Figure 2
Figure 2

Circ_0105346 knockdown inhibited OS cell proliferation, metastasis and glycolysis, while promoted cell apoptosis. (A) QRT-PCR assay for the expression level of circ_0105346 in hFOB, Saos-2 and SOSP-9607 cells. (B–R) Saos-2 and SOSP-9607 cells were transfected with si-NC or si-circ_0105346. (B) QRT-PCR assay for the expression level of circ_0105346 in transfected cells. (C and D) MTT assay for the cell viability of transfected cells. (E and F) Flow cytometry assay for the cell cycle of transfected cells. (G) Western blot assay for the protein levels of PCNA, Cyclin D1, Bax and Bcl-2 in transfected cells. (H) Flow cytometry assay for the cell apoptosis of transfected cells. (I and J) Wound healing assay for the migrated rate of transfected cells. (K and L) Transwell assay for the cell migration and invasion of transfected cells. Bar represents 50 μm. (M) Western blot assay for the levels of EMT-like-related proteins in transfected cells (N–Q) ECAR assay for the transfected cells. (R) Western blot assay for the protein markers of glycolysis in transfected cells. *P<0.05.

Figure 3
Figure 3

Circ_0105346 could sponge miR-1182. (A) Venn diagram of the overlapping target miRNAs of circ_0105346 predicted by Circular RNA Interactome and circBank. (B) QRT-PCR assay for the expression levels of miR-1178-3p, miR-1182, miR-1253, miR-661 and miR-936 in OS tissues and normal tissues (N=3). (C) QRT-PCR assay for the expression levels of miR-1178-3p, miR-11822, miR-1253, miR-661 and miR-936 in Saos-2 and SOSP-9607 cells were transfected with si-NC or si-circ_0105346. (D) The putative binding sites between circ_0105346 and miR-1182 forecasted by Circular RNA Interactome. (E and F) Dual-luciferase reporter assay for the luciferase activity of circ_0105346-WT and circ_0105346-MUT in Saos-2 and SOSP-9607 cells co-transfected with miR-1182 or miR-NC. (G and H) RIP and qRT-PCR assays for the enrichment of circ_0105346 and miR-1182 in the samples bound to the anti-Ago2 or anti-IgG. (I) RNA pull-down and qRT-PCR assays for the enrichment of circ_0105346 pulled down by Bio-miR-1182 or Bio-miR-NC in Saos-2 and SOSP-9607 cells. (J) QRT-PCR assay for the expression level of miR-1182 in OS tissues and normal tissues (N=40). *P<0.05.

Figure 4
Figure 4

Circ_0105346 knockdown repressed OS progression by upregulating miR-1182 level. (A) QRT-PCR assay for the expression level of miR-1182 in hFOB, Saos-2 and SOSP-9607 cells. (B) QRT-PCR assay for the expression level of miR-1182 in Saos-2 and SOSP-9607 cells transfected with anti-miR-NC or anti-miR-1182. (C–N) Saos-2 and SOSP-9607 cells were transfected with si-NC, si-circ_0105346, si-circ_0105346+anti-miR-NC or si-circ_0105346+anti-miR-1182. (C and D) MTT assay for the cell viability of transfected cells. (E) Flow cytometry assay for the cell apoptosis of transfected cells. (F) Wound healing assay for the migrated rate of transfected cells. (G and H) Transwell assay for the cell migration and invasion of transfected cells. (I–L) ECAR assay for the transfected cells. (M and N) Western blot assay for the protein markers of glycolysis in transfected cells. *P<0.05.

Figure 5
Figure 5

WNT7B served as a target of miR-1182. (A) The possible complementary sequence between miR-1182 and WNT7B predicted by TargetScan. (B and C) Dual-luciferase reporter assay for the luciferase activity of WNT7B 3ʹUTR-WT and WNT7B 3ʹUTR-MUT in Saos-2 and SOSP-9607 cells co-transfected with miR-1182 or miR-NC. (D and E) RIP and qRT-PCR assays for the enrichment of miR-1182 and WNT7B in the samples bound to the anti-Ago2 or anti-IgG. (F) QRT-PCR assay for WNT7B mRNA expression in Saos-2 and SOSP-9607 cells transfected with anti-miR-NC or anti-miR-1182. (G) Western blot assay for WNT7B protein expression in Saos-2 and SOSP-9607 cells transfected with anti-miR-NC or anti-miR-1182. (H) QRT-PCR assay for the expression level of WNT7B mRNA expression in OS tissues and normal tissues. (I) Western blot assay for WNT7B protein expression in OS tissues and normal tissues (N=3). *P<0.05.

Figure 6
Figure 6

MiR-1182 overexpression repressed cell proliferation, metastasis and glycolysis, while facilitated cell apoptosis via targeting WNT7B. (A) QRT-PCR assay for the expression level of WNT7B mRNA expression in hFOB, Saos-2 and SOSP-9607 cells. (B) Western blot assay for WNT7B protein expression in hFOB, Saos-2 and SOSP-9607 cells. (C) QRT-PCR assay for the expression level of WNT7B mRNA expression in Saos-2 and SOSP-9607 cells transfected with pcDNA or pcDNA-WNT7B. (D) Western blot assay for WNT7B protein expression in Saos-2 and SOSP-9607 cells transfected with pcDNA or pcDNA-WNT7B. (E–P) Saos-2 and SOSP-9607 cells were transfected with miR-NC, miR-1182, miR-1182+pcDNA or miR-1182+pcDNA-WNT7B. (E and F) MTT assay for the cell viability of transfected cells. (G) Flow cytometry assay for the cell apoptosis of transfected cells. (H) Wound healing assay for the migrated rate of transfected cells. (I and J) Transwell assay for the cell migration and invasion of transfected cells. (K–N) ECAR assay for the transfected cells. (O and P) Western blot assay for the protein markers of glycolysis in transfected cells. *P<0.05.

Figure 7
Figure 7

Circ_0105346 upregulated WNT7B expression by sponging miR-1182. (A) QRT-PCR assay for the expression level of WNT7B mRNA expression in Saos-2 and SOSP-9607 cells transfected with si-NC, si-circ_0105346, si-circ_0105346+anti-miR-NC or si-circ_0105346+anti-miR-1182. (B) Western blot assay for WNT7B protein expression in Saos-2 and SOSP-9607 cells transfected with si-NC, si-circ_0105346, si-circ_0105346+anti-miR-NC or si-circ_0105346+anti-miR-1182. (C–E) Pearson correlation analysis for the expression levels of circ_0105346, miR-1182 and WNT7B in OS tissues (N=40). *P<0.05.

Figure 8
Figure 8

Circ_0105346 knockdown blocked tumor growth in vivo. Saos-2 cells stably transfected with sh-circ_0105346 or sh-NC were subcutaneously injected into nude mice. (A) The surviving rate of nude mice after injection. (B) The volume of generated tumors measured every 7 days. (C) The images and weight of generated tumors. (D) QRT-PCR assay for the expression levels of circ_0105346 and miR-1182 in generated tumors. (E) QRT-PCR assay for the expression level of WNT7B mRNA expression in generated tumors. (F) Western blot assay for protein expression of WNT7B, PCNA, Cyclin D1, Bax, Bcl-2, EMT-like-related proteins and glycolysis protein markers in generated tumors. *P<0.05. ***P<0.001.

Figure 9
Figure 9

Schematic diagram of circ_0105346 regulating the proliferation, migration, invasion and glycolysis of OS cells.

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