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. 2020 Oct 15:22:957-970.
doi: 10.1016/j.omtn.2020.10.010. eCollection 2020 Dec 4.

lncRNA SNHG10 Promotes the Proliferation and Invasion of Osteosarcoma via Wnt/β-Catenin Signaling

Shutao Zhu  1 , Yang Liu  1 , Xiao Wang  1 , Junyi Wang  1 , Guanghui Xi  1
Affiliations

lncRNA SNHG10 Promotes the Proliferation and Invasion of Osteosarcoma via Wnt/β-Catenin Signaling (VSports在线直播)

Shutao Zhu et al. Mol Ther Nucleic Acids. .

Abstract

Uncontrolled growth and an enforced epithelial-mesenchymal transition (EMT) process contribute to the poor survival rate of patients with osteosarcoma (OS). Long noncoding RNAs (lncRNAs) have been reported to be involved in the development of OS. However, the significant role of lncRNA SNHG1O on regulating proliferation and the EMT process of OS cells remains unclear. In this study, quantitative real-time PCR and fluorescence in situ hybridization (FISH) results suggested that SNHG10 levels were significantly increased in OS compared with healthy tissues. In vitro experiments (including colony formation, CCK-8, wound healing, and transwell assays) and in vivo experiments indicated that downregulation of SNHG10 significantly suppressed the proliferation and invasion of OS cells. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay confirmed that SNHG10 could regulate FZD3 levels through sponging microRNA 182-5p (miR-182-5p). In addition, the SNHG10/miR-182-5p/FZD3 axis could further promote the β-catenin transfer into nuclear accumulation to maintain the activation of the Wnt singling pathway. Together, our results established that SNHG10 has an important role in promoting OS growth and invasion VSports手机版. By sponging miR-182-5p, SNHG10 can increase FZD3 expression and further maintain the activation of Wnt/β-catenin singling pathway in OS cells. .

Keywords: EMT; SNHG10; Wnt/β-catenin; lncRNA; osteosarcoma V体育安卓版. .

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None
Graphical abstract
Figure 1
Figure 1
LncRNA SNHG10 Is Overexpressed in OS and Is Associated with Poor Prognosis (A) Relative expression of SNHG10 in OS and adjacent normal samples were quantified by quantitative real-time PCR. (B) Relative expression of SNHG10 in different OS cell lines and healthy bronchial epithelial cells were quantified by quantitative real-time PCR. (C) Relative expression of SNHG10 in OS samples was analyzed according to the tumor stage. (D) Relative expression of SNHG10 in OS samples was analyzed according to metastasis status. (E) Forty-five OS samples were divided into two groups according to whether expression of SNHG10 was high or low. (F) Kaplan-Meier analysis was used to determine the association between SNHG4 high or low expression and overall survival of patients with OS. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Figure 2
Figure 2
Knockdown of SNHG10 Restrains OS Progression In Vitro (A) Relative expression of SNHG10 in OS cells was quantified by quantitative real-time PCR after transfection of sh-control (sh-ctrl) or sh-SNHG10. (B) The proliferation of transfected OS cells was evaluated with a CCK-8 assay. (C) The proliferation of transfected OS cells was evaluated with a colony formation assay. (D) The expression of cyclin D1 and E1 was quantified by western blot after transfection of sh-ctrl or sh-SNHG10. (E) The migration of transfected OS cells was evaluated with a wound healing assay. (F) The invasion of transfected OS cells was evaluated with a transwell assay. (G) The expression of E-cadherin, N-cadherin, and vimentin was quantified by western blot after transfection of sh-ctrl or sh-SNHG10. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Figure 3
Figure 3
SNHG10 Serves as a Sponge for miR-182-5p (A) FISH analysis indicated a subcellular location for SNHG10 in OS cells (green). Nuclei were stained with DAPI (blue). (B) Relative SNHG10 expression levels in nuclear and cytosolic fractions of the OS cells were quantified by quantitative real-time PCR. (C) A schematic drawing of the screening procedure of candidate miRNAs. (D) The luciferase reporter plasmids carrying SNHG10 were co-transfected into HEK293T cells with two miRNA-coding plasmids. (E) A schematic representation of the miR-182-5p binding sites in SNHG10 and site mutagenesis. (F) The luciferase reporter plasmid carrying wild-type (WT) or mutant (MUT) SNHG10 was co-transfected into OS cells with miR-182-5p in parallel with an empty vector. Relative luciferase activity in OS cells was determined. (G) Ago2 RIP assay analysis of the enrichment of SNHG10 and miR-182-5p pulled down from the Ago2 protein in OS cells, and the expression levels of SNHG10 and miR-182-5p was examined by quantitative real-time PCR analysis. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Figure 4
Figure 4
SNHG10 Promotes OS Progression through miR-182-5p (A) The proliferation of OS cells after transfection with sh-SNHG10 or co-transfection with a miR-182-5p mimic was evaluated with a CCK-8 assay. (B) The proliferation of OS cells after transfection with sh-SNHG10 or co-transfection with a miR-182-5p mimic was evaluated using a colony formation assay. (C) The expression of cyclin D1 and E1 in OS cells after transfection with sh-SNHG10 or co-transfection with a miR-182-5p mimic was quantified by western blot. (D) The expressions of E-cadherin, N-cadherin, and vimentin were quantified using western blot after transfection of sh-SNHG10 or co-transfection with a miR-182-5p mimic. (E) The migration of OS cells after transfection with sh-SNHG10 or co-transfection with a miR-182-5p mimic was evaluated with a wound healing assay. (F) The invasion of OS cells after transfection with sh-SNHG10 or co-transfection with a miR-182-5p mimic was evaluated with a transwell assay. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Figure 5
Figure 5
FZD3 Is a Target of miR-182-5p (A) Schematic of the candidate genes of miR-182-5p using four prediction tools. (B) GO and KEGG pathway analysis of candidate targets of miR-182-5p. (C) Schematic of the common candidate genes involved in regulating the Wnt/β-catenin signaling pathway. (D) mRNA expression of PPP3R1, FZD3, and PPP3CA in OS cells transfected with miR-NC or a miR-182-5p mimic. (E) Predicted miR-182-5p target sequences in the 3′ UTRs of FZD3 genes. (F) Relative FZD3 reporter activities in OS cells co-transfected with miR-182-5p and luciferase reporters. (G) Immunoprecipitation of the Ago2/RISC (RNA-induced silencing complex) using the Pan-Ago2 antibody in OS cells overexpressing miR-NC or miR-182-5p. IgG was used as a negative control and β-actin was used as an internal control. (H) Quantitative real-time PCR analysis of miR-182-5p incorporated into RISC in OS cells overexpressing miR-182-5p compared with the levels in the control. (I) Quantitative real-time PCR of FZD3 incorporated into RISC in OS cells overexpressing miR-182-5p. (J) FZD3 expression levels in OS cells transfected with a miR-182-5p mimic or anti-miR-182-5p were quantified by western blot. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Figure 6
Figure 6
SNHG10 Promotes OS Progression through FZD3 (A) The proliferation of OS cells after transfection with sh-SNHG10 or co-transfection with FZD3 was evaluated with a CCK-8 assay. (B) The proliferation of OS cells after transfection with sh-SNHG10 or co-transfection with FZD3 was evaluated with a colony-formation assay. (C) The expression of cyclin D1 and E1 in OS cells after transfection with sh-SNHG10 or co-transfection with FZD3 was quantified by western blot. (D) The expressions of E-cadherin, N-cadherin, and vimentin were quantified using western blot after transfection of sh-SNHG10 or co-transfection with FZD3. (E) The migration of OS cells after transfection with sh-SNHG10 or co-transfection with FZD3 was evaluated with a wound-healing assay. (F) The invasion of OS cells after transfection with sh-SNHG10 or co-transfection with FZD3 was evaluated with a transwell assay. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Figure 7
Figure 7
SNHG10 Activates Wnt/β-Catenin Pathway to Promote OS Progression (A) β-catenin protein redistribution in different cellular compartments of transfected OS cells was quantified by western blot. (B) β-catenin protein redistribution in different cellular compartments of transfected OS cells was quantified using immunofluorescence staining. Scale bar, 100 μm. (C) Relative expression levels of Wnt/β-catenin pathway downstream target genes were quantified by real-time PCR. (D) Relative TCF/LEF reporter activity was measured after transfection with sh-SNHG10 or co-transfection with FZD3. (E) The β-catenin protein redistribution in different cellular compartments of transfected or XAV-939 treated OS cells was quantified using western blot. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Figure 8
Figure 8
SNHG10 Promotes OS Tumorigenesis In Vivo (A) Subcutaneous tumors were separated and imaged at the endpoint of the experiment. Scale bar, 1 cm. (B) Relative expression of SNHG10 in subcutaneous tumor tissues was quantified by quantitative real-time PCR. (C) Tumor growth curves of different subcutaneous tumor groups are shown. (D) Tumor weights of different tumor groups are shown. (E) The expression levels of FZD3, Ki-67, and vimentin in different groups of subcutaneous tumors were evaluated by IHC. In all experiments, bars represent means ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
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