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. 2024 Apr 7;5(4):e543.
doi: 10.1002/mco2.543. eCollection 2024 Apr.

miR-1246 promotes osteosarcoma cell migration via NamiRNA-enhancer network dependent on Argonaute 2

Shuai Yang  1 , Qingping Zou  1 , Ying Liang  1 , Dapeng Zhang  2 , Lina Peng  1 , Wei Li  1 , Wenxuan Li  1 , Mengxing Liu  1 , Ying Tong  1 , Lu Chen  1 , Peng Xu  1 , Zhicong Yang  1 , Kaicheng Zhou  1 , Jianru Xiao  3 , Hailin Wang  2 , Wenqiang Yu  1
Affiliations

miR-1246 promotes osteosarcoma cell migration via NamiRNA-enhancer network dependent on Argonaute 2

Shuai Yang et al. MedComm (2020). .

Abstract

High metastatic propensity of osteosarcoma leads to its therapeutic failure and poor prognosis. Although nuclear activation miRNAs (NamiRNAs) are reported to activate gene transcription via targeting enhancer and further promote tumor metastasis, it remains uncertain whether NamiRNAs regulate osteosarcoma metastasis and their exact mechanism. Here, we found that extracellular vesicles of the malignant osteosarcoma cells (143B) remarkably increased the migratory abilities of MNNG cells representing the benign osteosarcoma cells by two folds, which attributed to their high miR-1246 levels. Specially, miR-1246 located in nucleus could activate the migration gene expression (such as MMP1) to accelerate MNNG cell migration through elevating the enhancer activities via increasing H3K27ac enrichment. Instead, MMP1 expression was dramatically inhibited after Argonaute 2 (AGO2) knockdown. Notably, in vitro assays demonstrated that AGO2 recognized the hybrids of miR-1246 and its enhancer DNA via PAZ domains to prevent their degradation from RNase H and these protective roles of AGO2 may favor the gene activation by miR-1246 in vivo. Collectively, our findings suggest that miR-1246 could facilitate osteosarcoma metastasis through interacting with enhancer to activate gene expression dependent on AGO2, highlighting the nuclear AGO2 as a guardian for NamiRNA-targeted gene activation and the potential of miR-1246 for osteosarcoma metastasis therapy.

Keywords: Argonaute 2; enhancer; metastasis; miR‐1246; nuclear activation miRNAs; osteosarcoma.

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Conflict of interest statement

The authors declare no potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Extracellular vesicles facilitate the alteration in aggressive state of osteosarcoma cells. (A and B) Comparison of the cell proliferation (A) and migration (B) in 143B and MNNG cells. (C) Experimental scheme on the evaluation of MNNG cell migration under different conditions. Top: Coculture of MNNG cells with MNNG or 143B cells. Bottom: MNNG cells are cultured with medium supernatant (MS) or extracellular vesicles (EVs) from 143B cells. (D–F) Evaluation of the migratory ability in MNNG cells under different culture associated with 143B cells. Left: representative images of the MNNG cell migration under different culture conditions including noncontacting culture with MNNG cells (top) or 143B cells (bottom) in (D), incubation with the culture MS of MNNG cells (top) or 143B cells (bottom) in (E), and incubation with the extracellular vesicles derived from MNNG cells (top) or 143B cells (bottom) in (F). Scale bars, 200 µm. Right: quantification of the migrated MNNG cells under these conditions. Data are shown as mean ± Standard Deviation (SD). p alues are calculated using the Student's t‐test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
FIGURE 2
FIGURE 2
EVs‐derived miR‐1246 determines the migration potential of osteosarcoma cells. (A) Profile of miRNA enriched in extracellular vesicles of 143B cells. (B) Heatmap of the differentially enriched miRNAs between 143B and MNNG cells. (C) RT‐qPCR verifies the differential miRNAs between 143B and MNNG cells. (D–F) Detection of cell migration in 143B cells dealt with miR‐1246 inhibitors (D) or MNNG cells dealt with miR‐1246 mimics (E) or miR‐1246 overexpressed MNNG cells (F). Scale bars, 200 µm. The histograms showing the quantification of the migrated cell numbers of 143B or MNNG cells after these different treatments. Data in (C–F) are shown as mean ± SD. p values are calculated using the Student's t‐test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
FIGURE 3
FIGURE 3
miR‐1246 promotes the osteosarcoma aggression via activating gene transcription in relation to cell migration. (A) Transcriptome analysis of the differential genes in miR‐1246‐overpressed MNNG cells. (B) Gene Ontology (GO) analysis on the upregulated genes after miR‐1246 overexpression in MNNG cells. (C) Differential expression genes for the GO terms associated with cell migration. (D) RT‐qPCR detects the mRNA expression of migration‐related genes in miR‐1246‐overpressed MNNG cells. Data are shown as mean ± SD. p values are calculated using the Student's t‐test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
FIGURE 4
FIGURE 4
miR‐1246 in nucleus activates the transcription of migration genes in HEK293T cells. (A) Represented confocal images showing the location of miR‐1246 in cytoplasm and nucleus of HEK293T cells. The red arrows indicate the miR‐1246 mimics in the nucleus. Blue: DAPI; green: miR‐1246 mimics labeled with FAM. Scale bars, 50 µm. (B and C) RT‐qPCR detects the expression of nuclear miR‐1246 in miR‐1246 mimic transfected HEK293T cells (B) and MMP1 expression in miR‐1246‐overexpressed HEK293T cells (C). (D) Western blot verifies the expression of MMP1 in HEK293T cells after miR‐1246 overexpression. (E) RT‐qPCR assaying the MMP1 expression in HEK293T with the transfection of different plasmids. HEK293T cells are transfected with miR‐1246 expressed plasmid (WT), miR‐1246 expressed plasmids with deletion or mutation of miR‐1246, or empty plasmid (control). Data are shown as mean ± SD. p values are calculated using the Student's t‐test in (B and C) or one‐way ANOVA in (E). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
FIGURE 5
FIGURE 5
miR‐1246 activates gene transcription via targeting enhancer dependent on AGO2. (A) ChIP‐seq analysis for H3K27ac modification in miR‐1246‐overpressed MNNG cells. (B) Peak of H3K27ac modification at the genomic enhancer regions of miR‐1246 locus in miR‐1246‐overpressed MNNG cells. (C and D) ChIP‐qPCR confirms the enrichments of H3K27ac markers at the miR‐1246 (C) and MMP1 enhancer loci (D) in miR‐1246‐overpressed MNNG cells. IGV image showing the enrichment of H3K27ac at the corresponding regions. (E) RT‐qPCR determines the adjacent gene expression of miR‐1246 locus in miR‐1246‐overpressed MNNG cells. (F) Luciferase reporter assay assesses the enhancer activity of miR‐1246 and MMP1 loci in HEK293T cells using the indicated plasmids. (G) ChIP‐qPCR evaluates the AGO2 enrichment at the enhancer of miR‐1246 and MMP1 loci in miR‐1246‐overpressed MNNG cells. (H) RT‐qPCR determines the mRNA levels of genes activated by miR‐1246 in MNNG cells after knockdown of AGO2. Data are shown as mean ± SD in (C–H). p values are calculated using the Student's t‐test in (C–E and G) or one‐way ANOVA in (F and H). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
FIGURE 6
FIGURE 6
High affinity binding of AGO2 to miR‐1246/ssDNA hybrids blocks RNase H‐mediated degradation. (A–C) EMSA detects the affinity binding of AGO2 to miR‐1246 (A), miR‐1246/ssDNA hybrids (B), R loop analogs containing miR‐1246 (C). (D) The estimated dissociation constants (K d) for the binding of synthesized nucleic acid probes to hAGO2. (E and F) EMSA shows the affinity binding of PYZ domain of AGO2 to miR‐1246 (E), miR‐1246/ssDNA hybrids (F). (G) Left: schematic diagram of RNase H cleavage assay on the complex of AGO2 with miR‐1246/ssDNA hybrids. Right: EMSA evaluates the potential protective roles of AGO2 for the stability of miR‐1246/ssDNA hybrids against RNase H. In these EMSA experiments, miR‐1246 or ssDNA are labeled with CY5 to show their migration. miR1246/ssDNA hybrids are formed by annealing of miR‐1246 with its complementary ssDNA. R loop analogs are the miR‐1246/DNA loop mimics.
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