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MEG3 interacts with <t>CTCF</t> to restrain the transcriptional activation on CXCR4. (A) Venn diagram illustrates the proteins detected in our MEG3 mass spectrometry data, predicted MEG3 binding proteins from the AnnoLnc2 database, CXCR4-ChIP-seq data from the Cistrome toolkit database, and data from genetic perturbation similarity analysis for CXCR4 upstream regulators from the GPSAdb (Duplicated gene names were removed). (B) RT-PCR was performed to analyze MEG3 lncRNA precipitated from RIP experiment with a CTCF antibody, IgG antibody was used as a control. (C) RNA-FISH was used to demonstrate the co-localization of the full-length MEG3 transcript and the CTCF-EGFP protein. (D) Western blot analysis was conducted on MDA-MB-231 cells following the knockdown of CTCF using two siRNAs to assess the impact on CXCR4 expression (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Wound healing assay to evaluate the migratory capacity of MDA-MB-231 cells after the downregulation of CXCR4 caused by CTCF knockdown (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001) (the scale bar represents 500 μm). (F) Transwell assay for MDA-MB-231 after CTCF depletion (n = 3, ∗∗∗p < 0.001) (the scale bar represents 200 μm). (G&H) Wound healing assay and Transwell assay for MEG3 overexpression and CTCF depletion dual treatment (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: no significance) (the scale bar represents 500 μm for Wound healing assay and 200 μm for Transwell assay). (I) Western blot analysis showing the effect of MEG3 overexpression in combination with CTCF depletion on CXCR4 gene expression (n = 3, ∗∗∗p < 0.001, ns: no significance). (J) Western blot analysis showing the effect of dual overexpression of MEG3 and CTCF on CXCR4 gene expression (n = 3, ∗p < 0.05, ∗∗p < 0.01, ns: no significance).
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MEG3 interacts with <t>CTCF</t> to restrain the transcriptional activation on CXCR4. (A) Venn diagram illustrates the proteins detected in our MEG3 mass spectrometry data, predicted MEG3 binding proteins from the AnnoLnc2 database, CXCR4-ChIP-seq data from the Cistrome toolkit database, and data from genetic perturbation similarity analysis for CXCR4 upstream regulators from the GPSAdb (Duplicated gene names were removed). (B) RT-PCR was performed to analyze MEG3 lncRNA precipitated from RIP experiment with a CTCF antibody, IgG antibody was used as a control. (C) RNA-FISH was used to demonstrate the co-localization of the full-length MEG3 transcript and the CTCF-EGFP protein. (D) Western blot analysis was conducted on MDA-MB-231 cells following the knockdown of CTCF using two siRNAs to assess the impact on CXCR4 expression (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Wound healing assay to evaluate the migratory capacity of MDA-MB-231 cells after the downregulation of CXCR4 caused by CTCF knockdown (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001) (the scale bar represents 500 μm). (F) Transwell assay for MDA-MB-231 after CTCF depletion (n = 3, ∗∗∗p < 0.001) (the scale bar represents 200 μm). (G&H) Wound healing assay and Transwell assay for MEG3 overexpression and CTCF depletion dual treatment (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: no significance) (the scale bar represents 500 μm for Wound healing assay and 200 μm for Transwell assay). (I) Western blot analysis showing the effect of MEG3 overexpression in combination with CTCF depletion on CXCR4 gene expression (n = 3, ∗∗∗p < 0.001, ns: no significance). (J) Western blot analysis showing the effect of dual overexpression of MEG3 and CTCF on CXCR4 gene expression (n = 3, ∗p < 0.05, ∗∗p < 0.01, ns: no significance).
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MEG3 interacts with <t>CTCF</t> to restrain the transcriptional activation on CXCR4. (A) Venn diagram illustrates the proteins detected in our MEG3 mass spectrometry data, predicted MEG3 binding proteins from the AnnoLnc2 database, CXCR4-ChIP-seq data from the Cistrome toolkit database, and data from genetic perturbation similarity analysis for CXCR4 upstream regulators from the GPSAdb (Duplicated gene names were removed). (B) RT-PCR was performed to analyze MEG3 lncRNA precipitated from RIP experiment with a CTCF antibody, IgG antibody was used as a control. (C) RNA-FISH was used to demonstrate the co-localization of the full-length MEG3 transcript and the CTCF-EGFP protein. (D) Western blot analysis was conducted on MDA-MB-231 cells following the knockdown of CTCF using two siRNAs to assess the impact on CXCR4 expression (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Wound healing assay to evaluate the migratory capacity of MDA-MB-231 cells after the downregulation of CXCR4 caused by CTCF knockdown (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001) (the scale bar represents 500 μm). (F) Transwell assay for MDA-MB-231 after CTCF depletion (n = 3, ∗∗∗p < 0.001) (the scale bar represents 200 μm). (G&H) Wound healing assay and Transwell assay for MEG3 overexpression and CTCF depletion dual treatment (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: no significance) (the scale bar represents 500 μm for Wound healing assay and 200 μm for Transwell assay). (I) Western blot analysis showing the effect of MEG3 overexpression in combination with CTCF depletion on CXCR4 gene expression (n = 3, ∗∗∗p < 0.001, ns: no significance). (J) Western blot analysis showing the effect of dual overexpression of MEG3 and CTCF on CXCR4 gene expression (n = 3, ∗p < 0.05, ∗∗p < 0.01, ns: no significance).
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MEG3 interacts with <t>CTCF</t> to restrain the transcriptional activation on CXCR4. (A) Venn diagram illustrates the proteins detected in our MEG3 mass spectrometry data, predicted MEG3 binding proteins from the AnnoLnc2 database, CXCR4-ChIP-seq data from the Cistrome toolkit database, and data from genetic perturbation similarity analysis for CXCR4 upstream regulators from the GPSAdb (Duplicated gene names were removed). (B) RT-PCR was performed to analyze MEG3 lncRNA precipitated from RIP experiment with a CTCF antibody, IgG antibody was used as a control. (C) RNA-FISH was used to demonstrate the co-localization of the full-length MEG3 transcript and the CTCF-EGFP protein. (D) Western blot analysis was conducted on MDA-MB-231 cells following the knockdown of CTCF using two siRNAs to assess the impact on CXCR4 expression (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Wound healing assay to evaluate the migratory capacity of MDA-MB-231 cells after the downregulation of CXCR4 caused by CTCF knockdown (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001) (the scale bar represents 500 μm). (F) Transwell assay for MDA-MB-231 after CTCF depletion (n = 3, ∗∗∗p < 0.001) (the scale bar represents 200 μm). (G&H) Wound healing assay and Transwell assay for MEG3 overexpression and CTCF depletion dual treatment (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: no significance) (the scale bar represents 500 μm for Wound healing assay and 200 μm for Transwell assay). (I) Western blot analysis showing the effect of MEG3 overexpression in combination with CTCF depletion on CXCR4 gene expression (n = 3, ∗∗∗p < 0.001, ns: no significance). (J) Western blot analysis showing the effect of dual overexpression of MEG3 and CTCF on CXCR4 gene expression (n = 3, ∗p < 0.05, ∗∗p < 0.01, ns: no significance).
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MEG3 interacts with <t>CTCF</t> to restrain the transcriptional activation on CXCR4. (A) Venn diagram illustrates the proteins detected in our MEG3 mass spectrometry data, predicted MEG3 binding proteins from the AnnoLnc2 database, CXCR4-ChIP-seq data from the Cistrome toolkit database, and data from genetic perturbation similarity analysis for CXCR4 upstream regulators from the GPSAdb (Duplicated gene names were removed). (B) RT-PCR was performed to analyze MEG3 lncRNA precipitated from RIP experiment with a CTCF antibody, IgG antibody was used as a control. (C) RNA-FISH was used to demonstrate the co-localization of the full-length MEG3 transcript and the CTCF-EGFP protein. (D) Western blot analysis was conducted on MDA-MB-231 cells following the knockdown of CTCF using two siRNAs to assess the impact on CXCR4 expression (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Wound healing assay to evaluate the migratory capacity of MDA-MB-231 cells after the downregulation of CXCR4 caused by CTCF knockdown (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001) (the scale bar represents 500 μm). (F) Transwell assay for MDA-MB-231 after CTCF depletion (n = 3, ∗∗∗p < 0.001) (the scale bar represents 200 μm). (G&H) Wound healing assay and Transwell assay for MEG3 overexpression and CTCF depletion dual treatment (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: no significance) (the scale bar represents 500 μm for Wound healing assay and 200 μm for Transwell assay). (I) Western blot analysis showing the effect of MEG3 overexpression in combination with CTCF depletion on CXCR4 gene expression (n = 3, ∗∗∗p < 0.001, ns: no significance). (J) Western blot analysis showing the effect of dual overexpression of MEG3 and CTCF on CXCR4 gene expression (n = 3, ∗p < 0.05, ∗∗p < 0.01, ns: no significance).
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MEG3 interacts with CTCF to restrain the transcriptional activation on CXCR4. (A) Venn diagram illustrates the proteins detected in our MEG3 mass spectrometry data, predicted MEG3 binding proteins from the AnnoLnc2 database, CXCR4-ChIP-seq data from the Cistrome toolkit database, and data from genetic perturbation similarity analysis for CXCR4 upstream regulators from the GPSAdb (Duplicated gene names were removed). (B) RT-PCR was performed to analyze MEG3 lncRNA precipitated from RIP experiment with a CTCF antibody, IgG antibody was used as a control. (C) RNA-FISH was used to demonstrate the co-localization of the full-length MEG3 transcript and the CTCF-EGFP protein. (D) Western blot analysis was conducted on MDA-MB-231 cells following the knockdown of CTCF using two siRNAs to assess the impact on CXCR4 expression (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Wound healing assay to evaluate the migratory capacity of MDA-MB-231 cells after the downregulation of CXCR4 caused by CTCF knockdown (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001) (the scale bar represents 500 μm). (F) Transwell assay for MDA-MB-231 after CTCF depletion (n = 3, ∗∗∗p < 0.001) (the scale bar represents 200 μm). (G&H) Wound healing assay and Transwell assay for MEG3 overexpression and CTCF depletion dual treatment (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: no significance) (the scale bar represents 500 μm for Wound healing assay and 200 μm for Transwell assay). (I) Western blot analysis showing the effect of MEG3 overexpression in combination with CTCF depletion on CXCR4 gene expression (n = 3, ∗∗∗p < 0.001, ns: no significance). (J) Western blot analysis showing the effect of dual overexpression of MEG3 and CTCF on CXCR4 gene expression (n = 3, ∗p < 0.05, ∗∗p < 0.01, ns: no significance).

Journal: Non-coding RNA Research

Article Title: LncRNA MEG3/CTCF-CXCR4 axis functions in the regulation of breast cancer cell migration

doi: 10.1016/j.ncrna.2025.05.014

Figure Lengend Snippet: MEG3 interacts with CTCF to restrain the transcriptional activation on CXCR4. (A) Venn diagram illustrates the proteins detected in our MEG3 mass spectrometry data, predicted MEG3 binding proteins from the AnnoLnc2 database, CXCR4-ChIP-seq data from the Cistrome toolkit database, and data from genetic perturbation similarity analysis for CXCR4 upstream regulators from the GPSAdb (Duplicated gene names were removed). (B) RT-PCR was performed to analyze MEG3 lncRNA precipitated from RIP experiment with a CTCF antibody, IgG antibody was used as a control. (C) RNA-FISH was used to demonstrate the co-localization of the full-length MEG3 transcript and the CTCF-EGFP protein. (D) Western blot analysis was conducted on MDA-MB-231 cells following the knockdown of CTCF using two siRNAs to assess the impact on CXCR4 expression (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Wound healing assay to evaluate the migratory capacity of MDA-MB-231 cells after the downregulation of CXCR4 caused by CTCF knockdown (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001) (the scale bar represents 500 μm). (F) Transwell assay for MDA-MB-231 after CTCF depletion (n = 3, ∗∗∗p < 0.001) (the scale bar represents 200 μm). (G&H) Wound healing assay and Transwell assay for MEG3 overexpression and CTCF depletion dual treatment (n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: no significance) (the scale bar represents 500 μm for Wound healing assay and 200 μm for Transwell assay). (I) Western blot analysis showing the effect of MEG3 overexpression in combination with CTCF depletion on CXCR4 gene expression (n = 3, ∗∗∗p < 0.001, ns: no significance). (J) Western blot analysis showing the effect of dual overexpression of MEG3 and CTCF on CXCR4 gene expression (n = 3, ∗p < 0.05, ∗∗p < 0.01, ns: no significance).

Article Snippet: Antibodies targeted to CTCF (Proteintech, 30428-1-AP, 1:1000), GAPDH (Proteintech, 10494-1-AP, 1:10000), MYH9 (Proteintech, 14844-1-AP, 1:500), PES1 (Proteintech, 13553-1-AP, 1:1000), IKBIP (Proteintech, 14589-1-AP, 1:500), PHB (Proteintech, 10787-1-AP, 1:1000) and CXCR4 (ABclonal, A19035, 1:1000) were used in Western blot.

Techniques: Activation Assay, Mass Spectrometry, Binding Assay, ChIP-sequencing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Knockdown, Expressing, Wound Healing Assay, Transwell Assay, Over Expression, Gene Expression